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WO2025014933A1 - Targeting tim-3 in mapk-driven glioma - Google Patents

Targeting tim-3 in mapk-driven glioma Download PDF

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Publication number
WO2025014933A1
WO2025014933A1 PCT/US2024/037178 US2024037178W WO2025014933A1 WO 2025014933 A1 WO2025014933 A1 WO 2025014933A1 US 2024037178 W US2024037178 W US 2024037178W WO 2025014933 A1 WO2025014933 A1 WO 2025014933A1
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tim
inhibitor
glioma
cells
immune
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PCT/US2024/037178
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French (fr)
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Amy Heimberger
Michael DECUYPERE
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Northwestern University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • compositions and methods for the treatment of glioma by targeting TIM-3 are provided herein.
  • a subject suffering from a glioma is administered a TIM-3 inhibitor alone or in combination with other glioma therapies.
  • the location of the immune cells is also variable. For example, certain T cell populations in high- and low-grade pediatric gliomas are close to vasculature while others such as CD 103+ T cells reside further away from the vasculature. Immune cell infiltration is also different between primary vs. recurrent tumors. Differences in immune surveillance have been shown in studies comparing pediatric vs. adult gliomas including: (i) low-levels of expression of programmed cell death protein ligand 1 (PD-L1) and NKG2D ligand; and (ii) the lack of prognostic significance associated with immunosuppressive CD 163+ macrophage infiltration in pediatric tumors. As such, it has been postulated that the pediatric brain TME may reflect a failure of immune surveillance rather than the establishment of an immunosuppressive environment as is seen in adult tumors.
  • PD-L1 programmed cell death protein ligand 1
  • NKG2D ligand NKG2D ligand
  • Immunological reactivity is a function of age.
  • age related T cell anergy, exhaustion, and senescence defects in activation of the inflammasome, and decreased innate immunity (.
  • CD8 T cell recent thymic emigrants are at least one factor that accounts for the prognostic power of age on clinical outcome in adult glioblastoma patients.
  • CD8 T cell RTEs account for the majority of tumor antigen-binding cells in the peripheral blood and are expanded following vaccination and are associated with clinical outcomes.
  • Preclinical modeling in mutant (CD8beta(-/-)) mice display an age-specific decrease in glioma host survival as well as a correlation between host survival and thymus cellular production.
  • Pilocytic astrocytomas are the most common pediatric glioma. Surgical resection of PA is typically the first-line treatment and gross total resection is often curative. However, PAs that are not amenable to gross total resection have associated long-term morbidity and mortality. The ten-year progression-free survival for PA patients with radiologically visible residual tumor is less than 50%. Other treatment options are frequently necessary in gliomas with a midline location, which can be challenging to biopsy or resect safely. Some PA undergo spontaneous regression after partial resection, which suggests a predisposition to immune surveillance and eradiation. PA can have a single driver BRAF rearrangement.
  • BRAFV600 expressing melanoma has been shown to be responsive to immune checkpoint inhibitor therapy, especially those with PD-L1 expression.
  • PD-L1 expression and immune cell infiltration are independent of BRAF V600E mutational status.
  • tumor mutational burden TMB
  • ICI immune checkpoint inhibitors
  • BRAF alterations are not typically associated with high TMB but may still be highly immunogenic to a sufficient degree to induce spontaneous regression and/or high propensity for response to immune therapy.
  • scRNAseq data of PA also indicate that microglia may constitute a high frequency immune cell population in these tumors. If and how PA microglia influence tumor immunoreactivity is unknown.
  • compositions and methods for the treatment of glioma by targeting TIM-3 are provided herein.
  • a subject suffering from a glioma is administered a TIM-3 inhibitor alone or in combination with other glioma therapies.
  • glioma is a MAPK-driven glioma.
  • the glioma is an astrocytoma.
  • the astrocytoma is a pilocytic astrocytomas (PA).
  • PA pilocytic astrocytomas
  • the subject exhibits one or more glioma biomarkers.
  • the subject has tested positive for a BRAF fusion, increased IFN, and/or increased p-ERK.
  • the BRAF fusion is a KIAA1549-BRAF fusion.
  • the subject is a child, adolescent, or early adult. In some embodiments, the subject is an adult.
  • the TIM-3 inhibitor is an inhibitor of TIM-3 activity. In some embodiments, the TIM-3 inhibitor is an anti-TIM-3 antibody. In some embodiments, the TIM-3 inhibitor is selected from RMT3- 23, ATIK2a, MBG453 (sabatolimab), AZD-7789, BMS-986258, INCAGN-2390, TSR-022 (Cobolimab), LY3321367, RO-7121661 (Lomvastomig), TQB-2618. In some embodiments, the TIM-3 inhibitor is Sabatolimab (MBG453).
  • the TIM-3 inhibitor is an antibody that binds to a ligand of TIM-3. In some embodiments, the TIM-3 inhibitor binds to galectin-9, HMGB1, ceacam-1, or phosphatidyl serine. In some embodiments, the TIM-3 inhibitor is a small molecule or peptide capable of binding to TIM-3 and/or a TIM-3 ligand. In some embodiments, the small molecule TIM-3 inhibitor is SMI402. In some embodiments, the TIM-3 inhibitor is an inhibitor of TIM-3 expression. In some embodiments, the TIM-3 inhibitor is a nucleic acid-based agent that reduces expression of TIM-3.
  • the TIM-3 inhibitor comprises one or more agents that allow for alteration of the TIM-3 gene and thereby reduce TIM-3 expression or activity.
  • the TIM-3 inhibitor is coadministered with one or more additional glioma therapies.
  • the TIM-3 inhibitor is co-administered with surgical removal of all or a portion of the glioma.
  • the TIM-3 inhibitor is co-administered with radiation therapy, chemotherapy, and/or an immunotherapy.
  • methods further comprise detecting one or more glioma biomarkers in the subject.
  • the glioma biomarkers are selected from a BRAF fusion, increased IFN, and/or increased p-ERK.
  • the BRAF fusion is a KIAA1549-BRAF fusion.
  • a TIM-3 inhibitor in the treatment of glioma in a subject.
  • FIG. 2A-G Inflammatory TME present in BRAF-Fusion PA.
  • A Schematic diagram of the workflow associated with orthogonal analyses of pediatric gliomas (Created with BioRender.com). scRNA Seq, single-cell RNA sequencing; GEMM, genetically engineered murine model.
  • B UMAP plot of glioma infiltrating immune cells analyzed with scRNAseq across 16 patients (13 BRAF-Fusion PA and 3 adjacent normal brains (ANB)).
  • D scRNA seq UMAP plot of the intratumoral myeloid cells shown in brown on panel B.
  • E scRNA seq UMAP plot of the intratumoral lymphoid cells shown in purple in panel B.
  • F Dot plot of immune marker genes within the myeloid subtypes (including the MG- Act population from panel E) characterized based on four distinct immunological functions: cytotoxicity, immune suppression, antigen presentation cell (APC), and phagocytosis. Bubble size corresponds to the percent of cells expressing the gene and colors indicate average expression.
  • G Dot plot of immune marker genes of the cell clusters shown in panel E broken into the four distinct immunological functions: cytotoxicity, immune suppression, antigen presentation cell (APC), and phagocytosis. Bubble size corresponds to the percent of cells expressing gene marker; shade indicates average expression.
  • Figure 3A-G Dot plot of immune marker genes within the myeloid subtypes (including the MG- Act population from panel E) characterized based on four distinct immunological functions: cytotoxicity, immune suppression, antigen presentation cell (APC), and phagocytosis. Bubble size corresponds to the
  • D) Histogram of the percent of TIM3+ cells that co-express CD1 lc+, P2RY12+, CD163+, GFAP+, or CD3+ within PA (n 8). Mean and SEM are displayed.
  • F) Representative image of the P2RY 12+CD3+NKG7+ MG- Act population within the TME of PA (n 8). Scale bars at 20 pm.
  • MFI mean fluorescence intensity
  • FIG. 5A-M A) Schema of the treatment of immunocompetent GEMM mice with either anti-TIM3 (300 pg/ mouse) or IgG (100 pg/mouse) once per week or anti-PD-1 (200ug/mouse) 3 times per week stalling at day 28 after induction of glioma.
  • F) Multiplex immunofluorescent imaging of TIM3 expression on P2RY12 microglia in the GEMM model (n 4). GFAP: purple; P2RY12: green; TIM3: yellow. Scale bars at 50 pm.
  • G) Multiplex immunofluorescent imaging p-ERKl/2 expression in the GEMM mode (n 4). Scale bar at 50 pm.
  • the survival rate of low-grade glioma GEMM mice was estimated by the Kaplan-Meier method.
  • IgG Control 20 mice (MS: 110.5 d), anti-PDl: 20 mice (MS: 134.5d), anti-TIM3: 21 mice (MS: 253d).
  • the survival rate of low-grade glioma CX3CR1 KO GEMM mice was estimated by the Kaplan-Meier method.
  • IgG Control 20 mice (MS: 122d), anti-TIM3: 28 mice (MS: 129.5).
  • FSC-H vs FSC-A to determine singlets.
  • SSC-A vs FSC-A to determine cells.
  • SSC-A vs LD for negative selection of live cells. The percentage of indicated subpopulation is displayed in blue.
  • LD Live/Dead fixable viability stain.
  • Myeloid cells are defined as live CD45+, CD1 lb+.
  • Lymphoid cells are defined as CD45+, CD 11b-.
  • FIG 7A-C A) Western plot of CT-2A cells showing p-MAPK expression.
  • Figure 9 Representative spatial multiplex immunofluorescence images of the adjacent normal brain showing the absence of TIM-3 and CD163 expression.
  • the vessels and anatomical layers of the normal cerebellar cortex (granular and white matter layers) are annotated. Scale bars at 100 pm.
  • FIG. 10 mRNA expression of HAVCR2, STAT3, and PDCD1 of all tumor types from 3 public databases (Griesinger, Gump, and Henriquez). mRNA expression levels are displayed after the Log2 transformation.
  • FIG 11A-D In vivo therapeutic effects of anti-TIM3 in the CT-2A glioma model.
  • IgG Control 10 mice (MS: 39 d), anti-PD-1: 10 mice (MS: 47 d), anti-TIM3: 10 mice (MS: 47d).
  • IgG Control 14 mice (MS: 105d), anti-T!M3: 19 mice (MS: undefined).
  • a TIM-3 inhibitor is a reference to one or more TIM-3 inhibitors and equivalents thereof known to those skilled in the art, and so forth.
  • the term “comprise” and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc.
  • the term “consisting of” and linguistic variations thereof denotes the presence of recited feature(s), element(s), method step(s), etc. and excludes any unrecited feature(s), element(s), method step(s), etc., except for ordinarily-associated impurities.
  • the phrase “consisting essentially of’ denotes the recited feature(s), element(s), method step(s), etc. and any additional feature(s), element(s), method step(s), etc.
  • compositions, system, or method that do not materially affect the basic nature of the composition, system, or method.
  • Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed “consisting of’ and/or “consisting essentially of” embodiments, which may alternatively be claimed or described using such language.
  • the term “pharmaceutically acceptable carrier” refers to non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, excipient, or carrier conventional in the art for use with a therapeutic agent for administration to a subject.
  • a pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
  • the pharmaceutically acceptable carrier is appropriate for the formulation employed.
  • the carrier may be a gel capsule.
  • a “pharmaceutical composition” typically comprises at least one active agent (e.g., the copolymers described herein) and a pharmaceutically acceptable carrier.
  • an effective amount refers to the amount of a composition (e.g., pharmaceutical composition) sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.
  • the term “administration” refers to the act of giving a drug, prodrug, or other agent, or therapeutic treatment (e.g., pharmaceutical compositions of the present invention) to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs.
  • exemplary routes of administration to the human body can be through the eyes (e.g., intraocularly, intravitreally, periocularly, ophthalmic, etc.), mouth (oral), skin (transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like.
  • co-administration refers to the administration of at least two agent(s) or therapies to a subject.
  • the coadministration of two or more agents or therapies is concurrent (e.g., in the same or separate formulations).
  • a first agent/therapy is administered prior to a second agent/therapy.
  • the appropriate dosage for coadministration can be readily determined by one skilled in the art.
  • agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone.
  • co-administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s).
  • the term “subject” broadly refers to any animal, including but not limited to, human and non-human animals (e.g., dogs, cats, cows, horses, sheep, poultry, fish, crustaceans, etc.).
  • the term “patient” typically refers to a subject that is being treated for a disease or condition.
  • the term “neonate”, when used in reference to a human subject, refers to a subject under 30 days of age.
  • child when used in reference to a human subject, refers to a subject that has yet to reach puberty.
  • an “immune response” refers to the action of a cell of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, neutrophils, etc.) and soluble macromolecules produced by any of these cells or the liver (e.g., antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from a subject of invading pathogens, cells or tissues infected with pathogens, or cancerous cells or other abnormal/diseased-associated cells.
  • a cell of the immune system e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, neutrophils, etc.
  • soluble macromolecules produced by any of these cells or the liver e.g., antibodies, cytokines, and complement
  • immunotherapy refers to the treatment or prevention of a disease or condition by a method comprising inducing, enhancing, suppressing or otherwise modifying an immune response.
  • immunotherapeutic refers to any agent (e.g., small molecule, peptide, antibody, engineered cell, etc.) capable of stimulating a host immune system to generate an immune response to a tumor or cancer in the subject.
  • agent e.g., small molecule, peptide, antibody, engineered cell, etc.
  • T-cell-based therapy refers to any immunotherapy that acts through T cells.
  • T-cell-based therapies include the administration of exogenous T cells (e.g., CAR-T cell therapies) and therapies that act upon or through a subjects endogenous T cells (e.g., checkpoint inhibitors.
  • antibody refers to a whole antibody molecule or a fragment thereof (e.g., fragments such as Fab, Fab', and F(ab')2), unless specified otherwise; an antibody may be polyclonal or monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, etc.
  • antibody fragment refers to a portion of a full-length antibody, including at least a portion antigen binding region or a variable region.
  • Antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, Fd, diabodies, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. See, e.g., Hudson et al. (2003) Nat. Med. 9:129-134; herein incorporated by reference in its entirety.
  • antibody fragments are produced by enzymatic or chemical cleavage of intact antibodies (e.g., papain digestion and pepsin digestion of antibody) produced by recombinant DNA techniques, or chemical polypeptide synthesis.
  • CAR chimeric antigen receptor
  • CAR-T cell refers to a T cell that has been engineered to express a chimeric antigen receptor.
  • T cells e.g., from a subject
  • ACT adaptive cell transfer
  • the cells may have originated from the patient or from another individual or cell line.
  • the cells are most commonly derived from the immune system, with the goal of improving immune functionality or eliciting a desired immune response.
  • cells are extracted from a subject, genetically modified (e.g., to express a desired construct (e.g., CAR or endanger molecule)), cultured in vitro, and returned to the subject.
  • a desired construct e.g., CAR or endanger molecule
  • diagnosis refers to the detection, determination, or recognition of a health status or condition of an individual on the basis of one or more signs, symptoms, data, or other information pertaining to that individual.
  • diagnosis encompass, with respect to a particular disease or condition, the initial detection of the disease; the characterization or classification of the disease; the detection of the progression, remission, or recurrence of the disease; and the detection of disease response after the administration of a treatment or therapy to the individual.
  • the diagnosis of glioma includes distinguishing individuals who have glioma (or are likely to) from individuals who do not (or are unlikely to).
  • prognosis refers to risk prediction of the severity of disease or of the probable course and clinical outcome associated with a disease.
  • method of prognosis refers to methods by which the skilled person can estimate and/or determine a probability that a given outcome will occur.
  • marker and “biomarker” are used interchangeably to refer to a target molecule or condition that indicates or is a sign of a normal or abnormal process in an individual or of a disease or other condition in an individual. Biomarkers may be detectable and measurable by a variety of methods including home, clinic, and laboratory assays.
  • compositions and methods for the treatment of glioma by targeting TIM-3 are provided herein.
  • a subject suffering from a glioma is administered a TIM-3 inhibitor alone or in combination with other glioma therapies.
  • gliomas are the leading cause of childhood mortality among childhood cancers. The repurposing of immunotherapies has not been successful in the treatment of pediatric gliomas.
  • whole transcriptome sequencing, single-cell sequencing, and sequential multiplex immunofluorescence were used to identify an immunotherapy strategy evaluated in multiple preclinical glioma models.
  • MAPK-driven pediatric gliomas have a higher interferon signature relative to other molecular subgroups.
  • Single-cell sequencing identified an activated and cytotoxic microglia population designated MG- Act in BRAF-fused MAPK-activated PA, but not in high-grade gliomas or normal brain.
  • TIM3 is expressed on MG- Act and on the myeloid cells lining the tumor vasculature but not normal brain. TIM3 expression becomes upregulated on immune cells in the PA microenvironment and anti-T!M3 reprograms ex vivo immune cells from human PAs to a pro-inflammatory cytotoxic phenotype.
  • anti-TIM3 treatment increased median survival over IgG and anti- PD1 treated mice. ScRNA sequencing data during the therapeutic window of anti-TIM3 demonstrates enrichment of the MG- Act population. The therapeutic activity of anti-TIM3 is abrogated in the CX3CR1 microglia knockout background.
  • MAPK-driven gliomas exhibited immune reactive signatures as indicated by IFN-y expression, as well as by heightened pro-inflammatory Ml macrophage and T cell component.
  • the tumor microenvironment (TME) of BRAF-fusion PA was notable for antigen presentation and the interaction of CD1 lc+ cells with T cells through Lck+ immunological synapses.
  • PA CD1 lc+CD206+ dendritic cells localized to vessel walls and were positive for TIM-3 expression, an immune checkpoint marker.
  • a unique highly activated microglia subcluster was identified that morphologically resembled T cells expressing TNF and TIM-3.
  • Treatment of an ex vivo PA with anti-TIM3 antibodies triggered the induction of pro-inflammatory responses in autologous immune cells.
  • Glioma is an aberrant growth of cells that begins in the brain or spinal cord and initially resembles healthy glial cells but is capable of growing into tumors that press on the brain or spinal cord resulting in symptoms. Malignant gliomas grow quickly and can invade healthy brain tissue.
  • Gliomas are classified as astrocytoma, ependymoma, glioblastoma, or oligodendroglioma, depending upon the types of cells from which they originate and the aggressiveness of the malignancy (astrocytoma - astrocytes, ependymoma - ependymal cells, glioblastoma - astrocytes (rapidly malignant), or oligodendroglioma - oligodendrocytes).
  • PA are the most common pediatric glioma. Surgical resection of PA is typically the first- line treatment and gross total resection is often curative (15, 16). However, PAs that are not amenable to gross total resection have associated long-term morbidity and mortality. The ten- year progression-free survival for PA patients with radiologically visible residual tumor is less than 50%. Other treatment options are frequently necessary in gliomas with a midline location, which can be challenging to biopsy or resect safely.
  • results are consistent with the results of others in suggesting potential benefit in targeting tumors with elevated MAPK activity. Since the expression of other immune checkpoints such as Lag-3, PD-1, and TIGIT are minimally co-expressed, this indicates that the T cells are not in a state of exhaustion that has been defined in adult high-grade gliomas.
  • TIM3 expression was primarily observed in myeloid cells, which is consistent with findings from orthotopic implanted high-grade glioma mouse models. In such models the combination of radiation, anti-TIM3 and anti-PD- 1 appeared to be curative of tumor. Preclinical testing has also shown that anti-TIM3 therapy shows synergistic anti-tumor activity when combined with adoptive immunotherapy therapeutics such as CAR T cells. No significant toxicity has been noted in preclinical studies involving TIM-3 targeting and experiments conducted during development of embodiments herein found minimal expression of TIM-3 within the adjacent normal brain.
  • TIM3 expression is on the endothelial side of the blood vasculature.
  • the role of TIM3 in this context is an area for future investigation but does provide a target for large molecules such as antibodies that are typically excluded by the blood brain barrier.
  • Companion biomarkers such as TMB, IFN signatures, PD-1, and PD-L1 expression have been used for identifying patients who may respond to ICT. However, with the exceptions of the IFN signature and p-ERK, these markers have not been predictive of response to ICI for adult glioblastoma patients. The expression of PD-1 and PD-L1 is lower relative to TIM3 in BRAF- Fusion PA. As such, the use of ICI targeting PD-1 or PD-L1 are unlikely to be of therapeutic benefit in this setting. Of the various biomarkers used to potentially predict responses to ICIs, only the IFN-signature and p-ERK have been shown to correlate with therapeutic responses in gliomas. Published literature indicates that MAPK activation is the oncogenic mechanism for BRAF driven gliomas. The marked expression of p-ERK in PA indicates that these tumors might be responsive to ICI therapy.
  • the TIM-3 inhibitors described herein are administered systemically.
  • the brain intrinsic microglia that express TIM-3 are accessed with blood-brain- barrier opening ultrasound strategies (e.g., implantable ultrasound device for blood-brain barrier opening).
  • methods described herein employ a TIM-3 inhibitor or a composition/method for the inhibition of TIM-3 activity or expression.
  • an inhibitor is a small molecule, peptide, antibody, antibody fragment, a genome editing agent, etc. that upon administration to a subject, cell, etc. reduces the activity of TIM-3, for example, systemically, within a specific tissue, organ, or body system, within a specific cell population, etc.
  • a TIM-3 inhibitor is an antibody or antibody fragment. In some embodiments, a TIM-3 inhibitor is an antibody or antibody fragment that binds to TIM-3 and reduces TIM-3 activity. In some embodiments, an anti-TIM-3 antibody or antibody fragment is provided that prevents or reduces binding of TIM-3 to a ligand thereof.
  • a TIM-3 antibody for use in a treatment herein is selected from MBG453 (aka, “Sabatolimab”, Novartis Pharmaceuticals, Clinical trial NCT02608268), TSR-022 (Tesaro, Inc. Clinical trial NCT02817633), or LY3321367 (Eli Lilly, Clinical trial NCT03099109).
  • a TIM-3 antibody for use in a treatment herein is selected from RMT3-23, ATIK2a, MBG453 (sabatolimab), AZD-7789, BMS-986258, INCAGN-2390, TSR-022 (Cobolimab), LY3321367, RO-7121661 (Lomvastomig), TQB-2618.
  • an inhibitor is a small molecule, peptide, antibody, antibody fragment, a genome editing agent, etc.
  • a TIM-3 inhibitor is a nucleic acid-based inhibitor.
  • the inhibitor is a small molecule, an aptamer, a siRNA, a shRNA, a miRNA, a morpholino, a ribozyme, an antisense nucleic acid molecule, a CRISPR-Cas9-based construct, a CRISPR-Cpfl -based construct, a meganuclease, a zinc finger nuclease, a transcription activator-like (TAL) effector (TALE) nuclease, etc.
  • TAL transcription activator-like effector
  • the TIM-3 inhibitor is a small interfering RNA (siRNA), also known as short interfering RNA or silencing RNA.
  • siRNA is an 18 to 30 nucleotide, preferably 19 to 25 nucleotide, most preferred 21 to 23 nucleotide or even more preferably 21 nucleotide-long double- stranded RNA molecule.
  • siRNA is involved in the RNA interference (RNAi) pathway where the siRNA interferes with the expression of a specific gene (e.g., the TIM-3).
  • RNAi RNA interference pathway
  • siRNAs naturally found in nature have a well-defined structure: a short doublestrand of RNA (dsRNA) with 2-nt 3' overhangs on either end.
  • Each strand has a 5' phosphate group and a 3' hydroxyl (-OH) group.
  • This structure is the result of processing by dicer, an enzyme that converts either long dsRNAs or small hairpin RNAs into siRNAs.
  • siRNAs can also be exogenously (artificially) introduced into cells to bring about the specific knockdown of a gene of interest (e.g., the TIM-3).
  • a gene of interest e.g., the TIM-3
  • any gene for which the sequence is known can thus be targeted based on sequence complementarity with an appropriately tailored siRNA.
  • the double-stranded RNA molecule or a metabolic processing product thereof is capable of mediating target- specific nucleic acid modifications, particularly RNA interference and/or DNA methylation.
  • Exogenously introduced siRNAs may be devoid of overhangs at their 3' and 5' ends, however, in some embodiments at least one RNA strand has a 5'- and/or 3'-overhang.
  • one end of the double-strand has a 3'-overhang from 1 to 5 nucleotides, more preferably from 1 to 3 nucleotides and most preferably 2 nucleotides.
  • the other end may be blunt-ended or has up to 6 nucleotides 3'-overhang.
  • any RNA molecule suitable to act as siRNA and inhibit the TIM-3 is envisioned in the present invention.
  • siRNA duplexes are provided composed of 21-nt sense and 21-nt antisense strands, paired in a manner to have a 2-nt 3'-overhang.
  • the sequence of the 2-nt 3' overhang makes a small contribution to the specificity of target recognition restricted to the unpaired nucleotide adjacent to the first base pair.
  • 2'- deoxynucleotides in the 3' overhangs are as efficient as ribonucleotides, but are often cheaper to synthesize and probably more nuclease resistant.
  • siRNA may be accomplished using any of the methods known in the art, for example by combining the siRNA with saline and administering the combination intravenously or intranasally or by formulating siRNA in glucose (such as for example 5% glucose) or cationic lipids and polymers can be used for siRNA delivery in vivo through systemic routes either intravenously (IV) or intraperitoneally (IP).
  • siRNA molecules that target and inhibit the expression (e.g., knock down) of the TIM-3
  • a short hairpin RNA is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression (e.g., of TIM-3) via RNA interference.
  • shRNA uses a vector introduced into cells and utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited.
  • the shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • the RISC binds to and cleaves RNAs which match the siRNA that is bound to (e.g., comprising the sequence of the TIM-3 ).
  • si/shRNAs to be used in the present invention are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.
  • shRNA molecules that target and inhibit the expression (e.g., knock down) of the TIM-3 .
  • Said RNA species are single-stranded RNA molecules.
  • Endogenously present miRNA molecules regulate gene expression by binding to a complementary mRNA transcript and triggering of the degradation of said mRNA transcript through a process similar to RNA interference.
  • exogenous miRNA may be employed as an inhibitor of the TIM-3 after introduction into target cells.
  • miRNA molecules that target and inhibit the expression (e.g., knock down) of the TIM-3.
  • Morpholines are synthetic nucleic acid molecules having a length of about 20 to 30 nucleotides and, typically about 25 nucleotides. Morpholines bind to complementary sequences of target transcripts (e.g., the TIM-3 ) by standard nucleic acid base-pairing. They have standard nucleic acid bases which are bound to morpholine rings instead of deoxyribose rings and linked through phosphorodiamidate groups instead of phosphates. Due to replacement of anionic phosphates into the uncharged phosphorodiamidate groups, ionization in the usual physiological pH range is prevented, so that morpholines in organisms or cells are uncharged molecules.
  • morpholino The entire backbone of a morpholino is made from these modified subunits. Unlike inhibitory small RNA molecules, morpholines do not degrade their target RNA molecules. Rather, they sterically block binding to a target sequence within a RNA and prevent access by molecules that might otherwise interact with the RNA. In some embodiments, provided herein are morpholino oligonucleotides that target and inhibit the expression (e.g., knock down) of the TIM-3.
  • a ribozyme (ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA) is an RNA molecule that catalyzes a chemical reaction. Many natural ribozymes catalyze either their own cleavage or the cleavage of other RNAs, but they have also been found to catalyze the aminotransferase activity of the ribosome.
  • Non-limiting examples of well-characterized small self-cleaving RNAs are the hammerhead, hairpin, hepatitis delta virus, and in vitro- selected leaddependent ribozymes, whereas the group I intron is an example for larger ribozymes. The principle of catalytic self-cleavage is well established.
  • hammerhead structures can be integrated into heterologous RNA sequences and that ribozyme activity can thereby be transferred to these molecules
  • catalytic antisense sequences can be engineered for almost any target sequence can be created, provided the target sequence contains a potential matching cleavage site.
  • the basic principle of constructing hammerhead ribozymes is as follows: A region of interest of the RNA (e.g., a portion of the TIM-3), which contains the GUC (or CUC) triplet, is selected. Two oligonucleotide strands, each usually with 6 to 8 nucleotides, are taken and the catalytic hammerhead sequence is inserted between them.
  • ribozyme inhibitors oligonucleotides of the TIM-3.
  • TIM-3 is inhibited (and/or TIM-3 activity is inhibited) by modifying the TIM-3 sequence in target cells.
  • the alteration of the TIM-3 is carried out using one or more DNA-binding nucleic acids, such as alteration via an RNA- guided endonuclease (RGEN).
  • RGEN RNA- guided endonuclease
  • the alteration can be carried out using clustered regularly interspaced short palindromic repeats (CRISPR) and CRIS PR-associated (Cas) proteins.
  • CRISPR system refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a "direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a "spacer” in the context of an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR locus.
  • a tracr trans-activating CRISPR
  • tracr-mate sequence encompassing a "direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system
  • guide sequence also referred to as a "spacer” in the context of an endogenous CRISPR
  • the CRISPR/Cas nuclease or CRISPR/Cas nuclease system can include a non-coding RNA molecule (guide) RNA, which sequence-specifically binds to DNA, and a Cas protein (e.g., Cas9), with nuclease functionality (e.g., two nuclease domains).
  • a CRISPR system can derive from a type I, type II, or type III CRISPR system, e.g., derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes.
  • a Cas nuclease and gRNA are introduced into the cell.
  • target sites at the 5' end of the gRNA target the Cas nuclease to the target site, e.g., the TIM-3, using complementary base pairing.
  • the target site may be selected based on its location immediately 5' of a protospacer adjacent motif (PAM) sequence, such as typically NGG, or NAG.
  • PAM protospacer adjacent motif
  • the gRNA is targeted to the desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence (e.g., sequence within the TIM-3).
  • a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence.
  • target sequence generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex.
  • the CRISPR system can induce double stranded breaks (DSBs) at the SRC-3 target site, followed by disruptions or alterations as discussed herein.
  • Cas9 variants deemed “nickases,” are used to nick a single strand at the target site (e.g., within the TIM-3). Paired nickases can be used, e.g., to improve specificity, each directed by a pair of different gRNAs targeting sequences such that upon introduction of the nicks simultaneously, a 5' overhang is introduced.
  • catalytically inactive Cas9 is fused to a heterologous effector domain such as a transcriptional repressor or activator, to affect gene expression (e.g., to inhibit expression of the TIM-3).
  • the CRISPR system is used to alter the TIM-3, inhibit expression of the TIM-3, and/or to inactivate the expression product of the TIM-3.
  • antisense nucleic acid molecule or “antisense oligonucleotide” as used herein, refers to a nucleic acid which is complementary to a target nucleic acid.
  • An antisense molecule in accordance with the invention is capable of interacting with the target nucleic acid, more specifically it is capable of hybridizing with the target nucleic acid. Due to the formation of the hybrid, transcription of the target gene(s) and/or translation of the target mRNA is reduced or blocked. Standard methods relating to antisense technology have been described (see, e.g., Melani et al., Cancer Res. (1991) 51:2897-2901).
  • antisense oligonucleotides capable of inhibiting expression of TIM-3 when administered to cell or subject.
  • the antisense oligonucleotides are antisense DNA- and/or RNA-oligonucleotides.
  • modified antisense oligonucleotides such as, antisense 2'-O-methyl oligo-ribonucleotides, antisense oligonucleotides containing phosphorothiaote linkages, antisense oligonucleotides containing Locked Nucleic Acid LNA(R) bases, morpholino antisense oligonucleotides, PPAR-gamma agonists, antagomirs.
  • ASOs comprise Locked Nucleic Acid (LNA) or 2’- methoxyethyl (MOE) modifications (internucleotide linkages are phosphorothioates interspersed with phosphodiesters, and all cytosine residues are 5 ’-methylcytosines).
  • LNA Locked Nucleic Acid
  • MOE methoxyethyl
  • methods are provided herein for the treatment, diagnosis, prognosis, etc. of glioma in a subject.
  • a subject of the methods herein suffers from glioma or is suspected of suffering from glioma.
  • the glioma is astrocytoma, ependymoma, glioblastoma, or oligodendroglioma.
  • the glioma is an astrocytoma selected from pilocytic astrocytoma (grade 1), diffuse astrocytoma (grade 2), anaplastic astrocytoma (grade 3), glioblastoma (grade 4), a pineal astrocytic tumor, a brain stem glioma, etc.
  • the glioma is a grade I, II, or III ependymoma.
  • the glioma is a grade I, II, or III oligodendroglioma.
  • a subject suffers from or is suspected of suffering from pilocytic astrocytoma.
  • the subject is a child, adolescent, or early adult.
  • the subject is 25 years, 24 years, 23 years, 22 years. 21 years, 20 years, 19 years, 18 years, 17 years, 16 years, 15 years, 14 years, 13 years, 12 years, 11 years, 10 years, 9 years, 8 years, 7 years, 6 years, 5 years, 4 years, 3 years, 2 years, 12 months, 9 months, 6 months, 3 months old, or fewer, or ranges therebetween (e.g., 5-25 years of age).
  • the subject is an adult.
  • the subject’s immune system is not exhausted (e.g., not in a hypo-responsive T cell state), as is the case for an adult with glioblastoma (GBM).
  • GBM glioblastoma
  • the subject exhibits one or more risk factors for glioma (e.g., a family history, being in remission from glioma or another caner, environmental or behavioral risk factors, a mutation or susceptibility factor that places the subject at increased risk, etc.).
  • risk factors for glioma e.g., a family history, being in remission from glioma or another caner, environmental or behavioral risk factors, a mutation or susceptibility factor that places the subject at increased risk, etc.
  • provided herein are methods for diagnosis of glioma (e.g., PA) in a subject. In some embodiments, provided herein are methods of providing a glioma-related prognosis for a subject.
  • glioma e.g., PA
  • glioma or astrocytoma biomarkers such as platelet-derived growth factor receptor (PDGFR), neurofilament light chain (NF-L), epidermal growth factor receptor (EGFR), and CD44.
  • PDGFR platelet-derived growth factor receptor
  • NF-L neurofilament light chain
  • EGFR epidermal growth factor receptor
  • CD44 CD44.
  • methylation of one or more genes e.g., MGMT, PTEN, RBI, TP53, CDKN2A, PDGFB, EMP3, SOCS1, PCDHGA11, OLIIG1/2, etc.
  • MGMT methylation of one or more genes
  • one or more chromosomal aberrations e.g., gain of Iq, 19q, 20q, etc. or loss of 6q, 9p, 13q, 14q, or 22q, etc.
  • chromosomal aberrations e.g., gain of Iq, 19q, 20q, etc. or loss of 6q, 9p, 13q, 14q, or 22q, etc.
  • mutations in one or more genes e.g., PTEN, ATRX, TP53, RBI, IDH1/2, NF1, EGFR, etc.
  • amplifications of one or more genes is indicative of glioma and may serve as a detectable biomarker in the methods herein.
  • deletions of one or more genes is indicative of glioma and may serve as a detectable biomarker in the methods herein.
  • methods are provided for determining a treatment course of action. In some embodiments, methods are provided for determining whether TIM-3 inhibition is a useful or potentially-useful treatment for glioma (e.g., PA) in a subject.
  • glioma e.g., PA
  • Experiments conducted during development of embodiments herein demonstrate that a clinical indication for TIM-3 inhibition for the treatment of a glioma (e.g., PA) in a subject with a BRAF-fusion (e.g., KIAA1549-BRAF fusion).
  • IFN-signature and p-ERK are correlated with therapeutic responses in gliomas.
  • biomarkers useful in embodiments herein may be detected and/or quantified by any suitable techniques, including but not limited to nucleic acid analysis, sequencing, ELISA, mass spectrometry, immunohistochemistry, etc.
  • Some embodiments comprise use of nucleic acid sequencing to detect, quantify, and/or identify biomarkers.
  • sequence refers to a method by which the identity of at least 10 consecutive nucleotides (e.g., the identity of at least 20, at least 50, at least 100, or at least 200 or more consecutive nucleotides) of a polynucleotide are obtained.
  • non-generation sequencing refers to the so-called parallelized sequencing-by-synthesis, sequencing-by-ligation platforms, nanopore sequencing methods, or electronic-detection based methods that will be understood in the field.
  • Some embodiments utilize various protein, peptide, or small molecule detection/quantification techniques.
  • Analytical platforms e.g., High-throughput platforms, automated platforms, etc.
  • NMR nuclear magnetic resonance
  • GC gas chromatography
  • LC liquid chromatography
  • MS mass spectrometry
  • Mass spectrometry can accurately identify /quantify thousands of biomolecules within complex biological samples.
  • biomolecules are detected/quantified in a biological sample using MS techniques, such as MALD1/TOF (time-of-flight), SELDI/TOF, liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography-mass spectrometry (HPLC-MS), capillary electrophoresis-mass spectrometry, nuclear magnetic resonance spectrometry, tandem mass spectrometry (e.g., MS/MS, MS/MS/MS, ESI-MS/MS etc.), secondary ion mass spectrometry (SIMS), or ion mobility spectrometry (e.g.
  • MS/MS time-of-flight
  • SELDI/TOF SELDI/TOF
  • LC-MS liquid chromatography-mass spectrometry
  • GC-MS gas chromatography-mass
  • Mass spectrometry methods are well known in the art and have been used to quantify and/or identify biomolecules, such metabolites.
  • Antibody-based techniques such as ELISA, or immunohistochemistry may also be utilized for the detection/quantification of biomarkers.
  • the TIM-3 inhibitor is co-administered with a chemotherapeutic, immunotherapeutic, surgery, and/or radiation.
  • chemotherapeutics for co-administration with a TIM-3 inhibitor include temozolomide, etoposide, doxorubicin, cisplatin, paclitaxel, carmustine, lomustine, ceramide and/or phosphorylcholine.
  • Exemplary immunotherapeutic s for co-administration with a TIM-3 inhibitor include immune checkpoint inhibitor, STING agonists (e.g., IACS-8803, TAK-500, TAK-676, ADU- S100, etc.), ACT therapy, CAR-T cell therapy, monoclonal antibody therapy, and bispecific T- cell engager therapy.
  • a TIM-3 inhibitor is co-administered with an immune checkpoint inhibitor that binds to and inhibits the activity of an immune checkpoint protein is selected from the group consisting of CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, or VISTA.
  • the TIM-3 inhibitor is administered with an immune checkpoint inhibitor selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, STI-Al l 10, TSR-042, RG- 7446, BMS-936559, BMS-936558, MK-3475, MPDL3280A, MEDI-4736, MSB-0020718C, AUR-012 and STI-A1010.
  • an immune checkpoint inhibitor selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, STI-Al l 10, TSR-042, RG- 7446, BMS-936559, BMS-936558, MK-3475, MPDL3280A, MEDI-4736, MSB-0020718C, AUR-012 and STI-A1010.
  • a TIM-3 inhibitor is co-administered with radiation therapy.
  • Techniques for administering radiation therapy are known in the art, and these techniques can be used in the combination therapies described herein.
  • Radiation therapy can be administered through one of several methods, or a combination of methods, including without limitation external-beam therapy, internal radiation therapy, implant radiation, stereotactic radiosurgery, systemic radiation therapy, radiotherapy and permanent or temporary interstitial brachytherapy.
  • a TIM-3 inhibitor is administered before and/or after surgical removal of cell or a tumor related to the glioma.
  • NGS was performed on genomic DNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor samples using the NextSeq or NovaSeq 6000 platforms (Illumina, Inc., San Diego, CA).
  • FFPE formalin-fixed paraffin-embedded
  • NovaSeq 6000 platforms Illumina, Inc., San Diego, CA.
  • FFPE formalin-fixed paraffin-embedded
  • a custom-designed SureSelect XT assay was used to enrich 592 whole-gene targets (Agilent Technologies, Santa Clara, CA).
  • NovaSeq sequenced tumors more than 700 clinically relevant genes at high coverage and high read-depth were used, along with another panel designed to enrich for an additional >20,000 genes at lower depth. All variants were detected with >99% confidence based on allele frequency and amplicon coverage, with an average sequencing depth of coverage of >500 and an analytic sensitivity of 5%.
  • tumor enrichment was achieved by harvesting targeted tissue using manual microdissection techniques. Genetic variants identified were interpreted by board-certified molecular geneticists and categorized as ‘pathogenic,’ ‘likely pathogenic,’ ‘variant of unknown significance,’ ‘likely benign,’ or ‘benign,’ according to the American College of Medical Genetics and Genomics standards. When assessing mutation frequencies of individual genes, ’pathogenic,’ and ‘likely pathogenic’ were counted as mutations. The copy number alteration of each exon was determined by calculating the average depth of the sample along with the sequencing depth of each exon and comparing this calculated result to a pre-calibrated value.
  • TMB was measured by counting all non-synonymous missense, nonsense, inframe insertion/deletion and frameshift mutations found per tumor that had not been previously described as germline alterations in dbSNP151, Genome Aggregation Database (gnomAD) databases or benign variants identified by Caris geneticists
  • gnomAD Genome Aggregation Database
  • Caris Life Sciences is a participant in the Friends of Cancer Research TMB Harmonization Project. Whole Transcriptome Sequencing
  • RNA FFPE tissue extraction kit was used for extraction, and the RNA quality and quantity was determined using the Agilent TapeStation.
  • Biotinylated RNA baits were hybridized to the synthesized and purified cDNA targets and the bait-target complexes were amplified in a post capture PCR reaction.
  • the resultant libraries were quantified, normalized and the pooled libraries are denatured, diluted and sequenced; the reference genome used was GRCh37/hgl9 and analytical validation of this test demonstrated >97% Positive Percent Agreement (PPA), >99% Negative Percent Agreement and >99% Overall Percent Agreement with a validated comparator method.
  • Raw data was demultiplexed by Illumina Dragen BioIT accelerator, trimmed, counted, PCR-duplicates removed and aligned to human reference genome hgl9 by STAR aligner.
  • transcripts per million values were generated using the Salmon expression pipeline.
  • Transcriptomic signatures predictive of response to immunotherapy T cell inflamed score
  • RSRD replication stress response defect
  • TPM transcripts per million
  • Immune cell fractions were estimated using RNA deconvolution (quanTIseq). The total cell fraction consists of 10 immune cell populations and an 11 th group designated as ‘uncharacterized cells’ that includes both tumor and other stromal cells that are not one of the 10 cell populations adapted from.
  • IHC Immunohistochemistry
  • T2 or FLAIR abnormality was resected as part of the approach to create a corridor to a benign mass.
  • the specimen was aliquoted for parallel analysis by the operative neurosurgeon.
  • Approximately 1 cm 3 of the gadolinium-enhancing tumor was designated for scRNA sequencing analysis and was processed into a single-cell suspension after enrichment for the immune cells by Percoll gradient.
  • a second adjacent piece of tumor in continuity with the surrounding brain was processed for FFPE. This specimen was used for sequential multiplex immunohistochemistry.
  • Patient tumors were graded pathologically by the study neuropathologists (CMH, NW) according to the World Health Organization classification. At least 500 mg of viable, non- necrotic tumor was required to obtain sufficient quantities for analysis and was processed within 1 hour after resection.
  • Normal brain (NB) was obtained from subjects as part of the planned surgical approach to gain access to a low-grade non-infiltrating glioma or during a planned super-total resection of adjacent regions. The normal brain was sent for analysis separately from the tumor. The freshly resected tissue was processed in parallel as both for a single cell suspension and for FFPE analysis.
  • the FFPE was used for sequential multiplexing immunofluorescence (SeqIFTM) and NanoString nCounter analysis of a 770 gene panel after microdissection of the tissue (tumor area vs. adjacent normal brain) and RNA isolation (Qiagen kit).
  • the tissue was minced into small pieces using a scalpel, dissociated, and suspended using a Pasteur pipette in 10ml Iscove’s DMEM (IMDM; Iscove’s Modification of DMEM) IX (Coming) containing 2% inactivated Fetal Bovine Serum (FBS; Sigma Aldrich) and collagenase and DNase enzymes at the final concentrations of lOOpg/ml and 20 units/ml, respectively.
  • the prepared mixture was incubated for 35-40 mins at 37°C with agitation.
  • the tissue was filtrated using a 70- pm nylon cell strainer (BD Biosciences) and then underwent centrifugation at 4°C.
  • the pellet was either resuspended culture media for functional assays or in 20 ml mix of 5.4ml of PercollTM Plus (GE Healthcare) overlaid with 12 ml of IX Phosphate Buffered Saline (PBS) and 0.6ml of 10X PBS (Corning) for single-cell RNA- sequencing.
  • the tube was centrifuged at 800g for 10 minutes at 4°C, with 9 acceleration and 0 deceleration. After centrifugation, the immune enriched cell pellet was collected, washed, stained with Trypan blue dye (Sigma- Aldrich), and counted using Countess II FL automated cell counter in a Countess cell counting chamber (Invitrogen).
  • Single-cell sequencing was carried out using the chromium Next GEM Single Cell protocol (lOx genomics). Post library preparation cells were sequenced using the Illumina Novaseq. Raw data was preprocessed and aligned using Cell Ranger to obtain the matrix and count files. Seurat R Package using scRNA-seq SeuratlOx genomic workflow was then used for all subsequent analysis unless noted otherwise. After filtering using percent mitochondrial DNA threshold of 20% and UMI range of 200 to 15000, 186,317 cells were included for further analysis. Cells were then subject to Log Normalize, Scale Data, and PCA functions. Find Clusters and Find Markers functions was utilized for clustering and marker identification and non-linear dimensional reduction techniques were applied to visual data in UMAP plot format.
  • the top 100 DEGs were used for Gene Ontology enrichment analysis using the Bioconductor Package Cluster Profiler.
  • LR ligand receptor
  • Bioconductor package CellChat Only immune populations annotated as myeloid or lymphoid were included in the analysis.
  • CellChat objects for BRAF-Fusion and NB were created, and a comparison analysis was used to infer differentially enriched ligand receptor interactions between the tumor subtypes. This was performed using compareinteractions and RankNet functions. Results were displayed using heatmaps and circle diagrams to visualize significant interactions occurring within the TME.
  • FFPE slides were collected from the Neurological Surgery Tumor Bank of Northwestern University. 4pm thickness tissue slices were prepared, mounted on positively charged glass slides (Super Frost Plus microscope slides, ThermoFisher) and stored at room temperature for subsequent staining analysis. For each case, 1 H&E slide was reviewed, and the tissue segmented by a certified neuropathologist (CMH). FFPE slides were preprocessed for antigen retrieval using the PT Module (Epredia) with Dewax and HIER Buffer H (TA999- DHBH, Epredia) for 60 min at 102°C. Subsequently, slides were rinsed and stored in Multistaining Buffer (BU06, Lunaphore Technologies) till use.
  • PT Module Epredia
  • Dewax and HIER Buffer H TA999- DHBH, Epredia
  • the protocol template was generated using the COMETTM Control Software, and reagents were loaded onto the device to perform the sequential immunofluorescence (seqIFTM) protocol.
  • the markers used for this analysis were: CD31 (endothelial cells), GFAP (glioma tumor cells), CD4 (helper T cells), CD8 (cytotoxic T cells), CD20, and CD19 (B cells), P2RY12, CX3CR1, and TMEM119 (microglia), CD68 (pan- monocyte/macrophage marker), CDl lc (antigen presenting cells), CD163 (macrophage scavenger receptor), CD205 (dendritic cells), NKG2D (NK cells), p-STAT3 (nuclear hub of immune suppression), TIM3, LAG3, PD-1, and PD-L1 (immune checkpoints), HLA-DR (MHC class II), Lek (immune synapse), FOXP3 (T regulatory cells), p-ERKl
  • DAPI 6-diamidino-2-pheynlindole
  • Elution step lasted 2min for each cycle and was performed with Elution Buffer (BU07-L, Lunaphore Technologies) at 37°C. Quenching step lasted for 30s and was performed with Quenching Buffer (BU08-L, Lunaphore Technologies). The imaging step was performed with Imaging Buffer (BU09, Lunaphore Technologies).
  • the seqIFTM protocol in COMETTM resulted in a multi-stack OME.tiff file where the imaging outputs from each cycle were stitched and aligned.
  • COMETTM OME.tiff contains DAPI image, intrinsic tissue autofluorescence in TRITC and Cy5 channels, and a single fluorescent layer per marker.
  • PBMCs peripheral blood mononuclear cells
  • TICs tumorinfiltrating cells
  • FACS buffer DPBS, Corning, 21-031-CM, and 1% FBS
  • Fc blocker Invitrogen, 14-9161-73
  • DBPS Fixable Live/Dead
  • mice were then randomly assigned to control and treatment groups: (i) IgG isotype control i.v. 300 pg; (ii) anti-TIM3 i.v. 300 pg (15 mg/kg, within the range of human dosing NCT03489343); and (iii) anti-PD-1 i.p. 100 pg.
  • the genetic low-grade model used was RCAS/Ntv-a which induces low-grade gliomas (58).
  • the vector constructs are propagated in DF-1 chicken fibroblasts. Live viruses are produced by transfecting plasmid versions of RCAS vectors into DF-1 cells using FuGene6 (Roche). DF-1 cells senesce 1-2 days after injection.
  • 2 x 10 4 DF-1 producer cells transfected with the RCAS vectors in 1-2 pL of PBS are injected into the frontal lobes of neonatal GEMM mice which carry the Ntv-a transgene using a 26G 10 pL Hamilton syringe.
  • Gliomas were induced in 3 different genetic backgrounds: RCAS/Ntv-a wildtype, Ntv-a/CD8 -/_ , and Ntv- a/CX3CRl -/_ , Ntv-a/CD8 -/_ generation is described in our prior study (59).
  • RCAS- Ntv- a/CX3CRl -/_ RCAS-CX3CR1 were created by cloning human CX3CR1 (V249) cDNA into a gateway-compatible RCAS vector using LR recombination (Invitrogen) and verified by sequencing. To verify that the appropriate immune cell populations have been eliminated, the CX3CR1 KO and CD8 KO mice were genotyped before breeding. TIM3 expression within the tumors of these models was confirmed by single-cell sequencing and immunofluorescence. Mice were randomized to the following treatment groups: anti-T!M3 antibody (300 pg i.v. once per week for four weeks); anti-PD (200 pg i.p.
  • mice In the CX3CR1 and CD8 KO background mice, the IgG control was administered at 300 pg i.v. once per week for four weeks starting at approximately day 28, identical to the dose and schedule for the anti-TIM3. The mice were observed daily for survival, and when they showed signs of neurological deficit (lethargy, hypothermia, failure to ambulate, lack of feeding, body condition score ⁇ 2.0, or loss of >20% body weight), they were compassionately euthanized.
  • neurological deficit lethargy, hypothermia, failure to ambulate, lack of feeding, body condition score ⁇ 2.0, or loss of >20% body weight
  • Two-sided Wilcoxon rank-sum test was used to calculate p-value for all pairwise comparisons.
  • Two-stage step-up (Benjamini correction) method was used and False Discover Rate (Q) ⁇ 0.01 was used.
  • P-values displayed in box and whisker plots are reported as adjusted p-values and using the following designations: * ⁇ 0.05, ** ⁇ 0.01 ,*** ⁇ 0.001 .
  • All box and whiskey plots show all individual points with the box showing 25th percentile, median, and 75th percentile and whiskers at minimum and maximum value.
  • GraphPad Prism version 9.2.0 was used to analyze the data.
  • IDH-WT High Grade (HG), H3F3A, MAPK-Driven, and IDH-MT included both glioblastoma and diffuse pediatric high-grade gliomas.
  • T cells were rare in gliomas regardless of molecular type classification. In contrast, dendritic cells and macrophages were more frequent in the MAPK-Driven tumors ( Figure 1). TMB and RSRD scores were low for all glioma groups, but MAPK-Driven tumors showed the highest 1FN expression signatures.
  • the analysis of immune markers in the MAPK-Driven group revealed elevated expressions of CD86 and HAVCR2 (TIM3), but a relatively low-level expression of IDO1 , PDCD1 (PD-1), LAG3, CTLA4, and CD274 (PD-L1).
  • BRAE -Fusion PA commonly expresses II M3
  • Phagocytic microglia and antigen presentation are present in BRAF-Fusion PA
  • Tumor-associated myeloid cells which include peripherally originating cells and brainresident microglia (MG) are a dominant immune population in the glioma TME ( Figure 2D).
  • the MG are identified using three canonical markers (TMEM119, CX3CR1, and P2RY 12).
  • the MG cells were further categorized into distinct subtypes: inflammatory groups 1 and 2 (CCL4L2, TMEM107, and TNF expression); phagocytic (C1QA, TMEM176B, and VSIG4 expression); HSP expressing (HSPA1A, HSPA1B, and HSPB1); perivascular (LYVE1, FOLR2, and MRC1 expression) and homeostatic (P2RY12, CSF1R).
  • CCL4L2, TMEM107, and TNF expression phagocytic
  • C1QA, TMEM176B, and VSIG4 expression phagocytic
  • HSP expressing HSPA1A, HSPA1B, and HSPB1
  • perivascular LYVE1, FOLR2, and MRC1 expression
  • P2RY12 homeostatic
  • TAM tumor-associated macrophage
  • CSTB lysosomal
  • LYZ lysosomal
  • LIPA lysosomal
  • IFI6 IFIT1, ISG15, CCL4
  • CCL4 APC-like
  • CSD APC-like
  • MS44A4A MS4A6A
  • RNA Splicing MALAT1, SLC1A3, SLC38A2
  • type 2 conventional dendritic cells CLC10A, FCER1A
  • plasmacytoid dendritic cells CLC4C, IL3RA
  • monocytes VACN, FCN1, S100A8
  • neutrophils LY6G6D, JMJD1C, CD117
  • CD4+ When the T/NK cell population was analyzed, the following clusters were identified: CD4+, three CD8+, double negative memory-like (CD4-CD8-.Tm), Tregs, three NK, y8-T cells, and a unique CD3E+CD4A+P2RY 12+ cluster defined as MG- Act ( Figure 2E).
  • the CD4+ T cells expressed central memory (cm) markers IL7R, CCR7, TCF7, and CD40LG.
  • CD8+ cells were subclassified as IL7R+, CD69+, TCF7+ and two early activated GZMA/K/H high GZMB low clusters were identified (CD8.Tearly.act.l and CD.Tearly.act.2).
  • NK cells were subclassified as XCL1/2+, GZMB hlgh PRFl+TIM3+CXC3CRl+, and memory-like due to expression of IL7R, CD69 and TCF7.
  • Most T cell effector populations did not express markers of immune exhaustion such as PD-1, TIGIT, and LAG3 on the scRNAseq data (Figure 2G).
  • T cells in the TME of PA could not be identified expressing these markers with the SeqIF Tm staining.
  • the MG- Act population is notable for having genes related to immune cytotoxic functions (Figure 2F-G).
  • the top 40 up-regulated genes for the MG- Act cluster included C1QC, APOE, C1QB, C1QA, CST3, APOCI, HLA-DRA, C3, AIF1, CD74, MARCKS, FTL, TREM2, SPP1, APOC2, CD68, TYROBP, HLA-DRB5, HLA-DPA1, SP11, NPC2, CTSB, TMEM176B, SERP1NA1, HLA- DRB1, FCER1G, IFI30, GSN, MS4A64, CSF1R, GPR34, LY86, CD14, VSIG4, HLA-DPB1, TUBA1B, and SCIN indicating antigen presentation capability.
  • These MG-Act cells are enriched in the BRAF-Fusion PA compared to ANB and HGG (scRNA data obtained from GEO GSE249263) (Figure 3A).
  • TIM3-expressing immune cells are spatially localized to distinct niches of the TME
  • SeqIF Tm was performed.
  • TIM3 expression is dispersed throughout the TME with minimal expression found in ANB ( Figure 3B-C).
  • CD1 lc+ and P2RY 12+ cells are the predominant TIM3+ expressing population with minimal TIM3 expression on GFAP+ tumor cells or CD3+ lymphoid cells ( Figure 3D).
  • TIM3 expression is found on the myeloid cells lining the vessels in the glioma ( Figure 3E), but not in ANB ( Figure 9).
  • the scRNA seq data implicating cytotoxic functions of MG- Act were validated at the protein expression level including for P2RY12, CD3, CD4, CD8, NKG7, and TIM3 ( Figure 3F-G).
  • the TIM3 expression on the P2RY 12+ MG- Act population is dispersed throughout the TME PA but not in the ANB.
  • TIM3 expression in PA can be therapeutically manipulated
  • TIM3 and STAT3 expression are significantly increased in PA tumors. Additionally, the Henriquez et. al. dataset contains both fetal and adult NB and analysis demonstrated that fetal NB had lower TIM3 and STAT3 expression compared to adult NB.
  • PDCD1 (PD-1) had lower expression in PA compared to ANB ( Figure 4A, Figure 10). Ex vivo flow cytometry showed increased TIM3 expression on tumorinfiltrating immune cells (TICs) in both myeloid and lymphoid compartments compared to matched PBMCs and healthy control PBMCs ( Figure 4B).
  • TICs tumorinfiltrating immune cells
  • Anti-TJM3 exerts a therapeutic effect in preclinical models of glioma
  • the IgG control mice had a median survival time of 39 days
  • PD-l-treated mice had a median survival of 47 days
  • the RCAS-PDGFB GEMM glioma model was histologically characterized by a board-certified neuropathologist.
  • the RCAS-PDGFB GEMM model displayed the classic features of a low-grade glioma including a loose microcystic pattern (Figure 5B), heterogeneity in cellular density (Figure 5C), the absence of mitosis (Figure 5D), and perineuronal satellitosis at the infiltrating edges of the tumors (Figure 5E).
  • This model also shows TIM3 expression on P2RY12+ microglia and activation of the MAPK pathway as assessed based on p-ERKl/2 expression (Figure 5F-G).
  • Anti-TIM3 mediates its therapeutic effect through the immune system
  • Anti-TIM3 enhances the MG-ACT population in vivo
  • mice were terminated for immune assessments of the TME using scRNAseq after either 2 or 4 treatments with anti-TIM3 or IgG.
  • the IgG-treated mice had large gliomas while the anti-TIM3 -treated group either had small gliomas or the tumors were absent (Figure 5 J) .
  • the MG- Act population was identified in the wild- type GEMM MAPK-driven low-grade glioma preclinical model (Figure 5K-L) which became more abundant with anti-TIM3 treatment alongside other effector populations including CD8+ cytotoxic T cells (Figure 5M). Additional dosing of anti-TIM3 further increased the MG- Act population and T/NK cell populations (NK Act 1, CD8+ cytotoxic, CD4+ central memory) (Figure 5M).
  • TMB tumor mutational burden
  • PubMed PMID 32217756; PubMed Central PMCID: PMCPMC7 174078. Finotello F, Mayer C, Plattner C, Laschober G, Rieder D, Hackl H, et al. Molecular and pharmacological modulators of the tumor immune contexture revealed by deconvolution of RNA-seq data. Genome Med. 2019;l l(l):34. Epub 2019/201728. doi: 10.1186/s 13073-019- 0638-6. PubMed PMID: 31126321; PubMed Central PMCID: PMCPMC6534875. Hervey-Jumper SL, Berger MS.
  • Dann E Henderson NC, Teichmann SA, Morgan MD, Marioni JC. Differential abundance testing on single-cell data using k-nearest neighbor graphs. Nat Biotechnol. 2022;40(2):245- 53. Epub 2021/10/02. doi: 10.1038/s41587-021-01033-z. PubMed PMID: 34594043. Yu G, Wang LG, Han Y, He QY. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. 2012;16(5):284-7. Epub 2012/03/30. doi: 10.1089/omi.2011.0118.
  • Alzheimer's-associated PLCgamma2 is a signaling node required for both TREM2 function and the inflammatory response in human microglia. Nat Neurosci. 2020;23(8):927-38. Epub 2020/06/10. doi: 10.1038/s41593-020-0650-6. PubMed PMID: 32514138. 61. Jokubaitis VG, Gresle MM, Kemper DA, Doherty W, Perreau VM, Cipriani TL, et al.
  • PubMed PMID 30306125; PubMed Central PMCID: PMCPMC6 174845.
  • TIM-3 blockade in diffuse intrinsic pontine glioma models promotes tumor regression and antitumor immune memory. Cancer Cell. 2023. Epub 2023/10/07. doi: 10.1016/j.ccell.2023.09.001. PubMed PMID: 37802053. Woroniecka K, Chongsathidkiet P, Rhodin K, Kemeny H, Dechant C, Farber SH, et al. T- Cell Exhaustion Signatures Vary with Tumor Type and Are Severe in Glioblastoma. Clin Cancer Res. 2018;24(17):4175-86. Epub 2018/02/14. doi: 10.1158/1078-0432.CCR-17-1846.
  • Neoadjuvant anti-PD-1 immunotherapy promotes a survival benefit with intratumoral and systemic immune responses in recurrent glioblastoma. Nat Med. 2019;25(3):477-86. Epub 2019/02/12. doi: 10.1038/s41591-018-0337-7. PubMed PMID: 30742122; PubMed Central PMCID: PMCPMC6408961. Picard D, Felsberg J, Langini M, Stachura P, Qin N, Macas J, et al. Integrative multi-omics reveals two biologically distinct groups of pilocytic astrocytoma. Acta Neuropathol.
  • PubMed PMID 36686797; PubMed Central PMCID: PMCPMC9846085. He S, Lin Q, Chen J, Ma C, Liu Z, Sun Y, et al. Differential expression of Tim3 protein in colorectal cancer associated with MSI and Braf mutation. Histol Histopathol. 2022;37(5):441-8. Epub 2022/01/08. doi: 10.14670/HH-18-419. PubMed PMID: 34994395.

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Abstract

Provided herein are compositions and methods for the treatment of glioma by targeting TIM-3. In particular, a subject suffering from a glioma is administered a TIM-3 inhibitor alone or in combination with other glioma therapies.

Description

TARGETING TIM-3 IN MAPK-DRIVEN GLIOMA
CROSS-REFERENCE TO RELATED APPLICATIONS
The present invention claims the priority benefit of U.S. Provisional Patent Application 63/512,851, filed July 10, 2023, which is incorporated by reference in its entirety.
FIELD
Provided herein are compositions and methods for the treatment of glioma by targeting TIM-3. In particular, a subject suffering from a glioma is administered a TIM-3 inhibitor alone or in combination with other glioma therapies.
STATEMENT REGARDING GOVERNMENT INTEREST
This invention was made with government support under 5R01NC120547-03 awarded by the Department of Health and Human Services, National Institutes of Health, and National Institute of Neurological Disorders and Stroke, and 5R01CA120813-14 awarded by the Department of Health and Human Services, National Institutes of Health, and National Cancer Institute. The government has certain rights in the invention.
BACKGROUND
Immunological studies of adult gliomas have been numerous, whereas pediatric gliomas are relatively understudied despite their being a leading cause of childhood mortality. Among pediatric glioma immunology studies conducted to date, the largest consisting of 218 patients spanning seven histological types of childhood brain cancer (low-grade glioma, ependymoma, high-grade glioma, medulloblastoma, ganglioglioma, craniopharyngioma, and atypical teratoid rhabdoid tumor) analyzed using whole-genome sequencing, RNA sequencing, and proteomic profiling revealed markedly diverse immune composition in tumor microenvironments (TME), across and within histologic groups. The location of the immune cells is also variable. For example, certain T cell populations in high- and low-grade pediatric gliomas are close to vasculature while others such as CD 103+ T cells reside further away from the vasculature. Immune cell infiltration is also different between primary vs. recurrent tumors. Differences in immune surveillance have been shown in studies comparing pediatric vs. adult gliomas including: (i) low-levels of expression of programmed cell death protein ligand 1 (PD-L1) and NKG2D ligand; and (ii) the lack of prognostic significance associated with immunosuppressive CD 163+ macrophage infiltration in pediatric tumors. As such, it has been postulated that the pediatric brain TME may reflect a failure of immune surveillance rather than the establishment of an immunosuppressive environment as is seen in adult tumors.
Many studies have demonstrated that the immune microenvironment of adult glioblastoma is highly immunosuppressive, thereby effectively shielding the tumor from immunological surveillance and eradication. Immunological reactivity, including against cancer, is a function of age. In general, there are extensive and comprehensive changes in immune reactivity that include age related T cell anergy, exhaustion, and senescence, defects in activation of the inflammasome, and decreased innate immunity (. Specifically in the context of gliomas, CD8 T cell recent thymic emigrants (RTEs) are at least one factor that accounts for the prognostic power of age on clinical outcome in adult glioblastoma patients. CD8 T cell RTEs account for the majority of tumor antigen-binding cells in the peripheral blood and are expanded following vaccination and are associated with clinical outcomes. Preclinical modeling in mutant (CD8beta(-/-)) mice display an age-specific decrease in glioma host survival as well as a correlation between host survival and thymus cellular production.
Pilocytic astrocytomas (PA) are the most common pediatric glioma. Surgical resection of PA is typically the first-line treatment and gross total resection is often curative. However, PAs that are not amenable to gross total resection have associated long-term morbidity and mortality. The ten-year progression-free survival for PA patients with radiologically visible residual tumor is less than 50%. Other treatment options are frequently necessary in gliomas with a midline location, which can be challenging to biopsy or resect safely. Some PA undergo spontaneous regression after partial resection, which suggests a predisposition to immune surveillance and eradiation. PA can have a single driver BRAF rearrangement. BRAFV600 expressing melanoma has been shown to be responsive to immune checkpoint inhibitor therapy, especially those with PD-L1 expression. PD-L1 expression and immune cell infiltration are independent of BRAF V600E mutational status. Although tumor mutational burden (TMB) is associated with response to immune checkpoint inhibitors (ICI) for various cancer lineages, this is not the case for adult gliomas. BRAF alterations are not typically associated with high TMB but may still be highly immunogenic to a sufficient degree to induce spontaneous regression and/or high propensity for response to immune therapy. scRNAseq data of PA also indicate that microglia may constitute a high frequency immune cell population in these tumors. If and how PA microglia influence tumor immunoreactivity is unknown.
SUMMARY
Provided herein are compositions and methods for the treatment of glioma by targeting TIM-3. In particular, a subject suffering from a glioma is administered a TIM-3 inhibitor alone or in combination with other glioma therapies.
In some embodiments, provided herein are methods of treating glioma in a subject comprising administering to the subject an inhibitor of TIM-3. In some embodiments, the glioma is a MAPK-driven glioma. In some embodiments, the glioma is an astrocytoma. In some embodiments, the astrocytoma is a pilocytic astrocytomas (PA). In some embodiments, the subject exhibits one or more glioma biomarkers. In some embodiments, the subject has tested positive for a BRAF fusion, increased IFN, and/or increased p-ERK. In some embodiments, the BRAF fusion is a KIAA1549-BRAF fusion. In some embodiments, the subject is a child, adolescent, or early adult. In some embodiments, the subject is an adult. In some embodiments, the TIM-3 inhibitor is an inhibitor of TIM-3 activity. In some embodiments, the TIM-3 inhibitor is an anti-TIM-3 antibody. In some embodiments, the TIM-3 inhibitor is selected from RMT3- 23, ATIK2a, MBG453 (sabatolimab), AZD-7789, BMS-986258, INCAGN-2390, TSR-022 (Cobolimab), LY3321367, RO-7121661 (Lomvastomig), TQB-2618. In some embodiments, the TIM-3 inhibitor is Sabatolimab (MBG453). In some embodiments, the TIM-3 inhibitor is an antibody that binds to a ligand of TIM-3. In some embodiments, the TIM-3 inhibitor binds to galectin-9, HMGB1, ceacam-1, or phosphatidyl serine. In some embodiments, the TIM-3 inhibitor is a small molecule or peptide capable of binding to TIM-3 and/or a TIM-3 ligand. In some embodiments, the small molecule TIM-3 inhibitor is SMI402. In some embodiments, the TIM-3 inhibitor is an inhibitor of TIM-3 expression. In some embodiments, the TIM-3 inhibitor is a nucleic acid-based agent that reduces expression of TIM-3. In some embodiments, the TIM-3 inhibitor comprises one or more agents that allow for alteration of the TIM-3 gene and thereby reduce TIM-3 expression or activity. In some embodiments, the TIM-3 inhibitor is coadministered with one or more additional glioma therapies. In some embodiments, the TIM-3 inhibitor is co-administered with surgical removal of all or a portion of the glioma. In some embodiments, the TIM-3 inhibitor is co-administered with radiation therapy, chemotherapy, and/or an immunotherapy. In some embodiments, methods further comprise detecting one or more glioma biomarkers in the subject. In some embodiments, the glioma biomarkers are selected from a BRAF fusion, increased IFN, and/or increased p-ERK. In some embodiments, the BRAF fusion is a KIAA1549-BRAF fusion.
In some embodiments, provided herein is the use of a TIM-3 inhibitor in the treatment of glioma in a subject.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Estimated Immune Cell Fractions in pediatric gliomas based on RNA deconvolution. N: IDH-MT = 91, IDH-WT HG = 24, H3F3A = 39 and MAPK-Driven = 96. Bulk RNA-Seq Immune Marker Signatures of Pediatric Glioma Molecular Groups. TPM, transcripts per million. **p < 0.01; ***p < 0.001 (unpaired t-test with two-stage step-up (Benjamini correction) method).
Figure 2A-G. Inflammatory TME present in BRAF-Fusion PA. A) Schematic diagram of the workflow associated with orthogonal analyses of pediatric gliomas (Created with BioRender.com). scRNA Seq, single-cell RNA sequencing; GEMM, genetically engineered murine model. B) UMAP plot of glioma infiltrating immune cells analyzed with scRNAseq across 16 patients (13 BRAF-Fusion PA and 3 adjacent normal brains (ANB)). C) Dot plot of selected immune marker genes within the general immune populations. Bubble size corresponds to the percent of cells expressing gene marker; colors indicate average expression. D) scRNA seq UMAP plot of the intratumoral myeloid cells shown in brown on panel B. E) scRNA seq UMAP plot of the intratumoral lymphoid cells shown in purple in panel B. F) Dot plot of immune marker genes within the myeloid subtypes (including the MG- Act population from panel E) characterized based on four distinct immunological functions: cytotoxicity, immune suppression, antigen presentation cell (APC), and phagocytosis. Bubble size corresponds to the percent of cells expressing the gene and colors indicate average expression. G) Dot plot of immune marker genes of the cell clusters shown in panel E broken into the four distinct immunological functions: cytotoxicity, immune suppression, antigen presentation cell (APC), and phagocytosis. Bubble size corresponds to the percent of cells expressing gene marker; shade indicates average expression. Figure 3A-G. A) Histogram of % of MG- Act population within ANB (n = 3), PA (n = 13), and HGG (n = 5). *<0.05 **< 0.01 (2-tailed t test). Mean and SEM are displayed. B) Multiplex immunofluorescent imaging of the spatial distribution of TIM3 expression in BRAF- Fusion PA relative to ANB. Scale bar at 500 pm. Box 1 is a higher magnification image from the tumor and Box 2 is from the ANB. Scale bar at 100 pm. C) Histogram of the percent of TIM3+ cells based on anatomical location (PA: n=8, ANB: n=3). Each symbol represents a patient specimen. Mean and SEM are displayed. ***<0.001 (2-tailed T test). D) Histogram of the percent of TIM3+ cells that co-express CD1 lc+, P2RY12+, CD163+, GFAP+, or CD3+ within PA (n=8). Mean and SEM are displayed. E) Representative image of CD163+ macrophages lining the vasculature wall of a BRAF-Fusion PA (left panel) and expressing TIM3 (right panel). Scale bars at 100 pm. F) Representative image of the P2RY 12+CD3+NKG7+ MG- Act population within the TME of PA (n=8). Scale bars at 20 pm. G) Histogram of the percent of MG- Act cells between ANB (n=3) and PA (n=8). Mean and SEM are displayed. *<0.05, **<0.01, ***<0.001, ****<0.0001 (2-tailed T test).
Figure 4A-C. A) Comparison of TIM3 (HAVCR2), STAT3, and PDCD1 mRNA expression in PA and normal brain (ANB) samples. Data extracted from Gliovis. Log2 mRNA expression of selected gene markers. **<0.01, ****<0.0001 (Students t-test) B) TIM3 mean fluorescence intensity (MFI) values for matched tumor-infiltrating immune cells (TICs) and peripheral blood mononuclear cells (PBMCs) from patients with BRAF-Fusion PA (n=6) relative to healthy controls (n=3). *<0.05, ****<0.0001 (two-tailed paired t-test) C) Ratio of TNFa+:p- STAT3+ lymphocytes and myeloid cells after 48 hours of treatment ex vivo with either IgG, anti- TIM3, or anti-PDl in ex vivo PA samples (n=3). *<0.05, **<0.01, ***<0.001, ****<0.0001 (two-tailed t-test). Mean and SEM are displayed.
Figure 5A-M. A) Schema of the treatment of immunocompetent GEMM mice with either anti-TIM3 (300 pg/ mouse) or IgG (100 pg/mouse) once per week or anti-PD-1 (200ug/mouse) 3 times per week stalling at day 28 after induction of glioma. B) Histological evaluation by H&E of the GEMM model displaying representative features of a low-grade glioma such as a loose microcystic pattern. Scale bar at 100 pm. C) H&E of the GEMM model demonstrating heterogeneity in a region of greater cellular density. Scale bar at 100 pm. D) H&E demonstrating glioma cells are monotonous and lack mitosis. Scale bar at 50 pM. E) H&E demonstrating perineuronal satellitosis at the infiltrating edges of the tumors. Scale bar at 50 pm. F) Multiplex immunofluorescent imaging of TIM3 expression on P2RY12 microglia in the GEMM model (n=4). GFAP: purple; P2RY12: green; TIM3: yellow. Scale bars at 50 pm. G) Multiplex immunofluorescent imaging p-ERKl/2 expression in the GEMM mode (n=4). Scale bar at 50 pm. H) The survival rate of low-grade glioma GEMM mice was estimated by the Kaplan-Meier method. IgG Control: 20 mice (MS: 110.5 d), anti-PDl: 20 mice (MS: 134.5d), anti-TIM3: 21 mice (MS: 253d). Statistics (log-rank test): control versus anti-PD-1 p = 0.44; control versus anti-TIM3 p=0.01; anti-TIM3 versus anti-PD-1 p = 0.03. 1) The survival rate of low-grade glioma CX3CR1 KO GEMM mice was estimated by the Kaplan-Meier method. IgG Control: 20 mice (MS: 122d), anti-TIM3: 28 mice (MS: 129.5). Statistics (log-rank test): control versus anti- TIM3 p = 0.57. J) Representative multiplex immunofluorescent imaging of brains from murine LGG model. Tumors were demarcated using H&E by a neuropathologist. Scale bar at 100 pm. K) scRNA seq UMAP plot of the intratumoral myeloid cells. N=2-3 per group. L) scRNA seq UMAP plot of the intratumoral lymphoid cells. M) Strip plot showing the differential abundance of cell types in the wild-type GEMM TME with treatment (anti-TIM3 vs IgG and 4 doses vs 2 doses), log2(fold change (FC). Cell types are ranked by mean log2 FC of anti-TIM3 vs IgG.
Figure 6A-B. A) Human immunophenotyping of matched donor PBMC and TIC gating strategy: Top: Unstained pooled samples. Bottom: Representative tumor-infiltrating cells (TIC) from matched pediatric patients. FSC-H vs FSC-A to determine singlets. SSC-A vs FSC-A to determine cells. SSC-A vs LD for negative selection of live cells. The percentage of indicated subpopulation is displayed. LD = Live/Dead fixable viability stain. Myeloid cells are defined as live CD45+, CD1 lb+. Lymphoid cells are defined as CD45+, CD1 lb-. B) Ex vivo analysis of PA samples. Top: Representative gating of lymphocytes. Bottom: Representative gating of myeloid cells. FSC-H vs FSC-A to determine singlets. SSC-A vs FSC-A to determine cells. SSC-A vs LD for negative selection of live cells. The percentage of indicated subpopulation is displayed in blue. LD = Live/Dead fixable viability stain. Myeloid cells are defined as live CD45+, CD1 lb+. Lymphoid cells are defined as CD45+, CD 11b-.
Figure 7A-C. A) Western plot of CT-2A cells showing p-MAPK expression. B) Nanostring analysis of tumor and matched peritumoral PA samples. Gene Upregulated in Tumor vs. Peritumor using unsupervised hierarchical clustering method. C) Directed Enrichment Score (DES) of tumor vs. peritumor. Figure 8A-B. A) Gene Ontology analysis between BRAF-Fusion and ANB. Bubble plot depicting immune-related gene ontology (GO) analysis of intratumoral myeloid-derived cells from BRAF-Fusion compared to ANB. Each bubble represents a GO term, the bubble size corresponds to the gene ratio and the color indicates the P-value. B) Multiplex immunofluorescent imaging demonstrating an immunological synapse present in BRAF-Fusion PA. CD4: red; CDl lc: green; LCK: cyan blue. Scale bars at 50 pm.
Figure 9. Representative spatial multiplex immunofluorescence images of the adjacent normal brain showing the absence of TIM-3 and CD163 expression. The vessels and anatomical layers of the normal cerebellar cortex (granular and white matter layers) are annotated. Scale bars at 100 pm.
Figure 10. mRNA expression of HAVCR2, STAT3, and PDCD1 of all tumor types from 3 public databases (Griesinger, Gump, and Henriquez). mRNA expression levels are displayed after the Log2 transformation.
Figure 11A-D. In vivo therapeutic effects of anti-TIM3 in the CT-2A glioma model. A) UMAP and feature plot showing the HAVCR2 (TIM3) expression within the CT2A model. B) Schema of the treatment of immunocompetent C57BL/6 mice that underwent intracerebral implantation of CT-2A glioma cells. Mice were treated with either anti-TIM3 (300ug/mouse) or IgG (lOOug/mouse) once per week or anti-PDl (100 ug/mouse) 3 times weekly starting on Day 7 after implantation. C) The survival rate of high-grade glioma-bearing C57BL/6 mice implanted were estimated by the Kaplan-Meier method. IgG Control: 10 mice (MS: 39 d), anti-PD-1: 10 mice (MS: 47 d), anti-TIM3: 10 mice (MS: 47d). Statistics (log-rank test): control versus anti- PD-1 p = 0.11, control versus anti-TIM3 p=0.14, anti-PD-1 vs anti-TIM3 p = 0.95. D) The survival rate of low-grade glioma CD8 KO GEMM mice was estimated by the Kaplan-Meier method. IgG Control: 14 mice (MS: 105d), anti-T!M3: 19 mice (MS: undefined). Statistics (logrank test): control versus anti-TIM3 p = 0.66.
DEFINITIONS
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before the present materials and methods are described, it is to be understood that this invention is not limited to the particular molecules, compositions, methodologies or protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the embodiments described herein, the following definitions apply.
As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a TIM-3 inhibitor” is a reference to one or more TIM-3 inhibitors and equivalents thereof known to those skilled in the art, and so forth.
As used herein, the term “comprise” and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term “consisting of” and linguistic variations thereof, denotes the presence of recited feature(s), element(s), method step(s), etc. and excludes any unrecited feature(s), element(s), method step(s), etc., except for ordinarily-associated impurities. The phrase “consisting essentially of’ denotes the recited feature(s), element(s), method step(s), etc. and any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed “consisting of’ and/or “consisting essentially of” embodiments, which may alternatively be claimed or described using such language.
As used herein, the term “pharmaceutically acceptable carrier” refers to non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, excipient, or carrier conventional in the art for use with a therapeutic agent for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed. For example, if the therapeutic agent is to be administered orally, the carrier may be a gel capsule. A “pharmaceutical composition” typically comprises at least one active agent (e.g., the copolymers described herein) and a pharmaceutically acceptable carrier.
As used herein, the term “effective amount” refers to the amount of a composition (e.g., pharmaceutical composition) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.
As used herein, the term “administration” refers to the act of giving a drug, prodrug, or other agent, or therapeutic treatment (e.g., pharmaceutical compositions of the present invention) to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs. Exemplary routes of administration to the human body can be through the eyes (e.g., intraocularly, intravitreally, periocularly, ophthalmic, etc.), mouth (oral), skin (transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like.
As used herein, the terms “co-administration” and “co-administer” refer to the administration of at least two agent(s) or therapies to a subject. In some embodiments, the coadministration of two or more agents or therapies is concurrent (e.g., in the same or separate formulations). In other embodiments, a first agent/therapy is administered prior to a second agent/therapy. Those of skill in the ail understand that the formulations and/or routes of administration of the various agents or therapies used may vary. The appropriate dosage for coadministration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s).
As used herein, the term “subject” broadly refers to any animal, including but not limited to, human and non-human animals (e.g., dogs, cats, cows, horses, sheep, poultry, fish, crustaceans, etc.). As used herein, the term “patient” typically refers to a subject that is being treated for a disease or condition.
As used herein, the term “neonate”, when used in reference to a human subject, refers to a subject under 30 days of age. As used herein, the term “infant”, when used in reference to a human subject, refers to a subject under 1 year of age.
As used herein, the term “child”, when used in reference to a human subject, refers to a subject that has yet to reach puberty.
As used herein, the term “adolescent”, when used in reference to a human subject, refers to a subject in the years ranging from puberty to early adulthood (e.g., 11-18).
As used herein, the term “early adult”, when used in reference to a human subject, refers to a subject form 19-25 years of age.
As used herein, an “immune response” refers to the action of a cell of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, neutrophils, etc.) and soluble macromolecules produced by any of these cells or the liver (e.g., antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from a subject of invading pathogens, cells or tissues infected with pathogens, or cancerous cells or other abnormal/diseased-associated cells.
As used herein, the term “immunotherapy” refers to the treatment or prevention of a disease or condition by a method comprising inducing, enhancing, suppressing or otherwise modifying an immune response.
As used herein, the term “immunotherapeutic” refers to any agent (e.g., small molecule, peptide, antibody, engineered cell, etc.) capable of stimulating a host immune system to generate an immune response to a tumor or cancer in the subject.
As used herein, the term “T-cell-based therapy” refers to any immunotherapy that acts through T cells. T-cell-based therapies include the administration of exogenous T cells (e.g., CAR-T cell therapies) and therapies that act upon or through a subjects endogenous T cells (e.g., checkpoint inhibitors.
As used herein, the term “antibody” refers to a whole antibody molecule or a fragment thereof (e.g., fragments such as Fab, Fab', and F(ab')2), unless specified otherwise; an antibody may be polyclonal or monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, etc.
As used herein, the term “antibody fragment” refers to a portion of a full-length antibody, including at least a portion antigen binding region or a variable region. Antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, Fd, diabodies, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. See, e.g., Hudson et al. (2003) Nat. Med. 9:129-134; herein incorporated by reference in its entirety. In certain embodiments, antibody fragments are produced by enzymatic or chemical cleavage of intact antibodies (e.g., papain digestion and pepsin digestion of antibody) produced by recombinant DNA techniques, or chemical polypeptide synthesis.
The term “chimeric antigen receptor” (“CAR”) refers to a recombinant polypeptide construct comprising at least an extracellular antigen-recognition domain, a transmembrane domain and an intracellular signaling domain. Upon binding to their target (e.g., displayed on a cancer cell), CARs typically modify the immune response of the immune cells they are displayed upon.
As used herein, the term “CAR-T cell” refers to a T cell that has been engineered to express a chimeric antigen receptor. In particular embodiments, T cells (e.g., from a subject) are engineered to express a CAR that binds to a cancer- specific antigen of cancer cells, thereby allowing CAR-T cells to effectively recognize and kill cancer cells,
As used herein, the term “adoptive cell transfer” (“ACT”) is the transfer of cells into a patient. The cells may have originated from the patient or from another individual or cell line. The cells are most commonly derived from the immune system, with the goal of improving immune functionality or eliciting a desired immune response. In some embodiments, cells are extracted from a subject, genetically modified (e.g., to express a desired construct (e.g., CAR or endanger molecule)), cultured in vitro, and returned to the subject.
As used herein, the term "diagnosis" refers to the detection, determination, or recognition of a health status or condition of an individual on the basis of one or more signs, symptoms, data, or other information pertaining to that individual. The terms “diagnose”, “diagnosing”, “diagnosis”, etc., encompass, with respect to a particular disease or condition, the initial detection of the disease; the characterization or classification of the disease; the detection of the progression, remission, or recurrence of the disease; and the detection of disease response after the administration of a treatment or therapy to the individual. The diagnosis of glioma includes distinguishing individuals who have glioma (or are likely to) from individuals who do not (or are unlikely to). As used herein, the term "prognosis" refers to risk prediction of the severity of disease or of the probable course and clinical outcome associated with a disease. Thus, the term "method of prognosis" as used herein refers to methods by which the skilled person can estimate and/or determine a probability that a given outcome will occur.
As used herein, “marker” and “biomarker” are used interchangeably to refer to a target molecule or condition that indicates or is a sign of a normal or abnormal process in an individual or of a disease or other condition in an individual. Biomarkers may be detectable and measurable by a variety of methods including home, clinic, and laboratory assays.
DETAILED DISCUSSION
Provided herein are compositions and methods for the treatment of glioma by targeting TIM-3. In particular, a subject suffering from a glioma is administered a TIM-3 inhibitor alone or in combination with other glioma therapies.
Pediatric gliomas are the leading cause of childhood mortality among childhood cancers. The repurposing of immunotherapies has not been successful in the treatment of pediatric gliomas. In experiments conducted during development of embodiments herein whole transcriptome sequencing, single-cell sequencing, and sequential multiplex immunofluorescence were used to identify an immunotherapy strategy evaluated in multiple preclinical glioma models. MAPK-driven pediatric gliomas have a higher interferon signature relative to other molecular subgroups. Single-cell sequencing identified an activated and cytotoxic microglia population designated MG- Act in BRAF-fused MAPK-activated PA, but not in high-grade gliomas or normal brain. TIM3 is expressed on MG- Act and on the myeloid cells lining the tumor vasculature but not normal brain. TIM3 expression becomes upregulated on immune cells in the PA microenvironment and anti-T!M3 reprograms ex vivo immune cells from human PAs to a pro-inflammatory cytotoxic phenotype. In a genetically engineered murine model of MAPK- driven low-grade gliomas, anti-TIM3 treatment increased median survival over IgG and anti- PD1 treated mice. ScRNA sequencing data during the therapeutic window of anti-TIM3 demonstrates enrichment of the MG- Act population. The therapeutic activity of anti-TIM3 is abrogated in the CX3CR1 microglia knockout background. These data support the use of anti- TIM3 in the treatment of pediatric low-grade MAPK-driven gliomas. Experiments conducted during development of embodiments herein demonstrate that MAPK-driven gliomas display an immune reactive phenotype. In particular, analysis of BRAF- fusion PA identified a unique highly activated P2RY 12+ microglia population dispersed throughout the tumor microenvironment, active antigen presentation between a CDllc+ antigen presenting cells and T cells, and a therapeutic opportunity for targeting TIM-3.
MAPK-driven gliomas exhibited immune reactive signatures as indicated by IFN-y expression, as well as by heightened pro-inflammatory Ml macrophage and T cell component. The tumor microenvironment (TME) of BRAF-fusion PA was notable for antigen presentation and the interaction of CD1 lc+ cells with T cells through Lck+ immunological synapses. PA CD1 lc+CD206+ dendritic cells localized to vessel walls and were positive for TIM-3 expression, an immune checkpoint marker. A unique highly activated microglia subcluster was identified that morphologically resembled T cells expressing TNF and TIM-3. Treatment of an ex vivo PA with anti-TIM3 antibodies triggered the induction of pro-inflammatory responses in autologous immune cells.
Glioma is an aberrant growth of cells that begins in the brain or spinal cord and initially resembles healthy glial cells but is capable of growing into tumors that press on the brain or spinal cord resulting in symptoms. Malignant gliomas grow quickly and can invade healthy brain tissue. Gliomas are classified as astrocytoma, ependymoma, glioblastoma, or oligodendroglioma, depending upon the types of cells from which they originate and the aggressiveness of the malignancy (astrocytoma - astrocytes, ependymoma - ependymal cells, glioblastoma - astrocytes (rapidly malignant), or oligodendroglioma - oligodendrocytes).
PA are the most common pediatric glioma. Surgical resection of PA is typically the first- line treatment and gross total resection is often curative (15, 16). However, PAs that are not amenable to gross total resection have associated long-term morbidity and mortality. The ten- year progression-free survival for PA patients with radiologically visible residual tumor is less than 50%. Other treatment options are frequently necessary in gliomas with a midline location, which can be challenging to biopsy or resect safely.
Experiments have previously been conducted to characterize adult gliomas for potential immune therapeutic targets. These studies revealed, for example, in adult high-grade IDH-WT gliomas, that targeting the CD73/adenosine pathway could be of benefit in treating this patient population. These prior analyses were conducted to provide prioritization of available immune therapeutic strategies and to clarify if response biomarkers needed to be considered for stratification and/or enrollment for immunotherapy clinical trials. Experiments were conducted during development of embodiments herein toward identifying frequent immune modulatory targets that may be relevant to treating pediatric glioma patients. Using orthogonal strategies of high dimensional immunofluorescence multiplexing and scSeq within the TME, it was discovered that agents targeting TIM3 could benefit patients with BRAF-Fusion PA. In addition, results are consistent with the results of others in suggesting potential benefit in targeting tumors with elevated MAPK activity. Since the expression of other immune checkpoints such as Lag-3, PD-1, and TIGIT are minimally co-expressed, this indicates that the T cells are not in a state of exhaustion that has been defined in adult high-grade gliomas.
TIM3 expression was primarily observed in myeloid cells, which is consistent with findings from orthotopic implanted high-grade glioma mouse models. In such models the combination of radiation, anti-TIM3 and anti-PD- 1 appeared to be curative of tumor. Preclinical testing has also shown that anti-TIM3 therapy shows synergistic anti-tumor activity when combined with adoptive immunotherapy therapeutics such as CAR T cells. No significant toxicity has been noted in preclinical studies involving TIM-3 targeting and experiments conducted during development of embodiments herein found minimal expression of TIM-3 within the adjacent normal brain.
Experiments were conducted during development of embodiments herein to test immune activation and immune mediated cytotoxicity in autologous co-cultures incubated with anti- TIM3 and enhanced immune cell proinflammatory response was observed in the immune compartment. In contrast to other immune therapeutic targets, TIM3 expression is on the endothelial side of the blood vasculature. The role of TIM3 in this context is an area for future investigation but does provide a target for large molecules such as antibodies that are typically excluded by the blood brain barrier.
Companion biomarkers such as TMB, IFN signatures, PD-1, and PD-L1 expression have been used for identifying patients who may respond to ICT. However, with the exceptions of the IFN signature and p-ERK, these markers have not been predictive of response to ICI for adult glioblastoma patients. The expression of PD-1 and PD-L1 is lower relative to TIM3 in BRAF- Fusion PA. As such, the use of ICI targeting PD-1 or PD-L1 are unlikely to be of therapeutic benefit in this setting. Of the various biomarkers used to potentially predict responses to ICIs, only the IFN-signature and p-ERK have been shown to correlate with therapeutic responses in gliomas. Published literature indicates that MAPK activation is the oncogenic mechanism for BRAF driven gliomas. The marked expression of p-ERK in PA indicates that these tumors might be responsive to ICI therapy.
Because TIM-3 is expressed on a substantial number of bone marrow derived immune cells such as dendritic cells, including those that line the blood vasculature, in some embodiments, the TIM-3 inhibitors described herein are administered systemically. In some embodiments, the brain intrinsic microglia that express TIM-3 are accessed with blood-brain- barrier opening ultrasound strategies (e.g., implantable ultrasound device for blood-brain barrier opening).
I. TIM-3 inhibition
In some embodiments, methods described herein employ a TIM-3 inhibitor or a composition/method for the inhibition of TIM-3 activity or expression.
In some embodiments, provided herein are inhibitors of TIM-3 activity. In some embodiments, an inhibitor is a small molecule, peptide, antibody, antibody fragment, a genome editing agent, etc. that upon administration to a subject, cell, etc. reduces the activity of TIM-3, for example, systemically, within a specific tissue, organ, or body system, within a specific cell population, etc.
In some embodiments, a TIM-3 inhibitor is an antibody or antibody fragment. In some embodiments, a TIM-3 inhibitor is an antibody or antibody fragment that binds to TIM-3 and reduces TIM-3 activity. In some embodiments, an anti-TIM-3 antibody or antibody fragment is provided that prevents or reduces binding of TIM-3 to a ligand thereof. In some embodiments, a TIM-3 antibody for use in a treatment herein is selected from MBG453 (aka, “Sabatolimab”, Novartis Pharmaceuticals, Clinical trial NCT02608268), TSR-022 (Tesaro, Inc. Clinical trial NCT02817633), or LY3321367 (Eli Lilly, Clinical trial NCT03099109). In some embodiments, a TIM-3 antibody for use in a treatment herein is selected from RMT3-23, ATIK2a, MBG453 (sabatolimab), AZD-7789, BMS-986258, INCAGN-2390, TSR-022 (Cobolimab), LY3321367, RO-7121661 (Lomvastomig), TQB-2618.
In some embodiments, provided herein are inhibitors of TIM-3 expression. In some embodiments, an inhibitor is a small molecule, peptide, antibody, antibody fragment, a genome editing agent, etc. In particular embodiments, a TIM-3 inhibitor is a nucleic acid-based inhibitor. In some embodiments, the inhibitor is a small molecule, an aptamer, a siRNA, a shRNA, a miRNA, a morpholino, a ribozyme, an antisense nucleic acid molecule, a CRISPR-Cas9-based construct, a CRISPR-Cpfl -based construct, a meganuclease, a zinc finger nuclease, a transcription activator-like (TAL) effector (TALE) nuclease, etc.
In some embodiments, the TIM-3 inhibitor is a small interfering RNA (siRNA), also known as short interfering RNA or silencing RNA. In some embodiments, an siRNA is an 18 to 30 nucleotide, preferably 19 to 25 nucleotide, most preferred 21 to 23 nucleotide or even more preferably 21 nucleotide-long double- stranded RNA molecule. siRNA is involved in the RNA interference (RNAi) pathway where the siRNA interferes with the expression of a specific gene (e.g., the TIM-3). siRNAs naturally found in nature have a well-defined structure: a short doublestrand of RNA (dsRNA) with 2-nt 3' overhangs on either end. Each strand has a 5' phosphate group and a 3' hydroxyl (-OH) group. This structure is the result of processing by dicer, an enzyme that converts either long dsRNAs or small hairpin RNAs into siRNAs. siRNAs can also be exogenously (artificially) introduced into cells to bring about the specific knockdown of a gene of interest (e.g., the TIM-3). Essentially any gene for which the sequence is known can thus be targeted based on sequence complementarity with an appropriately tailored siRNA. The double-stranded RNA molecule or a metabolic processing product thereof is capable of mediating target- specific nucleic acid modifications, particularly RNA interference and/or DNA methylation. Exogenously introduced siRNAs may be devoid of overhangs at their 3' and 5' ends, however, in some embodiments at least one RNA strand has a 5'- and/or 3'-overhang. Preferably, one end of the double-strand has a 3'-overhang from 1 to 5 nucleotides, more preferably from 1 to 3 nucleotides and most preferably 2 nucleotides. The other end may be blunt-ended or has up to 6 nucleotides 3'-overhang. In general, any RNA molecule suitable to act as siRNA and inhibit the TIM-3 is envisioned in the present invention. In some embodiments, siRNA duplexes are provided composed of 21-nt sense and 21-nt antisense strands, paired in a manner to have a 2-nt 3'-overhang. The sequence of the 2-nt 3' overhang makes a small contribution to the specificity of target recognition restricted to the unpaired nucleotide adjacent to the first base pair. 2'- deoxynucleotides in the 3' overhangs are as efficient as ribonucleotides, but are often cheaper to synthesize and probably more nuclease resistant. Delivery of siRNA may be accomplished using any of the methods known in the art, for example by combining the siRNA with saline and administering the combination intravenously or intranasally or by formulating siRNA in glucose (such as for example 5% glucose) or cationic lipids and polymers can be used for siRNA delivery in vivo through systemic routes either intravenously (IV) or intraperitoneally (IP). In some embodiments, provided herein are siRNA molecules that target and inhibit the expression (e.g., knock down) of the TIM-3
A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression (e.g., of TIM-3) via RNA interference. In some embodiments, shRNA uses a vector introduced into cells and utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). The RISC binds to and cleaves RNAs which match the siRNA that is bound to (e.g., comprising the sequence of the TIM-3 ). In some embodiments, si/shRNAs to be used in the present invention are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. In some embodiments, provided herein are shRNA molecules that target and inhibit the expression (e.g., knock down) of the TIM-3 .
Further molecules effecting RNAi (and useful herein for the inhibition of expression of the TIM-3) include, for example, microRNAs (miRNA). Said RNA species are single-stranded RNA molecules. Endogenously present miRNA molecules regulate gene expression by binding to a complementary mRNA transcript and triggering of the degradation of said mRNA transcript through a process similar to RNA interference. Accordingly, exogenous miRNA may be employed as an inhibitor of the TIM-3 after introduction into target cells. In some embodiments, provided herein are miRNA molecules that target and inhibit the expression (e.g., knock down) of the TIM-3.
Morpholines (or morpholino oligonucleotides) are synthetic nucleic acid molecules having a length of about 20 to 30 nucleotides and, typically about 25 nucleotides. Morpholines bind to complementary sequences of target transcripts (e.g., the TIM-3 ) by standard nucleic acid base-pairing. They have standard nucleic acid bases which are bound to morpholine rings instead of deoxyribose rings and linked through phosphorodiamidate groups instead of phosphates. Due to replacement of anionic phosphates into the uncharged phosphorodiamidate groups, ionization in the usual physiological pH range is prevented, so that morpholines in organisms or cells are uncharged molecules. The entire backbone of a morpholino is made from these modified subunits. Unlike inhibitory small RNA molecules, morpholines do not degrade their target RNA molecules. Rather, they sterically block binding to a target sequence within a RNA and prevent access by molecules that might otherwise interact with the RNA. In some embodiments, provided herein are morpholino oligonucleotides that target and inhibit the expression (e.g., knock down) of the TIM-3.
A ribozyme (ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA) is an RNA molecule that catalyzes a chemical reaction. Many natural ribozymes catalyze either their own cleavage or the cleavage of other RNAs, but they have also been found to catalyze the aminotransferase activity of the ribosome. Non-limiting examples of well-characterized small self-cleaving RNAs are the hammerhead, hairpin, hepatitis delta virus, and in vitro- selected leaddependent ribozymes, whereas the group I intron is an example for larger ribozymes. The principle of catalytic self-cleavage is well established. Since it was shown that hammerhead structures can be integrated into heterologous RNA sequences and that ribozyme activity can thereby be transferred to these molecules, catalytic antisense sequences can be engineered for almost any target sequence can be created, provided the target sequence contains a potential matching cleavage site. The basic principle of constructing hammerhead ribozymes is as follows: A region of interest of the RNA (e.g., a portion of the TIM-3), which contains the GUC (or CUC) triplet, is selected. Two oligonucleotide strands, each usually with 6 to 8 nucleotides, are taken and the catalytic hammerhead sequence is inserted between them. In some embodiments, provided herein are ribozyme inhibitors oligonucleotides of the TIM-3.
In some embodiments, TIM-3 is inhibited (and/or TIM-3 activity is inhibited) by modifying the TIM-3 sequence in target cells. In some embodiments, the alteration of the TIM-3 is carried out using one or more DNA-binding nucleic acids, such as alteration via an RNA- guided endonuclease (RGEN). For example, the alteration can be carried out using clustered regularly interspaced short palindromic repeats (CRISPR) and CRIS PR-associated (Cas) proteins. In general, "CRISPR system" refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a "direct repeat" and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a "spacer" in the context of an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR locus. The CRISPR/Cas nuclease or CRISPR/Cas nuclease system can include a non-coding RNA molecule (guide) RNA, which sequence-specifically binds to DNA, and a Cas protein (e.g., Cas9), with nuclease functionality (e.g., two nuclease domains). One or more elements of a CRISPR system can derive from a type I, type II, or type III CRISPR system, e.g., derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In some aspects, a Cas nuclease and gRNA (including a fusion of crRNA specific for the target sequence (e.g., a sequence within the TIM-3 ) and fixed tracrRNA) are introduced into the cell. In general, target sites at the 5' end of the gRNA target the Cas nuclease to the target site, e.g., the TIM-3, using complementary base pairing. The target site may be selected based on its location immediately 5' of a protospacer adjacent motif (PAM) sequence, such as typically NGG, or NAG. In this respect, the gRNA is targeted to the desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence (e.g., sequence within the TIM-3). In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence. Typically, "target sequence" generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. The CRISPR system can induce double stranded breaks (DSBs) at the SRC-3 target site, followed by disruptions or alterations as discussed herein. In other embodiments, Cas9 variants, deemed "nickases," are used to nick a single strand at the target site (e.g., within the TIM-3). Paired nickases can be used, e.g., to improve specificity, each directed by a pair of different gRNAs targeting sequences such that upon introduction of the nicks simultaneously, a 5' overhang is introduced. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain such as a transcriptional repressor or activator, to affect gene expression (e.g., to inhibit expression of the TIM-3). In some embodiments, the CRISPR system is used to alter the TIM-3, inhibit expression of the TIM-3, and/or to inactivate the expression product of the TIM-3. The term "antisense nucleic acid molecule" or “antisense oligonucleotide” as used herein, refers to a nucleic acid which is complementary to a target nucleic acid. An antisense molecule in accordance with the invention is capable of interacting with the target nucleic acid, more specifically it is capable of hybridizing with the target nucleic acid. Due to the formation of the hybrid, transcription of the target gene(s) and/or translation of the target mRNA is reduced or blocked. Standard methods relating to antisense technology have been described (see, e.g., Melani et al., Cancer Res. (1991) 51:2897-2901). In some embodiments, provided herein are antisense oligonucleotides capable of inhibiting expression of TIM-3 when administered to cell or subject. In some embodiments, the antisense oligonucleotides are antisense DNA- and/or RNA-oligonucleotides. In some embodiments, provided herein are modified antisense oligonucleotides, such as, antisense 2'-O-methyl oligo-ribonucleotides, antisense oligonucleotides containing phosphorothiaote linkages, antisense oligonucleotides containing Locked Nucleic Acid LNA(R) bases, morpholino antisense oligonucleotides, PPAR-gamma agonists, antagomirs. In some embodiments, ASOs comprise Locked Nucleic Acid (LNA) or 2’- methoxyethyl (MOE) modifications (internucleotide linkages are phosphorothioates interspersed with phosphodiesters, and all cytosine residues are 5 ’-methylcytosines).
II. Subject
In some embodiments, methods are provided herein for the treatment, diagnosis, prognosis, etc. of glioma in a subject. In some embodiments, a subject of the methods herein suffers from glioma or is suspected of suffering from glioma. In some embodiments, the glioma is astrocytoma, ependymoma, glioblastoma, or oligodendroglioma. In some embodiments, the glioma is an astrocytoma selected from pilocytic astrocytoma (grade 1), diffuse astrocytoma (grade 2), anaplastic astrocytoma (grade 3), glioblastoma (grade 4), a pineal astrocytic tumor, a brain stem glioma, etc. In some embodiments, the glioma is a grade I, II, or III ependymoma. In some embodiments, the glioma is a grade I, II, or III oligodendroglioma. In particular embodiments, a subject suffers from or is suspected of suffering from pilocytic astrocytoma.
In some embodiments, the subject is a child, adolescent, or early adult. In some embodiments, the subject is 25 years, 24 years, 23 years, 22 years. 21 years, 20 years, 19 years, 18 years, 17 years, 16 years, 15 years, 14 years, 13 years, 12 years, 11 years, 10 years, 9 years, 8 years, 7 years, 6 years, 5 years, 4 years, 3 years, 2 years, 12 months, 9 months, 6 months, 3 months old, or fewer, or ranges therebetween (e.g., 5-25 years of age). In other embodiments the subject is an adult. In some embodiments, the subject’s immune system is not exhausted (e.g., not in a hypo-responsive T cell state), as is the case for an adult with glioblastoma (GBM).
In some embodiments, the subject exhibits one or more risk factors for glioma (e.g., a family history, being in remission from glioma or another caner, environmental or behavioral risk factors, a mutation or susceptibility factor that places the subject at increased risk, etc.).
III. Diagnostics/prognostics
In some embodiments, provided herein are methods for diagnosis of glioma (e.g., PA) in a subject. In some embodiments, provided herein are methods of providing a glioma-related prognosis for a subject.
In some embodiments, provided herein is the detection and/or quantification of one or more glioma or astrocytoma biomarkers, such as platelet-derived growth factor receptor (PDGFR), neurofilament light chain (NF-L), epidermal growth factor receptor (EGFR), and CD44. In some embodiments, methylation of one or more genes (e.g., MGMT, PTEN, RBI, TP53, CDKN2A, PDGFB, EMP3, SOCS1, PCDHGA11, OLIIG1/2, etc.) is indicative of glioma and may serve as a detectable biomarker in the methods herein. In some embodiments, one or more chromosomal aberrations (e.g., gain of Iq, 19q, 20q, etc. or loss of 6q, 9p, 13q, 14q, or 22q, etc.) is indicative of glioma and may serve as a detectable biomarker in the methods herein. In some embodiments, mutations in one or more genes (e.g., PTEN, ATRX, TP53, RBI, IDH1/2, NF1, EGFR, etc.) is indicative of glioma and may serve as a detectable biomarker in the methods herein. In some embodiments, amplifications of one or more genes (e.g., MET, EGFR, PIK3CA, PDGFRA, CCND2, MDM2/4, etc.) is indicative of glioma and may serve as a detectable biomarker in the methods herein. In some embodiments, deletions of one or more genes (e.g., CDKN2A, CDKN2B, CDKN2C, PTEN, RBI, NFKB1A, etc.) is indicative of glioma and may serve as a detectable biomarker in the methods herein.
In some embodiments, methods are provided for determining a treatment course of action. In some embodiments, methods are provided for determining whether TIM-3 inhibition is a useful or potentially-useful treatment for glioma (e.g., PA) in a subject. Experiments conducted during development of embodiments herein demonstrate that a clinical indication for TIM-3 inhibition for the treatment of a glioma (e.g., PA) in a subject with a BRAF-fusion (e.g., KIAA1549-BRAF fusion).
There are numerous markers that have been used to predict responses to immune checkpoint inhibitors. Of the various biomarkers used to potentially predict responses to ICIs, IFN-signature and p-ERK are correlated with therapeutic responses in gliomas.
The biomarkers useful in embodiments herein may be detected and/or quantified by any suitable techniques, including but not limited to nucleic acid analysis, sequencing, ELISA, mass spectrometry, immunohistochemistry, etc.
Some embodiments comprise use of nucleic acid sequencing to detect, quantify, and/or identify biomarkers. The term “sequencing,” as used herein, refers to a method by which the identity of at least 10 consecutive nucleotides (e.g., the identity of at least 20, at least 50, at least 100, or at least 200 or more consecutive nucleotides) of a polynucleotide are obtained. The term “next-generation sequencing” refers to the so-called parallelized sequencing-by-synthesis, sequencing-by-ligation platforms, nanopore sequencing methods, or electronic-detection based methods that will be understood in the field.
Some embodiments utilize various protein, peptide, or small molecule detection/quantification techniques. Analytical platforms (e.g., High-throughput platforms, automated platforms, etc.) utilizing nuclear magnetic resonance (NMR) spectroscopy, gas chromatography (GC), liquid chromatography (LC), and/or mass spectrometry (MS) may be employed to measure biomarkers within a biological sample (e.g., blood, urine, biopsy, etc.) from a subject.
Mass spectrometry can accurately identify /quantify thousands of biomolecules within complex biological samples. In some embodiments, biomolecules are detected/quantified in a biological sample using MS techniques, such as MALD1/TOF (time-of-flight), SELDI/TOF, liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography-mass spectrometry (HPLC-MS), capillary electrophoresis-mass spectrometry, nuclear magnetic resonance spectrometry, tandem mass spectrometry (e.g., MS/MS, MS/MS/MS, ESI-MS/MS etc.), secondary ion mass spectrometry (SIMS), or ion mobility spectrometry (e.g. GC-IMS, IMS-MS, LC-IMS, LC-IMS-MS etc.). Mass spectrometry methods are well known in the art and have been used to quantify and/or identify biomolecules, such metabolites. Antibody-based techniques, such as ELISA, or immunohistochemistry may also be utilized for the detection/quantification of biomarkers.
IV. Co-administration
In some embodiments, the TIM-3 inhibitor is co-administered with a chemotherapeutic, immunotherapeutic, surgery, and/or radiation.
Exemplary chemotherapeutics for co-administration with a TIM-3 inhibitor include temozolomide, etoposide, doxorubicin, cisplatin, paclitaxel, carmustine, lomustine, ceramide and/or phosphorylcholine.
Exemplary immunotherapeutic s for co-administration with a TIM-3 inhibitor include immune checkpoint inhibitor, STING agonists (e.g., IACS-8803, TAK-500, TAK-676, ADU- S100, etc.), ACT therapy, CAR-T cell therapy, monoclonal antibody therapy, and bispecific T- cell engager therapy. In some embodiments, a TIM-3 inhibitor is co-administered with an immune checkpoint inhibitor that binds to and inhibits the activity of an immune checkpoint protein is selected from the group consisting of CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, or VISTA. In some embodiments, the TIM-3 inhibitor is administered with an immune checkpoint inhibitor selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, STI-Al l 10, TSR-042, RG- 7446, BMS-936559, BMS-936558, MK-3475, MPDL3280A, MEDI-4736, MSB-0020718C, AUR-012 and STI-A1010.
In some embodiments, a TIM-3 inhibitor is co-administered with radiation therapy. Techniques for administering radiation therapy are known in the art, and these techniques can be used in the combination therapies described herein. Radiation therapy can be administered through one of several methods, or a combination of methods, including without limitation external-beam therapy, internal radiation therapy, implant radiation, stereotactic radiosurgery, systemic radiation therapy, radiotherapy and permanent or temporary interstitial brachytherapy.
In some embodiments, a TIM-3 inhibitor is administered before and/or after surgical removal of cell or a tumor related to the glioma.
EXPERIMENTAL
Materials and methods Data collection
Human subjects were de-identified prior to analysis. Specimens are obtained from multiple research centers within the United States and have limited clinical annotation.
Next-Generation Sequencing (NGS) ofDNA
NGS was performed on genomic DNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor samples using the NextSeq or NovaSeq 6000 platforms (Illumina, Inc., San Diego, CA). For NextSeq sequenced tumors, a custom-designed SureSelect XT assay was used to enrich 592 whole-gene targets (Agilent Technologies, Santa Clara, CA). For NovaSeq sequenced tumors, more than 700 clinically relevant genes at high coverage and high read-depth were used, along with another panel designed to enrich for an additional >20,000 genes at lower depth. All variants were detected with >99% confidence based on allele frequency and amplicon coverage, with an average sequencing depth of coverage of >500 and an analytic sensitivity of 5%. Prior to molecular testing, tumor enrichment was achieved by harvesting targeted tissue using manual microdissection techniques. Genetic variants identified were interpreted by board-certified molecular geneticists and categorized as ‘pathogenic,’ ‘likely pathogenic,’ ‘variant of unknown significance,’ ‘likely benign,’ or ‘benign,’ according to the American College of Medical Genetics and Genomics standards. When assessing mutation frequencies of individual genes, ’pathogenic,’ and ‘likely pathogenic’ were counted as mutations. The copy number alteration of each exon was determined by calculating the average depth of the sample along with the sequencing depth of each exon and comparing this calculated result to a pre-calibrated value.
Tumor mutational burden assessment
TMB was measured by counting all non-synonymous missense, nonsense, inframe insertion/deletion and frameshift mutations found per tumor that had not been previously described as germline alterations in dbSNP151, Genome Aggregation Database (gnomAD) databases or benign variants identified by Caris geneticists A cutoff point of >=10 mutations per MB was used based on the KEYNOTE-158 pembrolizumab trial, which showed that patients with a TMB of > 10 mt/MB across several tumor types had higher response rates than patients with a TMB of <10 mt/MB (30). Caris Life Sciences is a participant in the Friends of Cancer Research TMB Harmonization Project. Whole Transcriptome Sequencing
Gene fusion detection was performed on mRNA isolated from a formalin-fixed paraffin- embedded tumor sample using the Illumina NovaSeq platform (Illumina, Inc., San Diego, CA) and Agilent SureSelect Human All Exon V7 bait panel (Agilent Technologies, f , CA). FFPE specimens underwent pathology review to diagnose percent tumor content and tumor size; a minimum of 10% of tumor content in the area for microdissection was required to enable enrichment and extraction of tumor-specific RNA. Qiagen RNA FFPE tissue extraction kit was used for extraction, and the RNA quality and quantity was determined using the Agilent TapeStation. Biotinylated RNA baits were hybridized to the synthesized and purified cDNA targets and the bait-target complexes were amplified in a post capture PCR reaction. The resultant libraries were quantified, normalized and the pooled libraries are denatured, diluted and sequenced; the reference genome used was GRCh37/hgl9 and analytical validation of this test demonstrated >97% Positive Percent Agreement (PPA), >99% Negative Percent Agreement and >99% Overall Percent Agreement with a validated comparator method. Raw data was demultiplexed by Illumina Dragen BioIT accelerator, trimmed, counted, PCR-duplicates removed and aligned to human reference genome hgl9 by STAR aligner. For transcription counting, transcripts per million values were generated using the Salmon expression pipeline. Transcriptomic signatures predictive of response to immunotherapy (T cell inflamed score) and replication stress response defect (RSRD) score were calculated on transcripts per million (TPM) values. Immune cell fractions were estimated using RNA deconvolution (quanTIseq). The total cell fraction consists of 10 immune cell populations and an 11th group designated as ‘uncharacterized cells’ that includes both tumor and other stromal cells that are not one of the 10 cell populations adapted from.
Immunohistochemistry
Immunohistochemistry (IHC) was performed on full FFPE sections of glass slides. Slides were stained using automated staining techniques, per the manufacturer’s instructions, and were optimized and validated per CLIA/CAP and ISO requirements. PD-L1 IHC (SP142, Spring
Biosciences) expression was evaluated in tumor cells The staining was regarded as positive if its intensity on the membrane of the tumor cells was >=2+ (on a semiquantitative scale of 0-3: 0 for no staining, 1+ for weak staining, 2+ for moderate staining, or 3+ for strong staining) and the percentage of positively stained cells was >5% as evaluated by a board-certified pathologist.
Human Subjects
Under STU00214485, approved by the institutional review board of Northwestern University and 2021-4677, approved by the Ann and Robert H. Lurie Children’s Hospital of Chicago, patients were identified with surgically resectable tumors. Pediatric patients with a presumed diagnosis of a central nervous system (CNS) lesions based on radiographic with a planned clinically indicated surgical resection were prospectively consented by their guardian. Peripheral blood based on the patient’ s weight was obtained at the time of initial anesthesia induction and transferred to the research laboratory for processing. No modifications were made regarding the standardized surgical resection and standard oncological principals of en bloc removal with a margin of surrounding adjacent brain if neurologically feasible. For some cases in which normal brain was obtained, T2 or FLAIR abnormality was resected as part of the approach to create a corridor to a benign mass. After the removal of the mass, the specimen was aliquoted for parallel analysis by the operative neurosurgeon. Approximately 1 cm3 of the gadolinium-enhancing tumor was designated for scRNA sequencing analysis and was processed into a single-cell suspension after enrichment for the immune cells by Percoll gradient. A second adjacent piece of tumor in continuity with the surrounding brain was processed for FFPE. This specimen was used for sequential multiplex immunohistochemistry.
Tissue Processing and preparation
Patient tumors were graded pathologically by the study neuropathologists (CMH, NW) according to the World Health Organization classification. At least 500 mg of viable, non- necrotic tumor was required to obtain sufficient quantities for analysis and was processed within 1 hour after resection. Normal brain (NB) was obtained from subjects as part of the planned surgical approach to gain access to a low-grade non-infiltrating glioma or during a planned super-total resection of adjacent regions. The normal brain was sent for analysis separately from the tumor. The freshly resected tissue was processed in parallel as both for a single cell suspension and for FFPE analysis. The FFPE was used for sequential multiplexing immunofluorescence (SeqIF™) and NanoString nCounter analysis of a 770 gene panel after microdissection of the tissue (tumor area vs. adjacent normal brain) and RNA isolation (Qiagen kit). For the single cell suspension, the tissue was minced into small pieces using a scalpel, dissociated, and suspended using a Pasteur pipette in 10ml Iscove’s DMEM (IMDM; Iscove’s Modification of DMEM) IX (Coming) containing 2% inactivated Fetal Bovine Serum (FBS; Sigma Aldrich) and collagenase and DNase enzymes at the final concentrations of lOOpg/ml and 20 units/ml, respectively. The prepared mixture was incubated for 35-40 mins at 37°C with agitation. The tissue was filtrated using a 70- pm nylon cell strainer (BD Biosciences) and then underwent centrifugation at 4°C. The pellet was either resuspended culture media for functional assays or in 20 ml mix of 5.4ml of Percoll™ Plus (GE Healthcare) overlaid with 12 ml of IX Phosphate Buffered Saline (PBS) and 0.6ml of 10X PBS (Corning) for single-cell RNA- sequencing. The tube was centrifuged at 800g for 10 minutes at 4°C, with 9 acceleration and 0 deceleration. After centrifugation, the immune enriched cell pellet was collected, washed, stained with Trypan blue dye (Sigma- Aldrich), and counted using Countess II FL automated cell counter in a Countess cell counting chamber (Invitrogen).
Single-cell RNA-Sequencing
Single-cell sequencing was carried out using the chromium Next GEM Single Cell protocol (lOx genomics). Post library preparation cells were sequenced using the Illumina Novaseq. Raw data was preprocessed and aligned using Cell Ranger to obtain the matrix and count files. Seurat R Package using scRNA-seq SeuratlOx genomic workflow was then used for all subsequent analysis unless noted otherwise. After filtering using percent mitochondrial DNA threshold of 20% and UMI range of 200 to 15000, 186,317 cells were included for further analysis. Cells were then subject to Log Normalize, Scale Data, and PCA functions. Find Clusters and Find Markers functions was utilized for clustering and marker identification and non-linear dimensional reduction techniques were applied to visual data in UMAP plot format. Harmony algorithm was used to regress batch effects. Cell clusters were annotated using three methods to produce robust cell assignments: 1 ) comparison against known cell markers; 2) examination of DEGs against the Human Protein Atlas; and 3) ScType R package, an automated cell assignment algorithm. For murine single-cell sequencing, human immune cell assignments and transcriptional profiles were utilized for reference mapping. The differential abundance of major cell types was assessed in partially overlapping local neighborhoods on a k nearest- neighbor graph using the novel statistical framework MiloR with parameters: k=30, p=0.1, and d=30..
Gene Ontology Enrichment Analysis
The top 100 DEGs were used for Gene Ontology enrichment analysis using the Bioconductor Package Cluster Profiler. Significantly enriched GO-BP (Gene Ontology- Biological processes) terms were retrieved by setting the threshold of FDR=3; queried genes were manually selected using immunological keywords. Results were displayed using bubble plots. Each bubble represents a GO term, the bubble size corresponds to the gene ratio and the color indicates P-value.
Interactome Analysis
Potential ligand receptor (LR) interactions were analyzed using Bioconductor package CellChat. Only immune populations annotated as myeloid or lymphoid were included in the analysis. CellChat objects for BRAF-Fusion and NB were created, and a comparison analysis was used to infer differentially enriched ligand receptor interactions between the tumor subtypes. This was performed using compareinteractions and RankNet functions. Results were displayed using heatmaps and circle diagrams to visualize significant interactions occurring within the TME.
Automated Hyperplex SeqIFTmStaining and Imaging on the COMET™ System
The FFPE slides were collected from the Neurological Surgery Tumor Bank of Northwestern University. 4pm thickness tissue slices were prepared, mounted on positively charged glass slides (Super Frost Plus microscope slides, ThermoFisher) and stored at room temperature for subsequent staining analysis. For each case, 1 H&E slide was reviewed, and the tissue segmented by a certified neuropathologist (CMH). FFPE slides were preprocessed for antigen retrieval using the PT Module (Epredia) with Dewax and HIER Buffer H (TA999- DHBH, Epredia) for 60 min at 102°C. Subsequently, slides were rinsed and stored in Multistaining Buffer (BU06, Lunaphore Technologies) till use. The protocol template was generated using the COMET™ Control Software, and reagents were loaded onto the device to perform the sequential immunofluorescence (seqIF™) protocol. The markers used for this analysis were: CD31 (endothelial cells), GFAP (glioma tumor cells), CD4 (helper T cells), CD8 (cytotoxic T cells), CD20, and CD19 (B cells), P2RY12, CX3CR1, and TMEM119 (microglia), CD68 (pan- monocyte/macrophage marker), CDl lc (antigen presenting cells), CD163 (macrophage scavenger receptor), CD205 (dendritic cells), NKG2D (NK cells), p-STAT3 (nuclear hub of immune suppression), TIM3, LAG3, PD-1, and PD-L1 (immune checkpoints), HLA-DR (MHC class II), Lek (immune synapse), FOXP3 (T regulatory cells), p-ERKl/2 (MAPK/ERK pathway predictive of response to immune checkpoint blockade), c-caspase 3 (marker of cellular apoptosis). Two secondary antibodies were used , Alexa Fluor Plus 647 goat anti-rabbit (Thermo Scientific, 1/400 dilution) and Alexa Fluor Plus 555 goat anti-mouse (Thermo Scientific, 1/200 dilution) associated with 4’,6-diamidino-2-pheynlindole (DAPI) counterstain (Thermo Scientific) by dynamic incubation for 2min. All antibodies were diluted in multi-staining Buffer (BU06, Lunaphore Technologies). For each cycle, the following exposure times were used: DAPI 80ms, TRITC 2min, Cy5 2min, and primary antibody 4min. Elution step lasted 2min for each cycle and was performed with Elution Buffer (BU07-L, Lunaphore Technologies) at 37°C. Quenching step lasted for 30s and was performed with Quenching Buffer (BU08-L, Lunaphore Technologies). The imaging step was performed with Imaging Buffer (BU09, Lunaphore Technologies). The seqIF™ protocol in COMET™ resulted in a multi-stack OME.tiff file where the imaging outputs from each cycle were stitched and aligned. COMET™ OME.tiff contains DAPI image, intrinsic tissue autofluorescence in TRITC and Cy5 channels, and a single fluorescent layer per marker. Visualization and analysis of the images was done using the Lunaphore Viewer software where virtual colors were assigned for each of the markers for better interpretation. Tissue segmentation, nuclei detection, and cell quantification were then conducted using the guided workflow and Phenoplex feature after the OME.tiff files were imported into the Visiopharm® software.
Ex Vivo Spectral Flow Cytometry of matched TICs and PBMCs
One million donor-matched peripheral blood mononuclear cells (PBMCs) and tumorinfiltrating cells (TICs) were isolated and frozen down in cell recovery media (Gibco, 12648010). Additionally, healthy PBMCs from FCD patients were included as a control. For Golgi inhibition and intracellular staining, cells were treated with lx STIM cocktail (Invitrogen 00-4970-93) in DMEM (Gibco, 11995-040) with 10% heat inactivated FBS (Gibco, 10082-147) at 37C for 2 hours. Following stimulation, cells were washed in FACS buffer (DPBS, Corning, 21-031-CM, and 1% FBS), blocked using Fc blocker (Invitrogen, 14-9161-73), washed an additional time, then stained with Fixable Live/Dead (Invitrogen, L34957) in DBPS (Corning, 21-031-CM) for 10 minutes. Cells were washed two additional times with FACS buffer and stained with a cocktail of surface antibodies diluted to 1:100, or as specified by the manufacturer, for 30 minutes. Cells were then permeabilized and fixed in Foxp3 transcription factor staining buffer (eBioscience, 00-5523-08) overnight, then stained with a cocktail of intracellular antibodies diluted to 1 :20, or as specified by the manufacturer, for 30 minutes. Sample acquisition was performed on a Cytek Aurora (Northern Lights) and analyzed using Cytobank V7.3.0. Initial gating for singlets was performed, and live (Aqua-) GFAP- CD45+ cells (GFAP-, CD45+) were identified. Myeloid cells (CD45+ CDl lb+) and Lymphoid cells (CD45+ CDl lb-) were assessed for indicated activation markers. Populations were then assessed for TIM3 expression (TIM3+) and mean fluorescence intensity (MFI) was analyzed and visualized using GraphPad.
Ex vivo PA Immune Functional Assessments
Single-cell suspensions of newly diagnosed PA containing the pre-existing immune cells in the TME were incubated for 24 hours at 38 °C in a flat bottom tissue culture treated plate at a concentration of 1 million cells per well in DMEM + 10% FBS. Cells were then treated with 300 ug of either isotype control (IgG4 Fc [MedChem Express, HY-P70771]), anti-TIM3 (Sabatolimab [MedChem Express, HY-P99044]), or anti-PD-1 (Nivolumab [MedChem Express, HY-P9903]) for another 48 hours. Flow cytometry was then used to assess the immune functions of the immune cells (Figure 6B) as a ratio of pro -inflammatory TNF-a to immune suppressive p- STAT3 expression.
Population-based Bioinformatics
Analysis from the pediatric brain tumor Griesinger, Gump, Henriquez, and Lambert glioma datasets were obtained from GlioVis (gliovis.bioinfo.cnio.es) with statistical analyses performed using Gliovis. Log2-transformed mRNA expression of selected markers (HAVCR2, STAT3, and PDCD1) were downloaded and visualized using box and whiskers plots. Murine Models of Glioma
Because there is no immune-competent low-grade glioma model that has a BRAF-Fusion available for preclinical testing (46-51), signals of response to anti-TIM3 were evaluated in a genetically engineered murine model (GEMM) of low-grade glioma triggered by PDGF which activates the MAPK pathway (52-57) and CT-2A in the C57BL/6J background that also activates the MAPK pathway. CT-2A cells show MAPK activation as shown by western blot (Figure 7A). The immune-competent high-grade glioma CT-2A was implanted in C57BL/6J mice at the tumorigenic cell number of I xlO5. Mice were then randomly assigned to control and treatment groups: (i) IgG isotype control i.v. 300 pg; (ii) anti-TIM3 i.v. 300 pg (15 mg/kg, within the range of human dosing NCT03489343); and (iii) anti-PD-1 i.p. 100 pg. The genetic low-grade model used was RCAS/Ntv-a which induces low-grade gliomas (58). The vector constructs are propagated in DF-1 chicken fibroblasts. Live viruses are produced by transfecting plasmid versions of RCAS vectors into DF-1 cells using FuGene6 (Roche). DF-1 cells senesce 1-2 days after injection. To transfer the gene via RCAS vectors, 2 x 104 DF-1 producer cells transfected with the RCAS vectors in 1-2 pL of PBS are injected into the frontal lobes of neonatal GEMM mice which carry the Ntv-a transgene using a 26G 10 pL Hamilton syringe. Gliomas were induced in 3 different genetic backgrounds: RCAS/Ntv-a wildtype, Ntv-a/CD8-/_, and Ntv- a/CX3CRl-/_, Ntv-a/CD8-/_ generation is described in our prior study (59). For the RCAS- Ntv- a/CX3CRl-/_, RCAS-CX3CR1 were created by cloning human CX3CR1 (V249) cDNA into a gateway-compatible RCAS vector using LR recombination (Invitrogen) and verified by sequencing. To verify that the appropriate immune cell populations have been eliminated, the CX3CR1 KO and CD8 KO mice were genotyped before breeding. TIM3 expression within the tumors of these models was confirmed by single-cell sequencing and immunofluorescence. Mice were randomized to the following treatment groups: anti-T!M3 antibody (300 pg i.v. once per week for four weeks); anti-PD (200 pg i.p. three times per week for five weeks); or the IgG control (200 pg i.p. three times per week for five weeks) starting at approximately day 28 in the Ntv-A model. In the CX3CR1 and CD8 KO background mice, the IgG control was administered at 300 pg i.v. once per week for four weeks starting at approximately day 28, identical to the dose and schedule for the anti-TIM3. The mice were observed daily for survival, and when they showed signs of neurological deficit (lethargy, hypothermia, failure to ambulate, lack of feeding, body condition score <2.0, or loss of >20% body weight), they were compassionately euthanized.
Statistical Assessments
Two-sided Wilcoxon rank-sum test was used to calculate p-value for all pairwise comparisons. For multiple T tests, Two-stage step-up (Benjamini correction) method was used and False Discover Rate (Q) < 0.01 was used. P-values displayed in box and whisker plots are reported as adjusted p-values and using the following designations: * <0.05, **<0.01 ,***<0.001 . All box and whiskey plots show all individual points with the box showing 25th percentile, median, and 75th percentile and whiskers at minimum and maximum value. GraphPad Prism version 9.2.0 was used to analyze the data.
Results
Immune Therapy Response Biomarker Profiles are Enriched in MAPK-Driven Gliomas
Gliomas from 250 pediatric and young adult (<25 years) patients were analyzed using whole transcriptome sequencing to examine immune microenvironment differences between molecular groups (IDH-WT High Grade (HG), H3F3A, MAPK-Driven, and IDH-MT). IDH-WT HG included both glioblastoma and diffuse pediatric high-grade gliomas. MAPK-Driven included PA with BRAF alterations (BRAFV600E, n=30; BRAF Fusion, n=38; BRAF MT other, n=20), diffuse low-grade gliomas with MAPK alterations (n =1), and rosette-forming glioneuronal tumors (n = 7). Based on QuantiSeq RNA deconvolution analysis, T cells were rare in gliomas regardless of molecular type classification. In contrast, dendritic cells and macrophages were more frequent in the MAPK-Driven tumors (Figure 1). TMB and RSRD scores were low for all glioma groups, but MAPK-Driven tumors showed the highest 1FN expression signatures. The analysis of immune markers in the MAPK-Driven group revealed elevated expressions of CD86 and HAVCR2 (TIM3), but a relatively low-level expression of IDO1 , PDCD1 (PD-1), LAG3, CTLA4, and CD274 (PD-L1).
BRAE -Fusion PA commonly expresses II M3
Because the BRAF-Fusion is expressed in 70% of PA, we prospectively collected patients to comprehensively characterize the unique immune biology of these tumors using nanostring profiling, scRNASeq, and SeqIF™ multiplex staining (Figure 2A). Molecular status was reported through the EHR. Nanostring analysis of tumor Direct Enrichment Scores (DES) revealed increases in leukocyte- and NK-associated genes, and expression of tumor necrosis factor (TNF) superfamily, interleukin (IL), and antigen presentation genes relative to adjacent brain (Figure 7B-C). Next we profiled PA at the single cell level by performing scRNAseq on 13 tumors and 3 adjacent normal brain (ANB) specimens that were resected separately during the approach to a brain lesion. We analyzed the transcriptome of a total of 134,576 cells from a total of 16 samples. After applying the scRNAseq integration pipeline, lymphoid, myeloid, and one CD45- cluster (platelets) were identified (Figure 2B). There was negligible expression of NOS2 (nitric oxide), Argl (Arginase), IDO1, and CD274 (PD-L1) in the BRAF-Fusion PA (Figure 2C). T cell effector functions such as PRF1 (perforin) and GZMB (granzyme B) showed strong expression while PD-1, TIGIT, and LAG3 were expressed to a lesser degree. STING (TMEM173) is expressed in innate and adaptive immune cells. Amongst various immune targets, TIM3 (HAVCR2) was commonly expressed across most immune cell lineages in BRAF-Fusion PA with the highest expression in the myeloid population (Figure 2C).
Phagocytic microglia and antigen presentation are present in BRAF-Fusion PA
Gene ontology (GO) enrichment analysis revealed that of the top 50 GO pathways, 80% were related to immune activation and antigen presentation. Consistent with GSE pathway analysis results, we confirmed that CDllc+ cells were interacting with either CD4 or CD8 T cells with LCK expression between the two cell populations (Figure 8A-B) which would be reflective of antigen presentation within the TME.
Tumor-associated myeloid cells, which include peripherally originating cells and brainresident microglia (MG) are a dominant immune population in the glioma TME (Figure 2D). The MG are identified using three canonical markers (TMEM119, CX3CR1, and P2RY 12). Using markers identified by other groups (41-45), the MG cells were further categorized into distinct subtypes: inflammatory groups 1 and 2 (CCL4L2, TMEM107, and TNF expression); phagocytic (C1QA, TMEM176B, and VSIG4 expression); HSP expressing (HSPA1A, HSPA1B, and HSPB1); perivascular (LYVE1, FOLR2, and MRC1 expression) and homeostatic (P2RY12, CSF1R). Single-cell analysis also indicated the presence of innate immune cells including four tumor-associated macrophage (TAM) populations: lysosomal (CSTB, LYZ, and LIPA), inflammatory (IFI6, IFIT1, ISG15, CCL4), APC-like (CTSD, MS44A4A, MS4A6A), and RNA Splicing (MALAT1, SLC1A3, SLC38A2); type 2 conventional dendritic cells (CLEC10A, FCER1A), plasmacytoid dendritic cells (CLEC4C, IL3RA); monocytes (VACN, FCN1, S100A8), and neutrophils (LY6G6D, JMJD1C, CD117) (Figure 2D). To begin to elucidate the immune functional roles in the innate immune cells within the BRAF-Fusion PA TME, the expression of key genes involved in tumor cytotoxicity, immune suppression, antigen presentation functions (APC), and phagocytosis were assessed. The dominant immune suppressive mechanisms were TGFp i, STAT3, LAIR1 , and TIM3 (Figure 2F).
Unique CD4+P2RY12+TIM3+ cluster identified in BRAF-Fusion PA
When the T/NK cell population was analyzed, the following clusters were identified: CD4+, three CD8+, double negative memory-like (CD4-CD8-.Tm), Tregs, three NK, y8-T cells, and a unique CD3E+CD4A+P2RY 12+ cluster defined as MG- Act (Figure 2E). The CD4+ T cells expressed central memory (cm) markers IL7R, CCR7, TCF7, and CD40LG. CD8+ cells were subclassified as IL7R+, CD69+, TCF7+ and two early activated GZMA/K/HhighGZMBlow clusters were identified (CD8.Tearly.act.l and CD.Tearly.act.2). NK cells were subclassified as XCL1/2+, GZMBhlghPRFl+TIM3+CXC3CRl+, and memory-like due to expression of IL7R, CD69 and TCF7. Most T cell effector populations did not express markers of immune exhaustion such as PD-1, TIGIT, and LAG3 on the scRNAseq data (Figure 2G). T cells in the TME of PA could not be identified expressing these markers with the SeqIFTm staining. The MG- Act population is notable for having genes related to immune cytotoxic functions (Figure 2F-G). The top 40 up-regulated genes for the MG- Act cluster included C1QC, APOE, C1QB, C1QA, CST3, APOCI, HLA-DRA, C3, AIF1, CD74, MARCKS, FTL, TREM2, SPP1, APOC2, CD68, TYROBP, HLA-DRB5, HLA-DPA1, SP11, NPC2, CTSB, TMEM176B, SERP1NA1, HLA- DRB1, FCER1G, IFI30, GSN, MS4A64, CSF1R, GPR34, LY86, CD14, VSIG4, HLA-DPB1, TUBA1B, and SCIN indicating antigen presentation capability. These MG-Act cells are enriched in the BRAF-Fusion PA compared to ANB and HGG (scRNA data obtained from GEO GSE249263) (Figure 3A).
TIM3-expressing immune cells are spatially localized to distinct niches of the TME To explore the spatial distribution of TIM3-expressing cells within the BRAF-Fusion PA TME, SeqIFTm was performed. TIM3 expression is dispersed throughout the TME with minimal expression found in ANB (Figure 3B-C). CD1 lc+ and P2RY 12+ cells are the predominant TIM3+ expressing population with minimal TIM3 expression on GFAP+ tumor cells or CD3+ lymphoid cells (Figure 3D). Notably, TIM3 expression is found on the myeloid cells lining the vessels in the glioma (Figure 3E), but not in ANB (Figure 9). The scRNA seq data implicating cytotoxic functions of MG- Act were validated at the protein expression level including for P2RY12, CD3, CD4, CD8, NKG7, and TIM3 (Figure 3F-G). The TIM3 expression on the P2RY 12+ MG- Act population is dispersed throughout the TME PA but not in the ANB.
TIM3 expression in PA can be therapeutically manipulated
Four publicly available datasets were queried to evaluate TIM3 and STAT3 expression in PA tumors compared to NB samples (6, 38-40). Both TIM3 and STAT3 expression is significantly increased in PA tumors. Additionally, the Henriquez et. al. dataset contains both fetal and adult NB and analysis demonstrated that fetal NB had lower TIM3 and STAT3 expression compared to adult NB. PDCD1 (PD-1) had lower expression in PA compared to ANB (Figure 4A, Figure 10). Ex vivo flow cytometry showed increased TIM3 expression on tumorinfiltrating immune cells (TICs) in both myeloid and lymphoid compartments compared to matched PBMCs and healthy control PBMCs (Figure 4B). To evaluate if anti-PD-1 or anti-TIM3 could reprogram the immune cells isolated from patient PA, three ex vivo tumors were placed into single-cell suspension and treated with antibodies. 48-hour treatment leads to an increased ratio of TNF-a:p-STAT3 in lymphocytes with both anti-TIM3 and anti-PDl treatment. However, only anti-TIM3 increased the TNF-a:p-STAT3 ratio in the myeloid cells (Figure 4C), indicating that anti-TlM3 can drive proinflammatory responses in both the adaptive and innate arms of the immune system.
Anti-TJM3 exerts a therapeutic effect in preclinical models of glioma
There is no immune-competent low-grade glioma model that has a BRAF-Fusion for preclinical testing (46-51). C57BL/6J mice bearing intracranial CT-2A high-grade gliomas were treated with anti-TIM3 or control once per week or anti-PD- 1 three times per week for 3 weeks starting on day 7 after tumor implantation. Additionally, we used a low-grade GEMM triggered by PDGF which activates the MAPK pathway (52-57). Ntva+/BL6 mice injected with RCAS- PDGFB were observed for 28 days and then randomized into treatment with anti-TIM3, anti-PD- 1, or IgG isotype control intravenously once per week for up to 4 weeks (Figure 5A). In the CT- 2A model which shows TIM3 expression in the myeloid compartment by scRNA, the IgG control mice had a median survival time of 39 days, PD-l-treated mice had a median survival of 47 days, and TIM3-treated mice had a median survival time of 47 days (log-rank test, P = 0.14; Figure 11A-C). To ascertain if these mono-therapeutic immunotherapies would have an impact on low-grade gliomas, the RCAS-PDGFB GEMM glioma model was histologically characterized by a board-certified neuropathologist. The RCAS-PDGFB GEMM model displayed the classic features of a low-grade glioma including a loose microcystic pattern (Figure 5B), heterogeneity in cellular density (Figure 5C), the absence of mitosis (Figure 5D), and perineuronal satellitosis at the infiltrating edges of the tumors (Figure 5E). This model also shows TIM3 expression on P2RY12+ microglia and activation of the MAPK pathway as assessed based on p-ERKl/2 expression (Figure 5F-G). In this low-grade GEMM glioma model, the median survival time in the IgG control group (n =20) was 110.5 days, the anti-PDl (n =20) median survival was 134.5 days (log-rank test, P = 0.64 vs IgG) and the anti-TIM3 (n =21) treated group was 253 days (log-rank test, P = 0.01 vs IgG; Figure 5H).
Anti-TIM3 mediates its therapeutic effect through the immune system
Because the GEMM models have a survival time of over 90 days, in vivo depletions are not a valid strategy. As such, we exploited CX3CR1 KO mice to identify the immune effector population (65). The anti-TIM3 antibody was administered using the same schedule as wild-type mice (Figure 5A). In contrast to the wild-type mice, the therapeutic effect of anti-TIM3 was lost in the CX3CR1 KO since this background eliminates cytotoxic effector functions (e.g., MG-Act) and is essential for the activation of adaptive immunity (e.g., CD8+ cytotoxic T cells) (log-rank test, P = 0.33) (Figure 51). The CD8 KO background similarly eliminated the therapeutic effect of anti-TIM3 treatment (Figure 1 ID).
Anti-TIM3 enhances the MG-ACT population in vivo
During the therapeutic window, wild-type mice were terminated for immune assessments of the TME using scRNAseq after either 2 or 4 treatments with anti-TIM3 or IgG. Upon termination, the IgG-treated mice had large gliomas while the anti-TIM3 -treated group either had small gliomas or the tumors were absent (Figure 5 J) . The MG- Act population was identified in the wild- type GEMM MAPK-driven low-grade glioma preclinical model (Figure 5K-L) which became more abundant with anti-TIM3 treatment alongside other effector populations including CD8+ cytotoxic T cells (Figure 5M). Additional dosing of anti-TIM3 further increased the MG- Act population and T/NK cell populations (NK Act 1, CD8+ cytotoxic, CD4+ central memory) (Figure 5M).
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Claims

1. A method of treating glioma in a subject comprising administering to the subject an inhibitor of TIM-3.
2. The method of claim 1, wherein the glioma is a MAPK-driven glioma.
3. The method of claim 2, wherein the glioma is an astrocytoma.
4. The method of claim 3, wherein the astrocytoma is a pilocytic astrocytomas (PA).
5. The method of claim 1, wherein the subject exhibits one or more glioma biomarkers.
6. The method of claim 5, wherein the subject has tested positive for a BRAF fusion, increased IFN, and/or increased p-ERK.
7. The method of claim 6, wherein the BRAF fusion is a KIAA1549-BRAF fusion.
8. The method of claim 1, wherein the TIM-3 inhibitor is an inhibitor of TIM-3 activity.
9. The method of claim 8, wherein the TIM-3 inhibitor is an anti-TIM-3 antibody.
10. The method of claim 9 wherein the TIM-3 inhibitor is selected from RMT3-23, ATIK2a,
MBG453 (sabatolimab), AZD-7789, BMS-986258, 1NCAGN-2390, TSR-022 (Cobolimab), LY3321367, RO-7121661 (Lomvastomig), TQB-2618.
11 . The method of claim 10, wherein the TIM-3 inhibitor is Sabatolimab (MBG453).
12. The method of claim 8, wherein the TIM-3 inhibitor is an antibody that binds to a ligand of TIM-3.
13. The method of claim 12, wherein the TIM-3 inhibitor binds to galectin-9, HMGB1, ceacam-1, or phosphatidyl serine.
14. The method of claim 1, wherein the TIM-3 inhibitor is a small molecule or peptide capable of binding to TIM-3 and/or a TIM-3 ligand.
15. The method of claim 14, wherein the small molecule TIM-3 inhibitor is SMI402.
16. The method of claim 1, wherein the TIM-3 inhibitor is an inhibitor of TIM-3 expression.
17. The method of claim 16, wherein the TIM-3 inhibitor is a nucleic acid-based agent that reduces expression of TIM-3.
18. The method of claim 1, wherein the TIM-3 inhibitor comprises one or more agents that allow for alteration of the TIM-3 gene and thereby reduce TIM-3 expression or activity.
19. The method of claim 1, wherein the TIM-3 inhibitor is co-administered with one or more additional glioma therapies.
20. The method of claim 19, wherein the TIM-3 inhibitor is co-administered with surgical removal of all or a portion of the glioma.
21. The method of claim 19, wherein the TIM-3 inhibitor is co-administered with radiation therapy, chemotherapy, and/or an immunotherapy.
22. The method of claim 1, further comprises detecting one or more glioma biomarkers in the subject.
23. The method of claim 22, wherein the glioma biomarkers arc selected from a BRAF fusion, increased IFN, and/or increased p-ERK.
24. The method of claim 23, wherein the BRAF fusion is a KIAA1549-BRAF fusion.
25. Use of a TIM-3 inhibitor in the treatment of glioma in a subject.
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Citations (5)

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