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WO2019202473A1 - Agents induisant ucp2 pour le traitement du cancer résistant au blocage des points de contrôle immunitaires - Google Patents

Agents induisant ucp2 pour le traitement du cancer résistant au blocage des points de contrôle immunitaires Download PDF

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WO2019202473A1
WO2019202473A1 PCT/IB2019/053090 IB2019053090W WO2019202473A1 WO 2019202473 A1 WO2019202473 A1 WO 2019202473A1 IB 2019053090 W IB2019053090 W IB 2019053090W WO 2019202473 A1 WO2019202473 A1 WO 2019202473A1
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ucp2
subject
tumor
immune checkpoint
cancer
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Ping-Chih HO
Wan-chen CHENG
Giovanni CIRIELLO
Yao-Chen TSUI
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Universite de Lausanne
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Universite de Lausanne
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/422—Oxazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/428—Thiazoles condensed with carbocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G01N33/57595—
    • 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

  • the present invention relates generally to the field of molecular biology and medicine. More particularly, the methods and compositions herein are useful for treating a subject resistant to immune checkpoint blockade treatment.
  • immunotherapy include antigen- specific vaccines, adoptive cell transfer of anti-tumor T cells, and chimeric antigen receptor cells (CARs).
  • Alternative immunotherapy approaches focus on modulating the activity of immune checkpoint proteins, which interact with specific ligands that induce signaling in the T cell and switch off or inhibit T cell function.
  • immune checkpoint proteins By expressing high levels of immune checkpoint proteins on their surface, cancer cells can control the function of T cells that enter the tumor microenvironment, thus suppressing the anticancer immune response.
  • therapies based on immune checkpoint blockade have proven to be a successful approach in a number of cancers, response rates remain relatively low, ranging from 15 to 40% depending on cancer type (EBioMedicine. 2018 Jul; 33: 18-19). Further, the development of secondary resistance with disease progression is common. As such, the majority of patients receiving immunotherapy does not benefit from the immunotherapeutic treatment or relapses after a period.
  • immunotherapy resistance occurs in immunologic ally“cold” tumors with little or no T cell infiltration of the tumor microenvironment, while“hot” tumors are enriched in immune cells within the tumor microenvironment (TME) and respond to the immunotherapy.
  • the disclosure provides a method of reducing resistance to immune checkpoint blockade therapy in a subject in need thereof, the method comprising
  • the disclosure provides a method for enhancing the potency of immune checkpoint blockade therapy administered in a subject in need thereof, the method comprising administering to the subject an effective amount of an uncoupling protein 2 (UCP2) inducing agent.
  • the disclosed methods further comprise administering an immune checkpoint modulator.
  • the disclosure provides a method of enhancing the anti-tumor response in a subject in need thereof, wherein the subject is resistant to immune checkpoint blockade therapy, the method comprising administering to the subject an effective amount of (i) a UCP2 inducing agent and (ii) an immune checkpoint modulator.
  • the disclosure provides a method of reducing tumor growth in a subject in need thereof, wherein the subject is resistant to immune checkpoint blockade therapy, the method comprising administering to the subject an effective amount of (i) a UCP2 inducing agent and (ii) an immune checkpoint modulator.
  • the disclosure provides a method of normalizing the tumor vasculature in a subject in need thereof, wherein the subject is resistant to immune checkpoint blockade therapy, the method comprising administering to the subject an effective amount of (i) a UCP2 inducing agent and (ii) an immune checkpoint modulator.
  • the disclosure provides a method of increasing lymphocyte infiltration of a tumor in a subject in need thereof, wherein the subject is resistant to immune checkpoint blockade therapy, the method comprising administering to the subject an effective amount of (i) a UCP2 inducing agent and (ii) an immune checkpoint modulator.
  • the disclosure provides a method of increasing T cell or NK cell infiltration.
  • the UCP2 inducing agent is selected from the group consisting of: metformin, a PPARy agonist, a thiazolinedione, DPP4 inhibitor, Fenofibrate, Stanniocalcin (STC1), and Irisin.
  • the thiazolinedione is selected from the group consisting of Lanifibranor, KRP-297, Efatutazone, Pioglitazone, Rosiglitazone, Troglitazone, Rivoglitazone, Reglitazar, and Netoglitazone.
  • the thiazolinedione is Rosiglitazone or KRP-297.
  • the UCP2 inducing agent is a nucleic acid molecule comprising a nucleic acid sequence encoding for UCP2.
  • the immune checkpoint modulator administered to the subject is an anti PD-l, anti PD-L1, anti PD-L2, anti CTLA-4, anti KIR, anti TIM-3, anti LAG-3, anti B7-H3, anti B7-H4, anti BTLA, or anti B7-H6 agent.
  • the immune checkpoint inhibitor is an anti PD-l, anti PD-L1, anti PD-L2, anti CTLA-4, anti KIR, anti TIM-3, anti LAG-3, anti B7-H3, anti B7-H4, anti BTLA, or anti B7-H6 antibody or fragment thereof.
  • the immune checkpoint modulator is an immune checkpoint inhibitor.
  • the disclosure provides methods for the administration of a UCP2 inducing agent to a subject, wherein the subject is resistant to immune checkpoint blockade therapy with an anti PD-l, anti PD-L1, anti PD-L2, anti CTLA-4, anti KIR, anti TIM-3, anti LAG-3, anti B7-H3, anti B7-H4, anti BTLA or anti B7-H6 agent.
  • the subject has a cancer with primary resistance to immune checkpoint blockade therapy.
  • the subject has a cancer select from the group consisting of melanoma, non-small cell lung cancer, uveal melanoma, breast cancer, ovarian cancer, head and neck cancer, prostate cancer, and pancreatic cancer. In one embodiment, the subject has melanoma.
  • the disclosure provides a method of treating a cancer resistant to immune checkpoint blockade therapy in a subject in need thereof, the method comprising administering to the subject an effective amount of (i) a UCP2 inducing agent and (ii) an immune checkpoint modulator.
  • the disclosure provides a method of selecting a subject for treatment with an immune checkpoint modulator, the method comprising:
  • the method further comprises administering to the subject an immune checkpoint modulator.
  • the disclosure provides a method of treating a cancer resistant to immune checkpoint blockade therapy in a subject in need thereof, the method comprising:
  • Figure 1 illustrates that UCP2 expression is associated with elevated T cell infiltration and prolonged survival rates.
  • Figure 1A Pearson correlation of UCP2 expression with CD8A, IFNG, GZMB, TNF, LCK and SYK.
  • FIG. 2 illustrates that UCP2 induction in melanoma cells promotes anti-tumor responses.
  • Figure 3 illustrates that UCP2 induction promotes CD8 + T cell infiltration of the tumor and normalization of the tumor vasculature.
  • Figure 3B Relative single-tumor vessel area (ANOVA P ⁇ 0.0001).
  • Figure 4 illustrates that genetic induction of UCP2 can overcome resistance to checkpoint blockade therapy.
  • Tumor growth Figure 4A
  • Kaplan-Meier survival curves Figure 4B
  • a PD-l, anti-PD-l monoclonal antibody mAh
  • Dox doxy cy cline
  • DOX + a PD-l, doxycycline plus anti-PD-l mAh (n 11).
  • Differences in survival times were analyzed by long-rank (Mantel-Cox) test ( Figure 4B).
  • Figure 5 illustrates that PPAR agonists can induce UCP2 expression.
  • Figure 5A Immunoblot analysis of indicated proteins in B16-OVA cells treated with either control vehicle or escalating doses of rosiglitazone.
  • Figure 5B Immunoblot analysis of Me290 melanoma cells treated with the indicated PPAR agonists. Cells were treated with 10 pg/ml of each PPAR agonist for 24 hr and collected for immunoblot analysis. Data represents a representative image of three independent experiments.
  • Ctrl Control. Rosiglitazone (PPAR- g agonist). Fanifibranor (PPAR-a/b/g agonist). KRP-297 (PPAR-a/g agonist). Reglitazar (PPAR-a/g agonist). Efatutazone (PPAR-g agonist). Pioglitazone (PPAR-g agonist).
  • Troglitazone (PPAR-g agonist).
  • Figure 6 illustrates that induction of UCP2 with a small molecule can overcome resistance to immune checkpoint blockade therapy.
  • Tumor growth (Figure 6A) and Kaplan- Meier survival curves (Figure 6B) of B16-OVA melanoma-bearing mice treated with indicated treatments.
  • Figure 6C Tumor growth of Batl3 ' mice engrafted with B16-OVA melanoma receiving indicated treatments.
  • the disclosure relates to methods for reducing resistance to immune checkpoint blockade and/or for enhancing the anti-tumor response in patients with resistance to immunotherapy, the methods comprising administering a UCP2 inducing agent to a subject in need thereof.
  • the methods further comprise
  • an immunomodulatory agent including, but not limited to, an immune checkpoint modulator.
  • an immune checkpoint modulator including, but not limited to, an immune checkpoint modulator.
  • methods of treating a cancer resistant to immune checkpoint blockade therapy are also disclosed.
  • Uncoupling proteins are a family of inner mitochondrial membrane proteins that allow the re-entry of protons to the mitochondrial matrix, by dissipating the proton gradient and, subsequently, decreasing membrane potential and production of reactive oxygen species (ROS). While UCP2 is a key regulator of cellular metabolism, its role in tumorigenesis is not well understood: UCP2 has been implicated in both carcinogenesis and cancer chemo-resistance, but anti-tumor activity of UCP2 has been observed as well.
  • ROS reactive oxygen species
  • the disclosure relates to the novel finding that induction of UCP2 can reduce resistance to immunotherapy, including, but not limited to resistance to immune checkpoint blockade.
  • immunotherapy resistance or“immunotherapy resistant” refer to a clinical scenario in which a cancer a) does not respond to an immunotherapy (primary resistance); b) is recognized by the immune system but it protects itself by adapting to the immune attack (adaptive immune resistance); or c) initially responds to immunotherapy but after a period of time relapses and progresses (acquired resistance).
  • Adaptive immune resistance may manifest clinically as primary resistance, a mixed response, or acquired resistance.
  • the disclosure provides a method of reducing primary resistance to immunotherapy in a subject in need thereof. In one embodiment, the disclosure provides a method of reducing acquired resistance to immunotherapy in a subject in need thereof. In some embodiments, the subject is resistant to treatment with one or more immune checkpoint modulators. Resistant cancer may also be referred to as refractory cancer.
  • A“UCP2 inducing agent” as used herein is an agent that increases the expression, production, available amount, solubility, stability and/or activity of UCP2 in vitro and/or in vivo.
  • An "agent” as used herein may refer, for example, to a small molecule, chemical compound, protein, peptide, antibody, nucleic acid molecule, or a fatty acid.
  • the UPC2 inducing agent may increase the expression or biological activity of a protein upstream of UCP2 in a UCP2 pathway.
  • the UCP2 inducing agent is a peroxisome proliferator- activated receptor (PPAR) agonist.
  • PPAR peroxisome proliferator- activated receptor
  • the term“PPAR agonist” refers to a molecule that can induce and/or upregulate the transcriptional activity of the PPAR.
  • a PPAR agonist upregulates PPAR gene expression.
  • a PPAR agonist may regulate at least one of PPAR-a, PPAR-b, or PPAR-g.
  • Exemplary PPAR agonists include, without limitation, fatty acids, hormones, biguanides, glitazars, thiazolidinediones, and fibrates.
  • the UCP2 inducing agent is a peroxisome proliferator- activated receptor g (PPAR-g) agonist.
  • PPAR-g agonist is a synthetic PPAR-g agonist, a dual PPAR-a/g agonists, or a pan PPAR-a/bd/g agonist.
  • Non limiting example of PPAR-g agonists include thiazolidinediones, glitazars, and noncanonical PPAR-g agonists.
  • Thiazolidinediones can include, without limitation, pioglitazone, rosiglitazone, lobeglitazone, ciglitazone, darglitazone, netoglitazone, englitazone,
  • glitazars include saroglitazar, aleglitazar, farglitazar, muraglitazar, tesaglitazar, sodelglitazar, ragaglitazar, naveglitazar, imiglitazar, netoglitazar, chiglitazar, indeglitazar, sipoglitaza.
  • Noncanonical PPAR-g agonists or partial agonists can include, without limitation, MRL-24, telmisartan, SR1664, SR1824, mesalamine, olsalazine, amorfrutins, amorfrutin A, and amorfrutin B.
  • PPAR-g agonists include CLX-0921, R-483, NIP-221, NIP-223, DRF-2189, 2-Cyano-3,l2- dioxooleana-l,9-dien-28-oic acid, (2S)-((2-Benzoylphenyl)amino-3[4-[2-(methylpyridin-2- ylamino)ethoxy]phenyl)-propionic acid (GW1929), indomethacin, fenprofen, ibuprofen, and flufenamic acid.
  • the UCP2 inducing agent is a fibrate, including, but not limited to Aluminium clofibrate, Bezafibrate, Ciprofibrate, Choline fenofibrate, Clinofibrate, Clofibrate, Clofibride, Fenofibrate, Gemfibrozil, Ronifibrate, and Simfibrate.
  • the UCP2 inducing agent is a fatty acid, including, but not limited to, Palmitic acid, Erucic acid, Oleic acid, Petroselinic acid, Linoleic acid, a-Linolenic acid, g-Linoleic acid, Why acid, Arachidonic acid, Docosahexaenoic acid, Eicosapentaenoic acid, and Palmitoleic acid.
  • the UCP2 inducing agent is an Eicosanoid, including, but not limited to, 8-(R) Hydroxyeicosatetraenoic acid, 8-(S) Hydroxyeicosatetraenoic acid, 15- Hydroxyeicosatetraenoic acid, 9-(R/S) Hydroxyoctadecadienoic acid;, l3-(R/S)
  • the UCP2 inducing agent is a dipeptidyl peptidase-4 (DPP4) inhibitor, including, but not limited to, Sitagliptin, Vildagliptin, Saxagliptin, Linagliptin, Gemigliptin, Anagliptin, Teneligliptin, Alogliptin, Trelagliptin, Omarigliptin (MK-3102), Evogliptin, Gosogliptin, Dutogliptin, and Berberine.
  • DPP4 dipeptidyl peptidase-4
  • the UCP2 inducing agent is an inhibitor of arachidonate 5- lipoxygenase, including, but not limited to meclofenamate sodium and zileuton.
  • the UCP2 inducing agent is an inhibitor of a
  • the UCP2 inducing agent is an AMPK activator.
  • the UCP2 inducing agent is a biguanide.
  • the UCP2 inducing agent is an agent that inhibits UCP2 degradation, including, but not limited to a protease inhibitor.
  • the UCP2 inducing agent is a protein or a peptide.
  • the UCP2 inducing agent maybe be a proteic hormone that increases UCP2 expression.
  • the UCP2 inducing agent is an antibody or fragment thereof.
  • proteic UCP2 inducing agents include, but are not limited to, a naturally occurring, a synthetic, or a modified incretin (non-limiting examples include glucagon-like peptide- 1 (GLP-l), gastric inhibitory peptide (GIP)), irisin, fibroblast growth factor 21, fibronectin type III domain-containing protein 5f, stanniocalcin, and adiponectin.
  • the UCP2 inducing agent is a nucleic acid, including, but not limited to DNA, RNA, a naturally occurring or a synthetic nucleic acid, in vitro transcribed
  • the nucleic acid comprises a sequence encoding UCP2.
  • the UCP2 gene sequence may be codon optimized.
  • plasmids or vectors comprising the UCP2 inducing nucleic acid.
  • the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • A“vector” includes, but is not limited to, a viral vector, a plasmid, a RNA vector or a linear or circular DNA or RNA molecule, which may comprise a chromosomal, non-chromosomal, semi- synthetic or synthetic nucleic acid.
  • the employed vectors are those capable of autonomous replication (episomal vector) and/or expression of nucleic acids to which they are linked (expression vectors).
  • Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno associated viruses, AAV), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.
  • rabies and vesicular stomatitis virus paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses such as picomavirus and alphavirus, and double- stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox).
  • Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.
  • retroviruses examples include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, and spumavirus.
  • the disclosure also provides for methods of enhancing UCP2 expression by expressing a recombinant UCP2 gene, introducing multiple copies of a UCP2 gene into a cell, placing a UCP2 gene under control of a more potent promoter instead of the original promoter, increasing the stability of mature UCP2 RNA transcripts, enhancing translation of UCP2 messenger RNA derived from a UCP2 gene, enabling or enhancing the transport of mature UCP2 transcripts out of the nucleus to the cytoplasm, and/or facilitating the assembly of complete ribosome with large and small subunits at the AUG initiation codon, thereby allowing efficient translation.
  • the UCP2 inducing agent is a transcription factor that increases UCP2 expression.
  • a CRISPR/Cas9 system is used to increase expression of UCP2. Additional methods of increasing the expression of a target gene contemplated by the disclosure are known in the art.
  • UCP2 expression, production, available amount, solubility, stability and/or activity of genes and proteins are well known in the art. Such methods can include measuring the level of transcription of the gene into a messenger RNA (mRNA) molecule; and/or measuring the level of translation of the mRNA into protein (i.e. measuring the level of protein per se).
  • mRNA messenger RNA
  • UCP2 expression, production, available amount, solubility, stability and/or activity is determined by measuring the amount of UCP2 messenger RNA.
  • mRNA levels can be measured by Northern blotting, RNA in situ hybridization, reverse-transcriptase PCR (RT- PCR), real-time (quantitative) RT-PCR, microarrays, single-cell sequencing, or“tag based” technologies such as SAGE (serial analysis of gene expression).
  • RT- PCR reverse-transcriptase PCR
  • microarrays single-cell sequencing, or“tag based” technologies
  • SAGE serial analysis of gene expression.
  • Primers or probes may be designed based on nucleotide sequences of the genes or transcripts thereof.
  • UCP2 expression, production, available amount, solubility, stability and/or activity is determined by measuring the amount of UCP2 protein levels.
  • protein levels can be determined by affinity chromatography, column chromatography, displacement chromatography, electrochromatography, gas chromatography, high-performance liquid chromatography, capillary chromatography, ion chromatrography, micellar electrokinetic chromatography, normal-phase chromatography, paper chromatography, reversed-phase chromatography, size-exclusion chromatography, thin-layer chromatography, two- dimensional chromatography, gel electrophoresis, direct immunofluorescence, indirect immunofluorescence, immunoblotting, western blotting, mass spectrometry, enzyme-linked immunosorbent assay (ELISA), FACS analysis, protein immunoprecipitation,
  • immunoelectrophoresis isoelectric focusing, surface plasmon resonance, multi-parametric surface plasmon resonance, nuclear magnetic resonance spectroscopy of proteins, peptide mass fingerprinting, protein fingerprinting, SDS-PAGE, SDS-AGE, secretion assays, stable isotope standards and capture by anti-peptide antibodies
  • the person skilled in the art may employ other methods to measure the expression, production, available amount, solubility, stability, and/or activity of UCP2, such as methods disclosed in standard molecular biology text books such as Molecular Cloning A Laboratory Manual, Green M.R. & Sambrook J. (2012) CSH Press.
  • the disclosure provides methods for blocking immunosuppressive features of the TME and/or for sensitizing cancer cells to treatment with an immunomodulatory agent in a subject, wherein the subject is resistant to immunotherapy, the method comprising administering to the subject an effective amount of a UCP2 inducing agent.
  • the disclosure provides methods for increasing infiltration of a tumor by immune cells (including, but not limited to, lymphocytes such as T cells or NK cells) in a subject, wherein the subject is resistant to immunotherapy, the method comprising administering to the subject an effective amount of a UCP2 inducing agent.
  • the disclosure provides methods for improving cytokine expression and/or shifting the cytokine milieu in the TME of a subject, wherein the subject is resistant to immunotherapy, the method comprising administering to the subject an effective amount of a UCP2 inducing agent.
  • the disclosure provides methods for increasing production of cytokines involved in migration of dendritic cells and T cells, including, but not limited to, CCL4, CCL5, CXCR3, CXLC9, and CXCL10.
  • the disclosure provides methods for increasing the activity of immune cells (including, but not limited, to T cell and NK cells) in a subject, wherein the subject is resistant to
  • the method comprising administering to the subject an effective amount of a UCP2 inducing agent.
  • the disclosure provides methods for
  • the disclosure provides methods for increasing the CD8:Treg ratio in a subject, wherein the subject is resistant to immunotherapy, the method comprising administering to the subject an effective amount of a UCP2 inducing agent.
  • the methods further comprise administering an immunomodulatory agent to the subject, including, but not limited to, an immune checkpoint modulator.
  • the disclosure provides methods of treating cancer resistant to immunotherapy, the method comprising administering to the subject an effective amount of
  • the disclosure provides methods of reducing tumor growth and/or reducing metastasis in a subject resistant to immunotherapy, the method comprising administering to the subject an effective amount of (i) a UCP2 inducing agent and
  • an immunomodulatory agent including, but not limited to, an immune checkpoint modulator.
  • an immunomodulatory agent including, but not limited to, an immune checkpoint modulator.
  • the method further comprises administering to the subject an immune checkpoint modulator.
  • the disclosure provides a method of treating a cancer resistant to immune checkpoint blockade therapy in a subject in need thereof, the method comprising:
  • A“control level” of UCP2 in a“control sample” can refer to the level of UCP2 gene or protein expression in one or more individuals that are not resistant to immune checkpoint blockade therapy. The level may be measured on an individual-by-individual basis, or on an aggregate basis such as an average. In some embodiments, the control level of UCP2 expression from the same individual whose condition is being monitored, but is obtained at a different time. In certain embodiments, a“control” level can refer to a level obtained from the same patient at an earlier time, e.g., weeks, months, or years earlier. In some embodiment, the control level is obtained from a patient before the patient received any cancer therapy. In some embodiment, the control level is obtained from a patient before the patient received treatment with a checkpoint inhibitor.
  • immunomodulatory agent refers to agents (compounds or biologicals) that are effective in enhancing or potentiating the immune system response in a mammal.
  • Immunomodulatory agents can be, for example, a nucleic acid (e.g., DNA) or a polypeptide (e.g., protein, glycoprotein, peptide, and the like). Examples of a nucleic acid (e.g., DNA) or a polypeptide (e.g., protein, glycoprotein, peptide, and the like). Examples of a nucleic acid (e.g., DNA) or a polypeptide (e.g., protein, glycoprotein, peptide, and the like). Examples of a nucleic acid (e.g., DNA) or a polypeptide (e.g., protein, glycoprotein, peptide, and the like). Examples of a nucleic acid (e.g., DNA) or a polypeptide (e.g., protein, glycoprotein, peptide, and the like).
  • immunomodulatory agents include, but are not limited to monoclonal antibodies, cytokines, vaccines, immune cells, and chimeric antigen receptor cells.
  • An“immunomodulatory agent” may be an agonist or an antagonist.
  • an“immune checkpoint modulator” or “checkpoint modulator” refers to an agent that alters the activity of an immune checkpoint protein (e.g., any of the immune checkpoint proteins described herein) in a cell relative to a control vehicle.
  • the term“modulator” is used herein in the broadest sense, and includes any molecule that partially or fully alters a signaling pathway regulated by one or more immune checkpoint molecules, including the signaling pathways mediated by the molecules described herein.
  • an immune checkpoint modulator is an inhibitor of an immune checkpoint molecule that reduces, slows, halts, and/or prevents the activity mediated by that immune checkpoint molecule.
  • the term“immune checkpoint inhibitor” is used herein in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a signaling pathway regulated by one or more immune checkpoint molecules, including the modulatory pathways mediated by the molecules described herein.
  • An immune checkpoint inhibitor as described herein can be an inhibitory agent that interferes with the signaling in an immune cell that is mediated by an immune checkpoint protein, for example, either by decreasing transcription or translation of immune checkpoint protein-encoding nucleic acid, or by inhibiting or blocking the immune checkpoint protein activity, or both.
  • immune checkpoint inhibitors include, but are not limited to, antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, checkpoint protein- specific aptamers, anti-checkpoint protein antibodies (e.g., full-length antibodies or antigen-binding fragments thereof), checkpoint-binding small molecules, checkpoint-binding peptides, and other polypeptides that specifically bind a checkpoint protein (including, but not limited to, checkpoint-binding fragments of its cognate ligand, which may optionally be fused to one or more additional domains), such that the interaction between the checkpoint inhibitor and the checkpoint protein results in a reduction or cessation of the checkpoint protein activity or expression.
  • antisense polynucleotides include, but are not limited to, antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, checkpoint protein- specific aptamers, anti-checkpoint
  • a checkpoint protein inhibitor can antagonize or neutralize one checkpoint protein activity without affecting another checkpoint protein activity.
  • a desirable immune checkpoint inhibitor can disrupt binding interaction between the checkpoint protein and one cognate ligand without affecting or minimally affecting the interaction between the checkpoint protein and another cognate ligand, if any.
  • Suitable inhibitory molecules specifically include antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, antisense oligonucleotides, small organic molecules, recombinant proteins or peptides, etc.
  • the immune checkpoint inhibitor is an antibody that specifically binds to an immune checkpoint molecule and inhibits its bioactivity, e.g., via interfering its binding to the cognate ligand.
  • the term“antibody” as includes but is not limited to polyclonal, monoclonal, humanized, chimeric, Fab fragments, Fv fragments, F(ab') fragments and F(ab')2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody.
  • scFv single chain antibodies
  • an immune checkpoint modulator is an activator of a checkpoint molecule that enhances and improves the activity mediated by that checkpoint molecule.
  • the term“immune checkpoint activator” is used herein in the broadest sense, and includes any molecule that enhances a signaling pathway regulated by one or more immune checkpoint molecules, including the signaling pathways mediated by the molecules described herein. Suitable activators include agonistic antibodies or antibody fragments, small organic molecules, recombinant proteins or peptides, etc.
  • immunomodulatory agent administered to the subject is an inhibitor of an immune checkpoint protein, including, but not limited to, Programmed Death-l (PD-l), Programmed Death Ligand-l (PD-L1), Programmed Death Ligand-2 (PD-L2), or CTLA-4 (cytotoxic T lymphocyte associated protein 4), Killer Immunoglobulin rVReceptor (KIR),T cell immunoglobulin and mucin- domain containing-3 (TIM-3), Lymphocyte-activation gene 3 (LAG-3), Cluster of an immune checkpoint protein, including, but not limited to, Programmed Death-l (PD-l), Programmed Death Ligand-l (PD-L1), Programmed Death Ligand-2 (PD-L2), or CTLA-4 (cytotoxic T lymphocyte associated protein 4), Killer Immunoglobulin rVReceptor (KIR),T cell immunoglobulin and mucin- domain containing-3 (TIM-3), Lymphocyte-activation gene 3 (LAG-3), Cluster of PD-l
  • PD-l Programmed Death Ligand-
  • Differentiation 276 CD276, B7-H3
  • V-set domain-containing T cell activation inhibitor 1 V-set domain-containing T cell activation inhibitor 1
  • B- and T-lymphocyte attenuator B7-H6
  • Natural Cytotoxicity Triggering Receptor 3 Ligand 1 B7-H6
  • Additional immune checkpoint inhibitors include, but are not limited to, inhibitors of AMHRII, B7-1, B7-2, B7-DC, B7-H1, B7-H2, , B7-H5, , B7-H7, , BTNL2, Butyrophilin family, CD27, CD28, CD30, CD40, CD40L, CD47, CD48, CD70, CD80, CD86, CD155,
  • SEMA4D Siglec family
  • Examples of PD-l inhibitors include, but are not limited to, Pembrolizumab (MK- 3475), Nivolumab (MDX-1106), Cemiplimab-rwlc (REGN2810), Pidilizumab (CT-011), Spartalizumab (PDR001), tislelizumab (BGB-A317), PF-06801591, AK105, BCD-100, BI 754091, JS001, LZM009, MEDI0680, MGA012, Sym02l, TSR-042.
  • Examples of PD-L1 inhibitors include, but are not limited to, Atezolizumab (MPDL3280A), Durvalumab
  • CTLA-4 inhibitors include Ipilimumab and Tremelimumab.
  • a therapeutically effective amount of a UCP2 inducing agent is administered to a mammal in need thereof.
  • a UCP2 inducing agent and immunomodulatory agents set forth herein are particularly useful for
  • the term “mammal” as used herein is intended to include, but is not limited to, humans, laboratory animals, domestic pets, and farm animals.“Therapeutically effective amount” means an amount of an agent set forth herein that, when administered to a mammal, is effective in producing the desired therapeutic effect.
  • cancer refers to or describes the pathophysiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers, as well as dormant tumors or micrometastases. Accordingly, the term “cancer” as used herein refers to an uncontrolled growth of cells, which interferes with the normal functioning of the bodily organs and systems, including cancer stem cells and tumor vascular niches.
  • a subject that has a cancer is a subject having objectively measurable cancer cells present in the subject's body. Cancers that migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.
  • Hematopoietic cancers such as leukemia, are able to out-compete the normal hematopoietic compartments in a subject, thereby leading to hematopoietic failure (in the form of anemia, thrombocytopenia and neutropenia) ultimately causing death.
  • Cancers that may be treated by the agents, pharmaceutical compositions, and methods contemplated by the disclosure include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors.
  • the cancers may comprise nonsolid tumors (such as hematological tumors, for example, leukemias and lymphomas) or may comprise solid tumors.
  • Types of cancers to be treated include, but are not limited to benign and malignant tumors, and malignancies, e.g., sarcomas, carcinomas, and melanomas.
  • sarcomas e.g., sarcomas, carcinomas, and melanomas.
  • adults tumors/cancers and pediatric tumors/cancers are also included.
  • Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • Non-limiting examples of tumors treatable by the methods described herein include, for example, carcinomas, lymphomas, sarcomas, blastomas, and leukemias.
  • Non limiting specific examples include, for example, breast cancer, pancreatic cancer, liver cancer, lung cancer, prostate cancer, colon cancer, renal cancer, bladder cancer, head and neck carcinoma, thyroid carcinoma, soft tissue sarcoma, ovarian cancer, primary or metastatic melanoma, squamous cell carcinoma, basal cell carcinoma, brain cancers of all histopathologic types, angiosarcoma, hemangiosarcoma, bone sarcoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelio sarcoma, synovioma, testicular cancer
  • ependymoma pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, retinoblastoma, leukemia, neuroblastoma, small cell lung carcinoma, bladder carcinoma, lymphoma, multiple myeloma, medullary carcinoma, B cell lymphoma, T cell lymphoma, NK cell lymphoma, large granular lymphocytic lymphoma or leukemia, gamma- delta T cell lymphoma or gamma-delta T cell leukemia, mantle cell lymphoma, myeloma, leukemia, chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, hematopoietic neoplasias, thymoma, sarcoma
  • Cancers that may treated by methods and compositions described herein include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp;
  • adenocarcinoma familial polyposis coli
  • solid carcinoma carcinoid tumor, malignant
  • branchiolo-alveolar adenocarcinoma papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell
  • adenocarcinoma granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma;
  • endometroid carcinoma skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma;
  • cystadenocarcinoma papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma;
  • paraganglioma malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant
  • choriocarcinoma mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma;
  • osteosarcoma juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma;
  • astrocytoma protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor;
  • meningioma malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas;
  • a patient can have more than one type of cancer.
  • Reducing includes inhibiting and/or reversing and can refer to, for example, the symptoms of the disorder being treated, the presence or size of metastases or
  • micrometastases the size of the primary tumor, the presence or the size of the dormant tumor.
  • subject is meant a mammal, including, but not limited to, a human or non human mammal, such as a bovine, equine, canine, ovine, or feline, etc. Individuals and patients are also subjects herein.
  • the terms“treat,”“treated,”“treating,” or“treatment” as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease.
  • Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.
  • the disclosure also provides a method for treating metastasis, which relates to the spreading of cancer from its primary site to other places in the body.
  • Cancer cells can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body.
  • Metastasis can be local or distant. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor, traveling through the bloodstream, and stopping at a distant site. At the new site, the cells establish a blood supply and can grow to form a life -threatening mass. Both stimulatory and inhibitory molecular pathways within the tumor cell regulate this behavior, and interactions between the tumor cell and host cells in the distant site are also significant. Metastases are most often detected through the sole or combined use of magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, blood and platelet counts, liver function studies, chest X-rays and bone scans in addition to the monitoring of specific symptoms.
  • Cancer stemness may refer to the ability of a cell to self-renew and to generate an additional, phenotypic ally distinct cell type.
  • Cancer stem cells are cancer cells that exhibit stem cell like properties. CSCs often exhibit at least one hallmark of cancer, and is capable of generating at least one additional, phenotypically distinct cell type. Furthermore, cancer stem cells are capable of both asymmetric and symmetric replication. It is appreciated that a cancer stem cell may result from differentiated cancer cells that acquire sternness traits and/or stem cells that acquire phenotypes associated with cancer cells. Alternatively, cancer stem cells can reconstitute non- stromal cell types within a tumor.
  • the efficacy of the treatment methods disclosed herein can be measured by various endpoints commonly used in evaluating cancer treatments, including but not limited to, tumor regression, tumor weight or size shrinkage, time to progression, duration of survival, progression free survival, overall response rate, duration of response, and quality of life.
  • the methods disclosed herein may reduce the number of cancer cells, reduce the tumor size, inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs, inhibit (i.e., slow to some extent and preferably stop) tumor metastasis, inhibit, to some extent, tumor growth, and/or relieve to some extent one or more of the symptoms associated with the disorder.
  • immunomodulatory agent administered to the subject is an amount effective in treating at least one of the cancers.
  • the effect can be cytostatic and/or cytotoxic.
  • a T cell response is determined by measuring the expression of one or more genes from a gene set in Table 1.
  • a T cell response is determined by measuring the expression of one or more of CD8A, IFNG, GZMB, TNF, FCK or SYK.
  • tumor vasculature includes, but are not limited to immune-histological staining for CD8alpha, SMA (smooth muscle B-actin, a marker of tumor pericytes and vascular smooth muscle) and vascular endothelial cadherin (a marker of endothelial cells).
  • compositions that comprise a therapeutically effective amount of an UPC2 inducing agent described herein (and optionally of an immunomodulatory agent) formulated together with one or more pharmaceutically acceptable excipients.
  • the dosage of active agent(s) may vary, depending on the reason for use, the individual subject, and the mode of administration.
  • the dosage may be adjusted based on the subject's weight, the age and health of the subject, and tolerance for the compound(s) or composition.
  • compositions and dosage forms may be formulated into compositions and dosage forms according to methods known in the art.
  • the pharmaceutical compositions of the present disclosure may be specially formulated in solid or liquid form, including those adapted for parenteral administration, for example, by subcutaneous, intratumoral, intramuscular or intravenous injection as, for example, a sterile solution or suspension.
  • compositions comprising a UPC2 inducing agent disclosed herein and optionally comprising an immunomodulatory agent may be formulated with one or more pharmaceutically-acceptable excipients, which can be a pharmaceutic ally- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, carrier,
  • manufacturing aid e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid
  • solvent or encapsulating material involved in carrying or transporting the therapeutic compound for administration to the subject, bulking agent, salt, surfactant and/or a preservative.
  • materials which can serve as pharmaceutically-acceptable excipients include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; gelatin; talc; waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as ethylene glycol and propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents; water; isotonic saline; pH buffered solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.
  • sugars such as lactose, glucose and sucrose
  • starches such as com starch and
  • a bulking agent is a compound which adds mass to a pharmaceutical formulation and contributes to the physical structure of the formulation in lyophilized form.
  • Suitable bulking agents according to the present disclosure include mannitol, glycine, polyethylene glycol and sorbitol.
  • a surfactant can reduce aggregation of the reconstituted protein and/or reduce the formation of particulates in the reconstituted formulation.
  • the amount of surfactant added is such that it reduces aggregation of the reconstituted protein and minimizes the formation of particulates after reconstitution.
  • Suitable surfactants according to the present disclosure include polysorbates (e.g. polysorbates 20 or 80); poloxamers (e.g.
  • poloxamer 188 Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-or stearyl- sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.
  • lauroamidopropyl myristamidopropyl-, palmidopropyl-, or isostearamidopropyl- dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; and polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g. Pluronics, PF68, etc.) ⁇
  • Preservatives may be used in formulations of the disclosure. Suitable preservatives for use in the formulation of the disclosure include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyl- dimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. Other suitable excipients can be found in standard pharmaceutical texts, e.g. in "Remington's Pharmaceutical Sciences", The Science and Practice of Pharmacy, l9th Ed. Mack Publishing Company, Easton, Pa., (1995).
  • compositions comprising the a UPC2 inducing agent disclosed herein, optionally the immunomodulatory agent, and the pharmaceutically acceptable carrier are lyophilized and provided in a composition for reconstitution prior to administration.
  • compositions comprising the UPC2 inducing agent disclosed herein optionally comprising an immunomodulatory agent may be administered in any convenient manner, including by injection, transfusion, implantation or transplantation.
  • compositions described herein may be administered to a patient orally, enterally,
  • the UPC2 inducing agent disclosed herein and optionally the immunomodulatory agent is administered to the mammal by intravenous infusion, i.e., introduction of the active agent(s) into the vein of a mammal over a certain period of time.
  • the period of time is about 5 minutes, about 10 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, or about 8 hours.
  • a dose of a compound or a composition is administered to a subject every day, every other day, every couple of days, every third day, once a week, twice a week, three times a week, once every two weeks, or once a month.
  • two, three or four doses of a compound or a composition is administered to a subject every day, every couple of days, every third day, once a week, once every two weeks or once a month.
  • a dose(s) of a compound or a composition is administered for 2 days, 3 days, 5 days, 7 days, 14 days, 21 days or 28 days.
  • a dose of a compound or a composition is administered for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months or more.
  • the disclosure provides UPC2 inducing agents that are administered with an additional therapeutic agent.
  • additional agents include, but are not limited to, cytotoxic agents, chemotherapeutic agents, growth inhibitory agents, anti-inflammatory agents, anti-cancer agents, anti-neurodegenerative agents, and anti-infective agents. Agents that are used in such combination therapies may fall into one or more of the preceding categories.
  • the UPC2 inducing agent is administered with an immune checkpoint modulator.
  • the UCP2 inducing agent is administered with a treatment targeting the b-catenin pathway, including, but not limited to, PRI-724, PRP, BBI-801.
  • the UCP2 inducing agent is administered with a PGE2 inducing agent.
  • the administration of the UPC2 inducing agent and the additional therapeutic agent may be concurrently or consecutively.
  • the administration of the UPC2 inducing agent and the additional therapeutic agent may be separately or as a mixture.
  • the methods of treatment contemplated by the disclosure can relate to a treatment in combination with one or more cancer therapies selected from the group of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy, and radiation therapy.
  • Exemplary additional therapeutic agents also include radionuclides with high- energy ionizing radiation that are capable of causing multiple strand breaks in nuclear DNA, and therefore suitable for inducing cell death (e.g., of a cancer).
  • Exemplary high-energy radionuclides include: 90 Y, 125 I, 131 I, 123 I, m In, 105 Rh, 153 Sm, 67 Cu, 67 Ga, 166 Ho, 177 Lu, 186 Re and 188 Re. These isotopes typically produce high energy a- or b-particles which have a short path length.
  • Such radionuclides kill cells to which they are in close proximity, for example neoplastic cells to which the conjugate has attached or has entered. They have little or no effect on non-localized cells and are essentially non-immunogenic.
  • Exemplary additional therapeutic agents also include cytotoxic agents such as cytostatics (e.g. alkylating agents, DNA synthesis inhibitors, DNA-intercalators or cross- linkers, or DNA-RNA transcription regulators), enzyme inhibitors, gene regulators, cytotoxic nucleosides, tubulin binding agents, hormones and hormone antagonists, anti-angiogenesis agents, and the like.
  • cytostatics e.g. alkylating agents, DNA synthesis inhibitors, DNA-intercalators or cross- linkers, or DNA-RNA transcription regulators
  • enzyme inhibitors e.g. alkylating agents, DNA synthesis inhibitors, DNA-intercalators or cross- linkers, or DNA-RNA transcription regulators
  • enzyme inhibitors e.g. alkylating agents, DNA synthesis inhibitors, DNA-intercalators or cross- linkers, or DNA-RNA transcription regulators
  • enzyme inhibitors e.g. alkylating agents, DNA synthesis inhibitors, DNA-intercalators or cross- linkers, or DNA-RNA transcription regulator
  • Exemplary additional therapeutic agents also include alkylating agents such as the anthracycline family of drugs (e.g. adriamycin, carminomycin, cyclosporin-A, chloroquine, methopterin, mithramycin, porfiromycin, streptonigrin, anthracenediones, and aziridines).
  • the chemotherapeutic moiety is a cytostatic agent such as a DNA synthesis inhibitor. Examples of DNA synthesis inhibitors include, but are not limited to, methotrexate and dichloromethotrexate, 3-amino- 1, 2, 4-benzotriazine 1, 4-dioxide,
  • DNA-intercalators or cross-linkers include, but are not limited to, bleomycin, carboplatin, carmustine, chlorambucil, cyclophosphamide, cis-diammineplatinum(II) dichloride (cisplatin), melphalan, mitoxantrone, and oxaliplatin.
  • Exemplary additional therapeutic agents also include transcription regulators such as actinomycin D, daunorubicin, doxorubicin, homoharringtonine, and idarubicin.
  • transcription regulators such as actinomycin D, daunorubicin, doxorubicin, homoharringtonine, and idarubicin.
  • Other exemplary cytostatic agents that are compatible with the present disclosure include ansamycin benzoquinones, quinonoid derivatives (e.g. quinolones, genistein, bactacyclin), busulfan, ifosfamide, mechlorethamine, triaziquone, diaziquone, carbazilquinone,
  • indoloquinone E09 diaziridinyl-benzoquinone methyl DZQ, triethylenephosphoramide, and nitrosourea compounds (e.g. carmustine, lomustine, semustine).
  • Exemplary additional therapeutic agents also include cytotoxic nucleosides such as, for example, adenosine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, floxuridine, ftorafur, and 6-mercaptopurine; tubulin binding agents such as taxoids (e.g. paclitaxel, docetaxel, taxane), nocodazole, rhizoxin, dolastatins (e.g., Dolastatin- 10, -11, or -15), colchicine and colchicinoids (e.g., ZD6126), combretastatins (e.g.,
  • cytotoxic nucleosides such as, for example, adenosine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, floxuridine, ftorafur, and 6-mercaptopurine
  • Combretastatin A-4, AVE-6032 Combretastatin A-4, AVE-6032
  • vinca alkaloids e.g., vinblastine, vincristine, vindesine, and vinorelbine (navelbine)
  • anti-angiogenesis compounds such as Angiostatin Kl-3, DL-a- difluoromethyl-ornithine, endostatin, fumagillin, genistein, minocycline, staurosporine, and ( ⁇ )-thalidomide.
  • Exemplary additional therapeutic agents also include hormones and hormone antagonists, such as corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone or medroprogesterone), estrogens, (e.g., diethylstilbestrol), antiestrogens (e.g., tamoxifen), androgens (e.g., testosterone), aromatase inhibitors (e.g., amino gluthetimide), 17- (allylamino)-l7-demethoxygeldanamycin, 4-amino- l,8-naphthalimide, apigenin, brefeldin A, cimetidine, dichloromethylene-diphosphonic acid, leuprolide (leuprorelin), luteinizing hormone-releasing hormone, pifithrin-a, rapamycin, sex hormone-binding globulin, and thapsigargin.
  • corticosteroids e.g
  • Exemplary additional therapeutic agents also include enzyme inhibitors such as, S(+)-camptothecin, curcumin, (-)-deguelin, 5, 6-dichlorobenz -imidazole 1-b- ⁇ - ribofuranoside, etoposide, formestane, fostriecin, hispidin, 2-imino-l-imidazolidineacetic acid (cyclocreatine), mevinolin, trichostatin A, tyrphostin AG 34, and tyrphostin AG 879.
  • enzyme inhibitors such as, S(+)-camptothecin, curcumin, (-)-deguelin, 5, 6-dichlorobenz -imidazole 1-b- ⁇ - ribofuranoside, etoposide, formestane, fostriecin, hispidin, 2-imino-l-imidazolidineacetic acid (cyclocreatine), mevinolin, trichostatin A, tyrphostin AG 34
  • Exemplary additional therapeutic agents also include gene regulators such as 5- aza-2'-deoxycytidine, 5-azacytidine, cholecalciferol (vitamin D3), 4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, trans-retinal (vitamin A aldehydes), retinoic acid, vitamin A acid, 9-cis-retinoic acid, l3-cis-retinoic acid, retinol (vitamin A), tamoxifen, and troglitazone.
  • gene regulators such as 5- aza-2'-deoxycytidine, 5-azacytidine, cholecalciferol (vitamin D3), 4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, trans-retinal (vitamin A aldehydes), retinoic acid, vitamin A acid, 9-cis-retinoic acid, l3-cis-
  • Exemplary additional therapeutic agents also include cytotoxic agents such as, for example, the pteridine family of drugs, diynenes, and the podophyllotoxins.
  • cytotoxic agents such as, for example, the pteridine family of drugs, diynenes, and the podophyllotoxins.
  • Particularly useful members of those classes include, for example, methopterin, podophyllotoxin, or podophyllotoxin derivatives such as etoposide or etoposide phosphate, leurosidine, vindesine, leurosine and the like.
  • auristatins e.g. auristatin E and monomethylauristan E
  • calicheamicin e.g. auristatin E
  • gramicidin D e.g. maytansanoids
  • neocarzinostatin topotecan
  • taxanes e.g. cytochalasin B
  • ethidium bromide e.g. emetine
  • tenoposide e.g. tenoposide
  • colchicin dihydroxy anthracindione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof.
  • the defined steps can be carried out in any order or simultaneously (except where the context excludes that possibility), and the method can include one or more other steps which are carried out before any of the defined steps, between two of the defined steps, or after all the defined steps (except where the context excludes those possibilities).
  • Flt3L-Tg Flt3L-Tg, BRaf A ; Tyr: CreER; Pten lox4 5 (Braf/Pten).
  • mice For tumor induction, 3-week-old Braf/Pten mice were topically treated with 1 pl 4- hydroxytamoxifen (8 mg m G 1 in ethanol) on the skin surface.
  • tumor cell lines were injected (l x 10 5 cells) subcutaneously into mice either as a single engraftment or co-engraftment as indicated in each experiment.
  • the mice were treated with Dox- containing water (2 mg ml -1 Dox with 5% sucrose) on day 7 or 10 after tumor engraftment, and changed every 2 days to maintain the potency of Dox.
  • the tumors were collected and analysis performed on day 20.
  • mice were treated with anti-CD8 antibody (200 m g per injection) twice weekly during the course of experiments.
  • Bl6-OVA-bearing mice were administrated either rosiglitazone (15 mg kg l , intra-tumorally, every 2 days) or control vehicle (dimethylsulfoxide), anti-PD-l antibody (200 m g per injection, intraperitoneally, every 3 days) from day 10 post-tumor engraftment.
  • mice were treated with rosiglitazone and/or anti-PD-l antibody as indicated above for a further 10 days.
  • B6 Cas9 mice were engrafted with 1 x 10 6 UCP2-deficient YUMM1.7 cells. After 10 days, mice were treated as described above.
  • Batf3 _/ mice were engrafted with 1 x 10 5 B16-OVA 3F-UCP2 cells. After 4 days of tumor injections, mice were treated with either control vehicle or Dox. Mice were transferred with 1 x 10 6 CD8 + DC and 1 x 10 6 OT-I T cells at days 9 and 12 post-tumor engraftment, respectively. Tumor samples were collected for flow cytometry analysis at day 14.
  • Cell lines YUMM1.7 melanoma cell line, B16-OVA mouse melanoma cell line.Flag tag- and flag tag-UCP2 B16-OVA or YUMM1.7 melanoma cell lines were established by stably transduced parental cell lines with lentivirus harboring Dox-inducible cassettes of indicated protein and selected by puromycin.
  • the B 16-OVA cell line containing dual-inducible expression cassettes of flag tag-UCP2 and FlIF-la -myc was generated by transducing the flag tag-UCP2 Bl 6-OVA cell line with lentivirus expressing HIF-la -myc and selected by G418.
  • the CXCL10KD and CCL5KD melanoma cell lines were established by stably transducing flag tag-UCP2 B 16-OVA with lentivirus harboring a short-hairpin RNA-expressing cassette (SMARTvector Mouse Lentiviral vector), and enriched by sorting with GFP-positive populations.
  • the YUMM1.7 The Cas9 cell line was derived from
  • YUMM1.7 transduced with pCW-Cas9, in which Dox can induce Cas9 expression.
  • Doxycycline-inducible protein expression plasmids were all created in the pCW-Cas9 backbone.
  • the murine UCP2 coding sequences were amplified by PCR and then cloned into pCW-Cas9 by Nhel and BamHl.
  • pCW-HIF-la-myc was created in two steps. First, the selection marker of pCW-Cas9 with blasticidin-resistant genes generated from pLX-sgRNA.
  • the HIF-Ia -myc-encoding sequence was amplified from pcDNA3 mHIF-la MYC
  • lentiviral vectors harboring shRNAi against CXCL10 and CCL5 were purchased from Dharmacon (SMARTvector, V3SM11241 and V3SM11244). Lentivirus was produced by transfecting 293T cells with the indicated expression plasmids and packing these using TurboFect (Thermo Fisher Scientific). For lentiviral transduction, cells were incubated with medium containing virus and 8 m g mF 1 polybrene for 24 h. Cells were allowed to recover for 48 h before antibiotic selection.
  • OT-I T cells were isolated from splenocytes of OT-I mice and cultured in Roswell Park Memorial Institute (RPMI) medium with 10% fetal bovine serum, 1% penicillin- streptomycin and b -mercaptoethanol.
  • RPMI Roswell Park Memorial Institute
  • OT-I splenocytes were treated with 1 m g mF 1 OVA257-264, 1 m g mF 1 anti-CD28 and 10 ng mF 1 IL-2 for 3 days, then cultured in the presence of IL-2 for another 2 days before adoptive transfer or in vitro effectontarget cell assay.
  • B6 mice received 50 m 1 of serum from Flt3L mice daily to enrich the CD8 + DC population.
  • CD11C+ DCs were isolated from the spleen. Isolated CD1 lc-i- dendritic cells were activated with 5 mg mF 1 poly(LC) for 24 h, then stimulated with 1 m g ml-l OVA257-264. For each dendritic cell preparation, activation marker expression was analyzed by flow cytometry with the majority of cells being CD1 lc + . After activation, cells showing high expression of MHCI and MHCII molecules were observed. Injection of CD8 + dendritic cells was based on the percentage of CD8 expression on CDl lc + DC; each mouse was administered 1 x 10 6 CD8 + CDl lc + DCs intravenously.
  • the single-sample Gene Set Enrichment analysis algorithm was used, implemented in R package GSVA, to calculate a T cell infiltration signature score for each sample. Default parameters from the GSVA package were used. Spearman correlation was used to quantify the association between UCP2 gene expression and T cell infiltration score, individually for each tumor type. The association between UCP2 expression and survival was evaluated by Cox regression and Kaplan-Meier analysis. For the latter, samples were stratified in three groups according to their UCP2 gene expression (low, intermediate, high). The 25th and 75th percentiles were used as cutoff thresholds. Survival analysis was performed separately for each tumor type.
  • Step 1 exploiting scRNA-Seq data, the set of genes predominantly expressed in malignant melanoma cells rather than in the other infiltrating cell types was pre selected. Then, the average expression of each of these genes in each cell type was quantified. All genes whose average expression in malignant cells exceeded that in infiltrating cells by at least 0.5 were retained (393 candidate genes in total).
  • Step 2 leveraging the sample size and statistical power of TCGA melanoma dataset, it was determined whether the expression of these 393 candidate genes selected in step 1 correlated with increased levels of UCP2 expression.
  • TCGA samples were stratified in three classes based on their UCP2 expression (UCP2 lG , UCP2 mid , UCP2 hl ), using 550 and 2,200 (roughly the 25th and 75th quantiles of UCP2 gene expression distribution) as cutoff thresholds.
  • UCP2 lG UCP2 mid , UCP2 hl
  • 550 and 2,200 roughly the 25th and 75th quantiles of UCP2 gene expression distribution
  • the differential expression of the candidate genes between UCP2 lG and UCP2 hl was tested by one-sided (right-sided) Wilcoxon rank-sum test. Genes with a nominal P > 0.05 were discarded, retaining 49 genes as UCP2 signature.
  • UCP2 signature was projected on the datasets of Tirosh et al, using this as a representative proxy of UCP2 expression in melanoma cells.
  • the UCP2 signature was quantified by summing the expression of all signature genes in malignant cells, thus ignoring the expression of these genes in other cell types.
  • Melanoma samples with either no T cells or no tumoral cells were excluded from this analysis. Each dot represents a sample from the dataset (with numbers corresponding to the original sample identity from Tirosh et al).
  • CD4 T cells CD45+/CD3+/CD4+
  • CD8 T cells CD45 + /CD3 + /CD8 +
  • Tregs CD45 + /CD3 + /CD4 + /FoxP3 +
  • B cells CD45 + /CD3- /CDl9 +
  • NK cells CD45V CD3-/NK1.1 +
  • CDl03 + DC CD45 + /Gr-l-/CDl lb- /CDl lc + /MHCII + /CDl03 + .
  • Cell pellets were lysed using RIPA lysis buffer (50 mM Tris- HC1, pH7.4, 2 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 50 mM NaF) containing EDTA-free Protease Inhibitor Cocktail. Protein lysates were mixed with SDS- PAGE loading dye and then subjected to SDS-PAGE for immunoblotting.
  • RIPA lysis buffer 50 mM Tris- HC1, pH7.4, 2 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 50 mM NaF
  • Protein lysates were mixed with SDS- PAGE loading dye and then subjected to SDS-PAGE for immunoblotting.
  • anti-UCP2 rabbit monoclonal Ab anti-EUF-l a rabbit monoclonal Ab
  • anti-rabbit IgG HRP-linked antibody
  • anti-mouse IgG HRP-linked antibody
  • anti- Phospho-Akt Ser473
  • anti- Phospho-Akt Thr308
  • rabbit monoclonal Ab anti-Akt (pan) mouse monoclonal Ab
  • anti-Phospho-S6 Ribosomal Protein Ser235/236
  • Tumor samples were fixed in 4% paraformaldehyde (PFA) and embedded in either paraffin or optimal cutting temperature compound; sections were stained using goat anti mouse VE-cadherin, mouse anti-mouse SMA-Cy3, rat anti-mouse CD8a, rabbit anti-mouse CD31 and goat anti-mouse VCAM-l primary antibodies and corresponding donkey secondary antibodies conjugated with Alexa 488, 555 and 647 fluorophores. Sections were mounted in Fluoromount G mounting medium supplemented with DAPI. A single section of each entire YUMM1.7 tumor from WT, Ragl 7 or WT mice treated with anti-CD8 monoclonal antibody was fully acquired using a Zeiss Axioscan.
  • PFA paraformaldehyde
  • B16-OVA tumors were acquired using a Zeiss LSM 880 with Airyscan confocal microscope. All data were analyzed using ImageJ software (NIH) and are presented as scatterplots, where each dot represent a single tumor calculated as the mean of entire tumor scans (Yumml.7) or three representative images (B 16-OVA).
  • NASH ImageJ software
  • VE-cadherin+ vessel density and area calculations were all automated using particle analysis and area measurements from each image in batches.
  • B16- OVA CD8a+ tumors cell infiltration and vessel density were analyzed based on manually selected tumor margins and cores.
  • Tumor-infiltrating CD8a + cells were quantified using particle analysis; eight random vessels per image were analyzed to quantify vessel length and diameter. Individual vessel surface area was calculated by multiplying average vessel length and width from each image. Mural cell co-localization with blood vessels was calculated by measuring the SMA intensity overlap over VE-cadherin (Yumml.7) or CD31 (B16-OVA) masks ⁇ 2 pixels, and normalized by the mask size itself. All data are presented as fold change relative to the corresponding controls. The density of VCAM-1+ and SMA+ vessels was quantified using the Cell Counter plug-in (ImageJ), and the data are presented as percentage of total number of CD3l + vessels. [0113] RNA purification, RT-PCR, quantitative PCR, RNA sequencing and bioinformatics analysis.
  • RNAs were then isolated using TRIzol reagent. Complementary DNA was converted from RNA using M-MLV Reverse Transcriptase. Indicated mRNA expression was performed in triplicate by quantitative real-time RT-PCR on a LightCycler 480 Instrument II machine using KAPA SYBR FAST qPCR Kit Master Mix. Relative expression was normalized by the expression of b -actin in each sample. For RNA sequencing, samples were collected after 3 days of treatment with Dox. DNA-free RNAs were extracted with Trizol and RNeasy Mini Kit. Messenger RNAs were then isolated for library construction. Libraries were sequenced on an Illumina HISEQ 2500. Mappable reads were analyzed using the DESeq2 package.
  • Chemokine expression levels in the culture supernatants were measured using the LEGENDplexTM mouse proinflammatory chemokine assay kit. This assay was used to quantify the concentration of chemokines secreted by tumor cells, according to the manufacturer’s instructions. The results were further normalized with protein concentration of tumor cell lysates in the same experiment.
  • proteome cytokine array tumor samples were frozen on dry ice immediately following resection from tumor-bearing mice. Tumor samples were then resuspended in phosphate buffered saline containing protease inhibitors and 1% Triton X-100 for three cycles of freezing and thawing.
  • B16-OVA 3F or B1A 3F-UCP2 cells were treated with either control vehicle or Dox for 3 days and were then collected and resuspended in phosphate buffered saline.
  • tumor cells were labeled with carboxyfluorescein succinimidyl ester (CFSE, 5 m M) for 30 min at 37 °C.
  • CFSE carboxyfluorescein succinimidyl ester
  • OT-I T cells in a 1:1 ratio at 37 °C.
  • cells were centrifuged at 200g for 2 min and fixed with 2% PFA for 10 min at room temperature. Cell pellets were then stained with anti-CD8a antibody and subjected to flow cytometric analysis.
  • the population of tumor cells recognized by OT-I T cells was determined according to double-positive CFSE and CD8a signals.
  • Example 1 UCP2 expression is associated with increased T cell anti-tumor response
  • TCGA Cancer Genome Atlas
  • the metabolic enzyme UCP2 was identified among the top gene hits upregulated in those patients with high T cell medicated anti-tumor responses. Further analyses revealed a positive correlation between UCP2 mRNA levels and individual gene transcripts related to T cell infiltration and anti-tumor immunity, including gene transcripts CD8A, IFNG, GZMB, TNF, LCK, and SYK (Figure 1A). Patients expressing higher UCP2 mRNA levels (UCP2 hl ) exhibited prolonged survival rates ( Figure IB). Of note, the expression of other members of the UCP family did not display strong association with gene transcripts related to T cell mediated anti-tumor responses.
  • UCP2 expression patterns were not caused by known melanoma driver mutations, increased neoantisen burden in tumors, or UCP2 expressing T cells
  • UCP2 expression patterns in melanomas were independent of any known melanoma driver mutations, indicating that UCP2 expression may not be controlled by these oncogenic pathways.
  • the genes were identified by cross-analyzing transcriptomes of UCP2 hl and UCP2 lG patients in the Cancer Genome Atlas and the scRNA-seq datasets.
  • the UCP2 gene signature in melanoma cells positively correlated with the abundance of CD8A mRNA in tumor-infiltrating lymphocytes, demonstrating that the positive correlation between UCP2 expression and the T cell anti-tumor signature was mediated by UCP2 expression in cancer cells, rather than in T cells.
  • Example 2 UCP2 expression is associated with an immunostimulatory chemokine profile and infiltration of CD8 + T cells and conventional type 1 dendritic cells (cDCl) in the TME
  • UCP2 expression is associated with the production of cytokines involved in anti tumor immunity
  • UCP2 mRNA was positively associated with genes controlling IFN-g signaling and leukocyte activation and migration.
  • UCP2 mRNA was strongly associated with gene transcripts that control migration of dendritic cells and T cell recruitment, including CCL4, CCL5, CXCR3, CXCL9 and CXCL10.
  • UCP2 expression is associated with cDCl infiltration of the tumor
  • CCL4 and CCL5 highly expressed in UCP2 hl patients, support the migration of conventional type 1 dendritic cells (cDCl) into tumors through stimulation of CCR5 expressed in cDC.
  • cDCl are dependent on basic leucine zipper transcriptional factor ATF- like 3 (BATF3) for development and are further important for priming CD8 + T cells against tumor antigens and attracting tumor- specific CD8 + T cells through the production of
  • BATF3 basic leucine zipper transcriptional factor ATF- like 3
  • UCP2 mRNA expression was strongly associated with cDCl tumor infiltration in melanoma patients from the transcriptome analysis.
  • cDCl tumor infiltration was computationally predicted based on the expression levels of a core gene set of cDCl, defined as the BATF3-DC signature score.
  • melanoma cells expressing UCP2 had higher frequencies of cDCl in tumors as indicated by XCR1 staining in samples obtained from the validation cohort.
  • Example 3 UCP2 induction suppresses tumor progression
  • a doxycycline (Dox)-inducible B 16-OVA melanoma cell line was established. This cell line stably expresses ovalbumin as a surrogate of tumor antigen and expresses either flag-tagged UCP2 (3F-UCP2) or flag tag (3F) upon Dox treatment. 3F and 3F-UCP2 B 16-OVA melanoma cells were co-engrafted into the left and right flanks of wild type mice, respectively.
  • mice were then treated with control vehicle as a mock treatment or Dox-containing water at day 7 post-tumor engraftment to induce either flag tag (control) or flag-tagged UCP2 expression in melanoma cells.
  • control control
  • flag-tagged UCP2 expression in melanoma cells.
  • 3F and 3F-UCP2 melanomas displayed similar tumor growth rates under mock treatment. Importantly, Dox treatment drastically suppressed the tumor growth and tumor burden of 3F-UCP2 melanomas, but not 3F melanomas ( Figures 2A and 2B). Of note, induction of UCP2 in the YUMM1.7 melanoma cell line, which harbors a Braf v600E mutation and PTEN deletion, using the same co-engraftment design, also suppressed in vivo tumor growth (see Figure 2C).
  • UCP2 induction promotes infiltration ofCD8 + T cells and NK cells
  • UCP2 induction increases CD8 + TIL infiltration of tumors and causes
  • CD8 + T cell infiltration, vascular morphology, and mural cell coverage were assessed by staining for CD8a, SMA (smooth muscle B -actin, a marker of tumor pericytes and vascular smooth muscle cells), and vascular endothelial cadherin (VE-cadherin, a marker of endothelial cells).
  • SMA smooth muscle B -actin, a marker of tumor pericytes and vascular smooth muscle cells
  • VE-cadherin vascular endothelial cadherin
  • VCAM-l on endothelial cells were increased in the core of UCP2-induced B16-OVA tumors.
  • CD8+ T cells are required for vessel normalization and reduction in tumor progression following UCP2 induction
  • mice were used as recipients for 3F- and 3F- UCP2 B16 OVA melanomas.
  • Ragl is required for somatic recombination of T cell receptor (TCR) and immunoglobulin (Ig) genes, and the absence of Ragl results in T and B cell deficiency.
  • TCR T cell receptor
  • Ig immunoglobulin
  • UCP2 induction in melanoma cells was sufficient to increase mural vascular coverage, but not vessel size, in those immunocompromised recipients.
  • these results demonstrate that UCP2 induction in melanomas facilitates recruitment of CD8 + T cells into the tumor core and normalizes tumor vessels, which are critical features of enhanced anti-tumor immunity and sensitivity to PD-l blockade treatment.
  • UCP2 induction in melanoma cells impedes tumor growth by facilitating CD8 + TIL recruitment and mounting an effective response against tumors.
  • Example 5 UCP2 induction promotes the production of anti-tumorigenic chemokines and facilitates cDCl infiltration in the TME
  • UCP2 induction promotes the production of cytokines involved in anti-tumor immunity and suppresses the expression of yro-tumorigenic factors
  • UCP2 expression in melanoma cells is associated with a reprogramming of the the cytokine milieu in the tumor.
  • cytokine profiles of tumors bearing UCP2 or control vector were determined in Dox-treated co engrafted mice.
  • UCP2 induction promoted the production of cytokines and molecules known to support anti-tumor immunity in vivo , including the production of CCL4, CCL5, CXCL9, CXCL10, IFN-g , IL-28 and ICAM-l.
  • UCP2 induction suppressed the expression of pro-tumorigenic factors IL-10 and M-CSF, and angiogenesis factors VEGF and angiopoietin-2.
  • CD8 + TIL infiltration was further examined in Batf3 / mice reconstituted with cDCl and activated OT-I cells.
  • OT-I cells are MHC class I-restricted, ovalbumin-specific, CD8 + T cells. These cells were designed to recognize ovalbumin peptide residues 257-264 in the context of H2Kb (CD8 co-receptor interaction with MHC class I). Similar levels of tumor infiltration of polyclonal CD8 + T cells and OT-I cells were observed in Dox-treated Bal/3 7 mice reconstituted with OT-I alone, and in control vehicle-treated Bal/3 7 mice reconstituted with OT-I and cDCl cells.
  • UCP2 induction enhanced tumor infiltration of polyclonal CD8 + T cells and OT-I cells in Bal/3 7 mice reconstituted with both OT-I and cDCl cells, supporting the notion that UCP2-induced CD8 + TIL infiltration is cDCl dependent.
  • CCR5 in cDCl plays a critical role in modulating tumor infiltration of cDCl. Since CCR5-deficient mice (Ccr5 7 ) have normal T cell priming, UCP2-mediated anti-tumor responses were examined in Ccr5 / mice. UCP2 induction failed to suppress tumor progression and promote recruitment of CD8 + TIL and cDCl in these mice, consistent with the notion that UCP2-induced CD8 + TIL infiltration is cDCl dependent.
  • UCP2 induction in cancer cells can boost the production of anti- tumorigenic chemokines and molecules to facilitate cDCl infiltration and anti-tumor immune responses.
  • Active b-catenin signaling can block tumor infiltration of cDCl via an ATF3- dependent mechanism.
  • Transcriptome analysis revealed that UCP2 expression correlated inversely, but moderately, with CTNNB1 score (based on the expression levels of six b- catenin signaling target genes).
  • UCP2 induction neither suppressed the expression of b-catenin signaling target genes, including EFNB3 and TCF12, nor inhibited ATF3 expression in melanoma cells, indicating that UCP2 induction promotes tumor infiltration of cDCl not through inhibition of the b-catenin-ATF3 pathway.
  • the results indicate that the regulation of UCP2 expression is independent of known oncogenic pathways and UCP2 expression in melanoma cells is not associated with activity of the b-catenin pathway or production of PGE2.
  • inducing UCP2 expression in melanoma cells will synergize with treatments targeting the b-catenin pathway and production of PGE2 to stimulate tumor infiltration by cDCl and CD8 + T cells through parallel regulatory mechanisms.
  • Example 7 UCP2 induction promotes engagement and amplification of the cDCl-CD8 + T cell anti-tumor immune cycle by stimulating IRF5-dependent CXCL10 production.
  • UCP2 induction in melanoma cells affects chemokine profiles in the TME
  • changes in the transcriptome of melanoma cells following UCP2 induction were assessed.
  • UCP2 induction promoted expression of CXCL10, but not CCL5 and CCL4, indicating that UCP2 induction in melanoma cells may enhance CCL5 and CCL4 levels in the TME by promoting CCL5 and CCL4 production by other cell types.
  • CXCL10 produced by melanoma cells attracts both CD8 + T and NK cells, which in turn have been reported to be the main producers of CCL5 in melanomas.
  • UCP2- overexpressing melanomas may facilitate a low level of CD8 + T cell recruitment through initial upregulation of CXCL10 production in melanoma cells.
  • This low-grade increase in CD8 + TILs in turn may lead to upregulation of CCL5 in tumors.
  • the increasing involvement of CCL5 supports cDCl tumor infiltration, which further enhances CD8 + TIL recruitment through additional CXCL10 production.
  • depletion of CD8 + T cells abrogated the induction of both CCL5 and CXCL10, and impaired cDCl tumor infiltration in UCP2-overexpressing melanomas.
  • CD8 + T cells appear to be the major producers of CCL5 following UCP2 induction, and the presence of CD8 + T cells appears to be important for maximizing cDCl infiltration.
  • UCP2 induction further stimulated expression of interferon regulatory factor 5 (IRF5), a transcription factor that stimulates CXCL10 expression and that has been suggested to affect immune responses in melanoma patients.
  • IRF5 interferon regulatory factor 5
  • melanoma cells expressing UCP2 showed a higher frequency of IRF5 expression in validation cohort of melanoma patients. Consistent with the notion that UCP2 induction in melanoma cells promotes CXCL10 production through IRF5-dependent transcriptional regulation, silencing of IRF5 abrogated UCP2-induced CXCL10 production.
  • Example 8 UCP2 induction reduces resistance to immune checkpoint blockade therapy and prolongs survival of tumor-bearing mice
  • mice were engrafted with 3F-UCP2 B16- OVA, a cell line resistant to anti-PD-l monoclonal antibody treatment.
  • Tumor-bearing mice were treated with either control vehicle or Dox, with or without anti-PD-l monoclonal antibody.
  • UCP2 induction sensitized B16-OVA melanomas to anti-PD-l therapy and prolonged the survival of tumor-bearing mice ( Figures 4A and 4B), showing that UCP2 induction can overcome primary resistance to PD-l blockade in melanomas.
  • a peroxisome yroliferator-activator receytor (PPAR) agonists can induce UCP2 in a dose-dependent manner
  • PPAR peroxisome proliferator-activator receptor
  • a combination treatment of a PPAR agonist and a checkpoint inhibitor can be used to successfully treat tumors resistant to checkpoint blockade
  • UCP2 expression in melanoma cells is important for rosiglitazone-mediated sensitization to PD-l blockade
  • a PPAR agonist sensitizes non-T cell-inflamed Braf/Pten for PD-l treatment
  • a non-T cell-inflamed Braf/Pten melanoma model was utilized. This cell lines conditionally expresses Braf kinase (BrafV 600E) mutation and PTEN deletion.
  • the anti-PD-l mAh failed to restrict melanoma progression; however, combined treatment with anti-PD-l monoclonal antibody and rosiglitazone stabilized tumor progression in Braf/Pten mice ( Figures 6G and H).

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Abstract

L'invention concerne des méthodes de réduction de la résistance au blocage des points de contrôle immunitaires et/ou de traitement d'un cancer résistant au traitement du blocage des points de contrôle immunitaires. Les méthodes de la présente invention comprennent l'administration à un sujet d'un agent induisant une protéine de découplage 2 (UCP2) et/ou d'un modulateur des points de contrôle immunitaires.
PCT/IB2019/053090 2018-04-16 2019-04-15 Agents induisant ucp2 pour le traitement du cancer résistant au blocage des points de contrôle immunitaires Ceased WO2019202473A1 (fr)

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WO2023104020A1 (fr) * 2021-12-07 2023-06-15 深圳先进技术研究院 Utilisation de metformine dans la préparation d'un médicament antitumoral à des fins d'amélioration de l'effet d'un inhibiteur des points de contrôle immunitaire
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WO2021165367A1 (fr) * 2020-02-20 2021-08-26 Worldwide Innovative Network Procédé pour améliorer le traitement avec une thérapie de blocage de point de contrôle immunitaire
WO2023104020A1 (fr) * 2021-12-07 2023-06-15 深圳先进技术研究院 Utilisation de metformine dans la préparation d'un médicament antitumoral à des fins d'amélioration de l'effet d'un inhibiteur des points de contrôle immunitaire
WO2024059183A1 (fr) * 2022-09-14 2024-03-21 President And Fellows Of Harvard College Méthodes et compositions pour la modulation de piézo1 dans le traitement du cancer

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