WO2020086440A1 - Immunomodulatory compounds - Google Patents

Immunomodulatory compounds Download PDF

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Publication number
WO2020086440A1
WO2020086440A1 PCT/US2019/057170 US2019057170W WO2020086440A1 WO 2020086440 A1 WO2020086440 A1 WO 2020086440A1 US 2019057170 W US2019057170 W US 2019057170W WO 2020086440 A1 WO2020086440 A1 WO 2020086440A1
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cells
antibody
cancer
6pgd
alkyl
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PCT/US2019/057170
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French (fr)
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Pankaj Seth
Saeed DANESHMANDI
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Beth Israel Deaconess Medical Center
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Publication of WO2020086440A1 publication Critical patent/WO2020086440A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K31/455—Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides
    • 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
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • the disclosure is directed to compounds and methods for modulating immune response, such as compounds that modulate 6-phosphogluconate dehydrogenase (6PGD).
  • 6PGD 6-phosphogluconate dehydrogenase
  • PPP pentose phosphate pathway
  • G6PD glucose-6-phosphate dehydrogenase
  • TEFF T effector
  • TM fatty acid oxidation
  • Inhibiting glycolysis or promoting FAO results in the generation of central memory (TCM) and stem cell memory (TSM) cells, which have improved longevity and anti-tumor function.
  • TCM central memory
  • TMS stem cell memory
  • identifying metabolic checkpoints that reprogram glucose utilization may provide a means to modulate metabolic intermediates that play a vital role in cellular differentiation.
  • the present disclosure provides novel immunomodulatory effects of Pentose Phosphate Pathway (PPP) and glycolytic metabolism and their impact on T cell function.
  • PPP Pentose Phosphate Pathway
  • the disclosure provides a method for modulating an immune response, comprising administering a compound to a subject in need thereof that binds to or interacts with 6- phosphogluconate dehydrogenase (6PGD).
  • 6PGD 6- phosphogluconate dehydrogenase
  • the disclosure provides a method for modulating an immune response, comprising administering a compound of Formula (I) or a pharmaceutically acceptable salt, hydrate or solvate thereof to a subject in need thereof:
  • the R 1 is -N(R 6 )2;
  • R 2 is H or (C1-C6)alkyl;
  • R 3 is H or (C1-C6)alkyl;
  • R 4 is - C(0)N(R 6 ) 2 ;
  • the R 1 is -N(R 6 )2; R 2 is H; R 3 is H; R 4 is -C(0)N(R 6 )2; R 5 is H; and each R 6 is independently H or (C1-C6)alkyl.
  • the compound is Formula (II) or a pharmaceutically acceptable salt, hydrate or solvate thereof.
  • the compound binds to or interacts with 6PGD.
  • the modulation of the immune response comprises stimulating an increase of cytotoxic T cells levels as compared to levels without administration of the compound.
  • the modulation of the immune response comprises stimulating an increase of cytotoxic T cells activity as compared to activity without administration of the compound.
  • the modulation of the immune response comprises stimulating an increase in levels or activity of a granzyme.
  • the granzyme is selected from granzyme A and granzyme B.
  • the modulation of the immune response comprises stimulating an increase in levels or activity of an interferon as compared to levels without administration of the compound.
  • the interferon is selected from IFNa, IFN , and IFNy.
  • the modulation of the immune response is within the tumor microenvironment. In some embodiments, the modulation of the immune response is a reduction or suppression of an immune inhibitory cell. In some embodiments, the modulation of the immune response is an increase or enhancing of an immune stimulatory cell.
  • the immune inhibitory cell is selected from myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), FoxP3 + T cells; tumor associated neutrophils (TANs), M2 macrophages, and tumor associated macrophages (TAMs).
  • the immune stimulatory cell is selected from T cells, cytotoxic T lymphocytes, T helper cells, natural killer (NK) cells, natural killer T (NKT) cells, B cells, and dendritic cells.
  • the modulation of the immune response increases a ratio of immune stimulatory cells to immune inhibitory cells.
  • the modulation of the immune response increases a ratio cytotoxic T cells (Tc) to regulatory T cell (Tregs).
  • the modulation of the immune response comprises a reduction in checkpoint inhibition.
  • the disclosure provides a method for treating or preventing cancer, comprising administering a compound of Formula (I) to a subject in need thereof:
  • R 1 , R 2 , R 3 R 4 and R 5 are defined as above.
  • the subject is undergoing treatment with one or more immunotherapies.
  • the immunotherapy is an agent that modulates one or more PD-1 , programmed death-ligand 1 (PD-L1 ), or programmed death-ligand 2 (PD-L2).
  • the method further comprises administering an agent that modulates one or more PD-1 , PD-L1 , PD-L2, CTLA-4, Tim-3, or LAG-3.
  • the administration is sequential or simultaneous.
  • the agent that modulates PD-1 is an antibody or antibody format specific for PD-1.
  • the antibody or antibody format specific for PD-1 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.
  • the antibody or antibody format specific for PD-1 is selected from nivolumab, pembrolizumab, and pidilizumab.
  • the agent that modulates PD-L1 is an antibody or antibody format specific for PD-L1.
  • the antibody or antibody format specific for PD-L1 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.
  • the antibody or antibody format specific for PD-L1 is selected from atezolizumab, avelumab, durvalumab, and BMS-936559.
  • the agent that modulates PD-L2 is an antibody or antibody format specific for PD-L2.
  • the antibody or antibody format specific for PD-L2 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.
  • the agent that modulates CTLA-4 is an antibody or antibody format specific for CTLA-4.
  • the antibody or antibody format specific for CTLA-4 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen binding portion of an antibody.
  • the antibody or antibody format specific for CTLA- 4 is selected from ipilimumab (YERVOY), tremelimumab, AGEN1884, and RG2077.
  • the administration is by intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or direct injection into cancer tissue. In some embodiments, the administration is intratumoral. In some embodiments, the subject is predicted to be poorly responsive or non-responsive to the immune checkpoint immunotherapy or has presented as poorly responsive or non-responsive to the immune checkpoint immunotherapy.
  • the method reduces and/or mitigates one or more side effects of the immune checkpoint immunotherapy.
  • the side effect is selected from decreased appetite, rashes, fatigue, pneumonia, pleural effusion, pneumonitis, pyrexia, nausea, dyspnea, cough, constipation, diarrhea, immune-mediated pneumonitis, colitis, hepatitis, endocrinopathies, hypophysitis, iridocyclitis, and nephritis.
  • the method reduces the dose of the immune checkpoint immunotherapy. In some embodiments, the method reduces number of administrations of the immune checkpoint immunotherapy.
  • the method increases a therapeutic window of the immune checkpoint immunotherapy.
  • the method elicits a potent immune response in less-immunogenic tumors.
  • the method converts a tumor with reduced inflammation (“cold tumor”) to a responsive, inflamed tumor (“hot tumor”).
  • the method makes the cancer responsive or more responsive to a combination therapy of the immune checkpoint immunotherapy and one or more chemotherapeutic agents and/or radiotherapy.
  • the subject is predicted to be poorly responsive or non-responsive to the immune checkpoint immunotherapy based on expression of one or more of PD-1 , PD-L1 , or PD-L2, in a patient's biological specimen.
  • the subject is predicted to be poorly responsive or non-responsive to an agent that modulates one or more of PD-1 , PD-L1 , and PD-L2 based on low on expression of PD-1 , PD- L1 , and PD-L2 in a tumor specimen.
  • the subject is predicted to be poorly responsive or non-responsive to an agent that modulates one or more of PD-1 , PD-L1 , and PD-L2 tumor proportion score (TPS) of less than about 49% for PD-L1 staining.
  • TPS tumor proportion score
  • the disclosure provides a method for treating or preventing an infection, comprising administering a compound of Formula (I) to a subject in need thereof:
  • R 1 , R 2 , R 3 R 4 and R 5 as defined above.
  • the infection is a microbial infection and/or chronic infection.
  • the infection is selected from Chagas disease, HIV/AIDS, tuberculosis, osteomyelitis, hepatitis B, hepatitis C, Epstein-Barr virus or parvovirus, T cell leukemia virus, bacterial overgrowth syndrome, fungal and parasitic infections.
  • the method is in combination with an anti-infective agent.
  • the anti-infective agent is an anti-viral agent including, but not limited to, abacavir, acyclovir, adefovir, amprenavir, atazanavir, cidofovir, darunavir, delavirdine, didanosine, docosanol, efavirenz, elvitegravir, emtricitabine, enfuvirtide, etravirine, famciclovir, and foscarnet.
  • abacavir acyclovir
  • adefovir amprenavir
  • atazanavir cidofovir
  • darunavir delavirdine
  • didanosine didanosine
  • docosanol efavirenz
  • elvitegravir emtricitabine
  • enfuvirtide etravirine
  • famciclovir famciclovir
  • the anti-infective agent is an anti-bacterial agent selected from cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics (cipro, Levaquin, floxin, tequin, avelox, and norflox); tetracycline antibiotics (tetracycline, minocycline, oxytetracycline, and doxycycline); penicillin antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, and methicillin); monobactam antibiotics (aztreonam); and carbapenem antibiotics (ertapenem, doripenem, imipenem/cilastatin, and meropenem).
  • cephalosporin antibiotics cephalexin,
  • the anti-infectives include anti-malarial agents (e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline, artemether/lumefantrine, atovaquone/proguanil and sulfadoxine/pyrimethamine), metronidazole, tinidazole, ivermectin, pyrantel pamoate, and albendazole.
  • anti-malarial agents e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline, artemether/lumefantrine, atovaquone/proguanil and sulfadoxine/pyrimethamine
  • metronidazole e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline, artemether/lumefantrine, atovaquone/proguanil and sulfa
  • the disclosure provides a method of making an immunomodulatory cancer treatment, comprising: (a) identifying an immunomodulatory anti-cancer agent by: (i) determining whether the agent binds to or interacts with 6-phosphogluconate dehydrogenase (6PGD); and (ii) classifying the agent as immunomodulatory based on an ability to bind to or interact with 6PGD; and (b) formulating the agent for cancer treatment.
  • 6PGD 6-phosphogluconate dehydrogenase
  • the compound binds to or interacts with 6PGD.
  • the agent stimulates an increase in levels or activity of a granzyme.
  • the granzyme is selected from granzyme A and granzyme B.
  • the agent stimulates an increase in levels or activity of an interferon as compared to levels without administration of the compound.
  • the interferon is selected from IFNa, IFN , and IFNy.
  • FIG. 1 A-H is an illustrative schematic representation of the metabolic pathway with X representing 6PGD inhibition.
  • Metabolic analysis confirming increased metabolite 6-Phosphogluconate (FIG. 1 C) accumulation in 6-PGD cd4-Cre mine when compared to Glucose (FIG. 1 B), Phosphoenolpyruvate (PEP) (FIG. 1 D), Pyruvate (FIG. 1 E), and Lactate (FIG. 1 F).
  • FIG. 1G and 1 FI show an increase in OCR in 6PGD 7 - compared to 6PGD +/+ treatments.
  • FIG. 2A-E are a series of flow cytometry images showing that 6PGD deficiency induces an activated T cell phenotype in thymus (FIG. 2A), spleen (FIG. 2B and 2D), and lymph nodes (FIG. 2C and 2E).
  • FIG. 3A-B are a pair of flow cytometry images showing that 6PGD deficiency in T cells induces a memory phenotype in 20 weeks old (FIG. 3A) and 18 weeks old (FIG. 3B).
  • FIG. 4A-I show that 6PGD 7 - CD8 + T cells display enhanced TEFF function. IFN-g production as determined by intracellular staining and by ELISA of culture supernatants are shown in Fig. 4A and B. Compared to OTI/6PGD +/+ control, OTI/6PGD 7 - CD8 + T cells showed higher CTL function (Fig. 4C). Analysis of the activated CD8+ T cells for expression of genes with cytolytic function showed higher expression in cytotoxic profile genes including Granzyme family ( Gzma , Gzmb, Gzmc, Gzmd, Gzme) and the cell death-inducing factor Fasl (Fig. 4D).
  • Gzma , Gzmb, Gzmc, Gzmd, Gzme Granzyme family
  • Fasl Fig. 4D
  • mRNA for IFN-g and the transcription factor Tbx21 (encoding for T-bet) were also elevated in 6PGD 7 - cells as determined by qPCR (Fig. 4D) and intracellular staining (Fig. 4H). Higher expression of granzyme B (Fig. 4E) and Fas/FasL (Fig. 4F and 4G) on CD8 + 6PGD 7 - T cells, which are consistent with their enhanced cytolytic function.
  • FIG. 5A-C show that 6-Aminonicotinamide (6-AN), a small molecule inhibitor of 6PGD phenocopies 6PGD 7 - results in vitro.
  • Blocking of 6PGD in T cells resulted in significant increase of IFN-y production (Fig. 5A), enhanced expression of granzyme B (Fig. 5B) and enhanced CTL function as determined by co-culture of 6-AN-treated OTI cells with EG7 targets (Fig. 5D).
  • 6-AN-treated CD8 cells also displayed higher fatty acid uptake (Fig. 5C), a hallmark of TEFF cells.
  • FIG. 6A is a microarray image showing that 6PGD 7 - CD8 + T cells have a robust gene expression signature of TEFF.
  • FIG. 6B is a microarray image showing that the Th2 gene signature predominates in 6PGD 7 - YFP +/+ T-regulatory cells (Tregs).
  • FIG. 6C shows a Real-Time PCR validation assay.
  • FIG. 7 is a microarray image showing chromatin accessibility of the 6PGD 7 - and 6PGD +/+ CD8 + T cells genome by ATAC-Sequencing without any stimulation.
  • FIG. 8A-D are a series of histograms and bar graphs showing altered utilization of glucose in 6PGD 7 - T cells.
  • FIG. 8A and 8C show glucose uptake and
  • FIG. 8B and 8D shows Glutl expression.
  • FIG. 9A-J show that deficiency in 6PGD 7 -T cells reprograms metabolic circuits.
  • PGD 7 - T altered the profile of glycolysis intermediates in response to stimulation with phosphoenolpyruvate (PEP) (FIG. 9D) being higher and pyruvate (FIG. 9F) lower compared to 6PGD +/+ cells.
  • PEP phosphoenolpyruvate
  • FIG. 9F pyruvate
  • activated 6PGD 7 - and 6PGD +/+ T cells contained comparable levels of malate (FIG. 9G) and fumarate (FIG. 91 ), 6PGD 7 - T cells had significantly higher levels of citrate compared to 6PGD +/+ control.
  • FIG. 10A-F show that 6PGD 7 -T cells demonstrate higher mitochondrial activity that supports anti tumor function.
  • Analysis of mitochondrial function demonstrated that CD8+ T cells from 6PGD 7 - mice develop significantly higher mitochondrial mass, membrane potential (DYiti) and production of reactive oxygen species (ROS) compared to 6PGD +/+ cells (Fig 10A-C).
  • 6PGD 7 T cells displayed higher oxygen consumption rate (OCR) at base line, a robust increase of mitochondrial metabolism in response to mitostress and elevated spare respiratory capacity (SRC) (Fig. 10D-F).
  • OCR oxygen consumption rate
  • SRC spare respiratory capacity
  • FIG. 11A-E shows that Glucose-Pyruvate-Acetyl-CoA axis modulates increased histone acetylation in 6PGD 7 CD8 + T cells. Elevated Acetyl-CoA levels are shown in Fig. 11 D and E. 6PGD 7 had H3K9/K27 acetylation, which was inhibited by the HK2 inhibitor 2DG and by the ACLY inhibitor BMS303141 (Fig. 11B and 11C).
  • FIG. 12A-B show the effective response of 6PGD 7 - CD8 + T cells to Listeria m. infection in vivo.
  • 6PGD 7 - cells were able to clear the Lm-Ova more effectively than control and Ag-specific T cells showed higher capacity in TNF-a production and expression of maturation markers (Fig. 12A).
  • Recipients of OTI/6PGD 7 - cells displayed higher rate of bacterial clearance compared to recipients of OTI/6PGD +/+ cells (Fig. 12B) and Ag specific OTI/6PGD 7 - cells also showed higher IFN-g production.
  • FIG. 13A-C shows that 6PGD 7 T cells have potent anti-tumor activity which can be recapitulated by and 6PGD small molecule inhibitor 6-Aminonicotinamide (6-AN) in vivo.
  • 6PGD small molecule inhibitor 6-Aminonicotinamide (6-AN) in vivo.
  • OTI/6PGD +/+ CD8 + T cells OTI/6PGD 7 - CD8 + T cells were more efficient in reducing tumor growth (Fig. 13A) and expressed higher levels of granzyme B in the tumor environment (Fig. 13B).
  • FIG. 14A-B shows 6PGD 7 - TIL are resistant to metabolic exhaustion and tumor microenvironment-induced mitochondrial dysfunction.
  • 6PGD 7 - mice had significantly smaller tumors compared to 6PGD +/+ control group (Fig. 14A) and Recipients of pmel-1/6PGD 7 - T cells had significantly smaller tumors compared to recipients of pmel-1/6PGD +/+ T cells (Fig. 14B).
  • FIG. 15A-D shows how 6PGD deficiency alters differentiation of naive CD4+TH subset Cells. IFNgamma expression was significantly elevated in 6PGD 7 - compared to control CD4+ T cells (Fig. 15A).
  • FIG. 15B an increased number of IL-17A + RORyt + CD4 + T cells in 6PGD 7 - mice compared to control (Fig. 15C and D).
  • FIG. 16 shows a high number of 6PGD 7 - T mice developed rectal prolapse between 4 to 5 months of age, strongly indicative of colitis.
  • FIG. 17 shows iodoacetate (IAA), which targets glyceraldehyde phosphate dehydrogenase (GAPDFI) and oxalate (OXA) which targets pyruvate kinase (PK).
  • IAA iodoacetate
  • GAPDFI glyceraldehyde phosphate dehydrogenase
  • OXA oxalate
  • PK pyruvate kinase
  • FIG. 18A-H are a series of flow cytometry images showing that 6PGD 7 - T cells not only display activated phenotype and elevated expression of PD-1 but lack expression of exhaustion markers in the Spleen for PD-1 (Fig. 18A), Tim-3 (Fig. 18B), LAG-3 (Fig. 18C) and CTLA-4 (Fig. 18D) and in the Lypm node, for PD-1 (Fig. 18E), Tim-3 (Fig. 18F), LAG-3 (Fig. 18G) and CTLA-4 (Fig. 18H).
  • FIG. 19A-B are a series of flow cytometry images showing Bone Marrow Myeloid Derived Stem Cells (MDSCs) in the presence of 6AN vs DMSO.
  • MDSCs Bone Marrow Myeloid Derived Stem Cells
  • FIG. 20A-B are a series of histograms showing bone marrow derived MDSCs suppression assay following 6AN and DMSO treatment.
  • MDSCs derived in presence of DMSO from bone marrow are more suppressive than 6-AN derived as measured by their effect on T cell proliferation, indicating that 6-AN reduces suppressive activity of MDSCs.
  • FIG. 21A-B are a series of immunohistochemical images comparing 6PGD f/f -FoxP3 cre (FIG. 21 A) and 6PGD f/f -FoxP3 WT (FIG. 21 B) in lung, pancreas and skin cells.
  • FIG. 22A-B are a series of immunohistochemical images comparing 6PGD f/f -FoxP3 cre (FIG. 22A) and 6PGD f/f -FoxP3 WT (FIG. 22B) in heart, intestine and liver cells.
  • FIG. 23A-B are graphs of a Treg in vitro suppression assay showing that 6PGD 7 - deficient T- regulatory cells lack in vitro suppressive activity.
  • FIG. 24A-D shows how 6PGD 7 - deficient T-regulatory cells abrogate in vivo suppressive activity.
  • FIG. 24A shows a schematic of the Rag1 7 - Colitis model.
  • FIG. 24B shows a immunohistochemical image of hematoxylin and eosin staining of Rag1 7 - mice.
  • FIG. 24C shows the colon of Rag1 7 - mice under different conditions.
  • FIG. 25A-D shows how 6-AN treatment drives lineage specific transcription factors.
  • FIG. 25A is a schematic showing how 6-AN treatment drives lineage specific transcription factors in Treg and Th2 cells.
  • FIG. 25B and FIG. 25C show isolated Tregs (YFP + ) from 6PGD fl/fl FoxP3 Cre mice having lower suppressive activity in a suppression assay.
  • FIG. 25D is a series of flow cytometry images showing isolated Tregs in the presence of 6-AN vs DMSO.
  • FIG. 26A-M shows deletion of 6PGD in Tregs results in early onset fatal autoimmune disorder.
  • FIG. 26A and FIG. 26B show expresion of 6PGD mRNA and protein in YFP + cells that were sorted from 6PGD +/+ FoxP3 Cre (WT) and 6PGD fl/fl FoxP3 Cre mice.
  • FIG. 26C is a representative image of 21 days old WT and 6PGD fl/fl FoxP3 Cre mice.
  • FIG. 26D is a representative image of lymphadenopathy in 6PGD fl/fl FoxP3 Cre compared to WT mice.
  • FIG. 26E shows absolute number of T cells per spleen and pLNs in WT and 6PGD fl/fl FoxP3 Cre mice. Results are representative of 12 mice per group.
  • FIG. 26F shows a survival curve of WT and 6PGD fl/fl FoxP3 Cre mice. Results are representative of 27 mice per group.
  • FIG. 26G show splenocytes from 19 days old WT and 6PGD fl/fl FoxP3 Cre mice that were harvested, and distribution of CD4 + vs CD8 + T cells were evaluated.
  • 26H shows that deletion of 6PGD induces enhanced effector phenotype (CD44 hi 9 h CD62L
  • FIG. 26I shows both CD4 + and CD8 + T cells that have elevated expression of CD69 activation marker in 6PGD fl/fl FoxP3 Cre compared to WT mice.
  • 26J shows splenocytes from 19 days old WT and 6PGD fl/fl FoxP3 Cre mice that were stimulated with PMA (50 ng/ml)/lonomycin (1 pg/ml) plus Golgiplug (1 mI/ml) for 4 hours and expression of IFN-g was evaluated by flow cytometry.
  • FIG. 26K shows granzyme B (top panel) and CD107a degranulation activation marker (bottom panel) expression was assessed on splenocytes of 19 days old WT and 6PGD fl/fl FoxP3 Cre mice by flow cytometry.
  • 26M shows serum collected from 20 days old WT and 6PGD fl/fl FoxP3 Cre mice and levels of serum antibodies (FIG. 26L), and IFN-g, IL-17A, IL-4 and IL-5 (FIG. 26M) was detected . Results are representative of 12 mice per group.
  • FIG. 27A shows YFP + cells that were isolated from 20 day old 6PGD +/+ FoxP3 Cre (WT) and 6PGD fl/fl FoxP3 Cre mice. FoxP3 mRNA levels were evaluated by real-time PCR.
  • FIG. 27B shows isolated Tregs (YFP + ) that were cultured in vitro in the presence of IL-2 (700 lU/ml) and anti-CD3/anti-CD28 coated beads (Treg:beads ratio 1 :3), and cell numbers were assessed at 24 and 48 hour time points. Results are representative of three independent experiments.
  • the present disclosure is based at least in part, on the surprising discovery that compounds that inhibit 6 Phosphogluconate dehydrogenase (6PGD) in a manner similar to 6-Aminonicotinamide (6AN) are able to activate T cell that can be used in cellular therapy. Moreover, immune modulation of T cells results in enhanced effector phenotype and function, with especial effect on PD-1 /PD-L1 pathway. These findings have potential clinical applicability as pharmacologic inhibition of 6PGD with a small molecule inhibitor recapitulates the metabolic, immunologic and functional features of 6PGD deficient T cells.
  • the disclosure provides a method for modulating an immune response, comprising administering a compound of Formula (I) or a pharmaceutically acceptable salt, hydrate or solvate thereof to a subject in need thereof:
  • R 1 , R 2 , R 3 R 4 and R 5 as defined above.
  • the compound is Formula (II) or a pharmaceutically acceptable salt, hydrate or solvate thereof:
  • the disclosure provides a method for treating or preventing cancer, comprising administering a compound of Formula (I) to a subject in need thereof:
  • R 1 , R 2 , R 3 R 4 and R 5 as defined above.
  • the disclosure provides a method for treating or preventing an infection, comprising administering a compound of Formula (I) to a subject in need thereof:
  • R 1 , R 2 , R 3 R 4 and R 5 as defined above.
  • the disclosure provides a method of making an immunomodulatory cancer treatment, comprising: (a) identifying an immunomodulatory anti-cancer agent by: (i) determining whether the agent binds to or interacts with 6-phosphogluconate dehydrogenase (6PGD); and (ii) classifying the agent as immunomodulatory based on an ability to bind to or interact with 6PGD; and (b) formulating the agent for cancer treatment.
  • 6PGD 6-phosphogluconate dehydrogenase
  • T cell differentiation has a key role in T cell differentiation.
  • the success of antitumor responses via adoptive T cell-based therapies not only requires effective cytolytic function but also the ability of T cells to persist as long-term memory cells.
  • TEFF T effector
  • TM memory
  • Glycolysis and PPP are two key metabolic routes that utilize glucose to fulfill the bioenergetics and biomass requirements of a cell.
  • PPP consists of two branches, (i) the largely irreversible NADPH-producing oxidative branch starting from glucose-6-phosphate (G6P) that produces ribulose-5-phosphate (Rib-5P) or (ii) the reversible non-oxidative branch that converts glyceraldehyde-3- phosphate (GAP) and fructose-6-phosphate (F6P) to Rib-5P and Xyl-5-P.
  • G6P glucose-6-phosphate
  • Rib-5P ribulose-5-phosphate
  • F6P fructose-6-phosphate
  • 6PGD is upregulated in human cancers and inactivation of 6PGD in tumor cells results in decreased tumorigenesis. Upregulation of 6PGD activity is a mechanism of tumor resistance. There is a potential clinical applicability for a small molecule inhibitor such as the compounds described herein which can recapitulate the metabolic and functional features of 6PGD deficiency resulting in potent antigen-specific CD4+ and CD8+ cells.
  • 6-PGD small molecule inhibitors include one or more compounds that inhibit one or more proteins upstream of 6-PGD in the PPP, such as a G6PD inhibitor, a 6-phosphogluconolactonase; one or more compounds that inhibit one or more proteins downstream of 6-PGD in the PPP.
  • An exemplary 6-PGD small molecule inhibitor further includes glucose 1 ,6-diphosphate.
  • 6-PGD antagonists include one or more agents or compounds that directly or indirectly inhibit 6- PGD gene expression, protein expression, or enzymatic activity.
  • Exemplary 6-PGD antagonists include an, an anti- 6-PGD antibody, and small molecule inhibitors. Additional 6-PGD antagonists can be identified by any useful method, such as by inhibiting or activating one or more proteins upstream of 6- PGD in the PPP that results in 6-PGD inhibition.
  • the disclosure provides a method for modulating an immune response, comprising administering a compound of Formula (I) or a pharmaceutically acceptable salt, hydrate or solvate thereof to a subject in need thereof:
  • the compound is Formula (II) or a pharmaceutically acceptable salt, hydrate or solvate thereof.
  • the compound binds to or interacts with 6PGD.
  • the binds to or interacts with 6PGD comprises Polydatin (3,4',5-trihydroxystilbene-3- -d-glucoside; trans- resveratrol 3- -mono-D-glucoside; piceid), Physcion (1 ,8-Dihydroxy-3-methoxy-6-methylanthraquinone, Emodin-3-methyl ether).
  • the modulation of the immune response comprises stimulating an increase in levels or activity of an interferon as compared to levels without administration of the compound.
  • the interferon is selected from IFNa, IFN , and IFNy.
  • the modulation of the immune response is within the tumor microenvironment. [0085] In some embodiments, the modulation of the immune response is a reduction or suppression of an immune inhibitory cell. In some embodiments, the modulation of the immune response is an increase or enhancing of an immune stimulatory cell.
  • the immune inhibitory cell is selected from MDSCs, Tregs, FoxP3+ T cells; TANs, M2 macrophages, and TAMs.
  • the immune stimulatory cell is selected from T cells, cytotoxic T lymphocytes, T helper cells, NK cells, NKT cells, B cells, and dendritic cells. In some embodiments, the modulation of the immune response increases a ratio of immune stimulatory cells to immune inhibitory cells. In some embodiments, the modulation of the immune response increases a ratio cytotoxic T cells (Tc) to Tregs.
  • Tc cytotoxic T cells
  • Some human tumors can be eliminated by a patient's immune system.
  • administration of a monoclonal antibody targeted to an immune "checkpoint” molecule can lead to complete response and tumor remission.
  • a mode of action of such antibodies is through inhibition of an immune regulatory molecule that the tumors have co-opted as protection from an anti-tumor immune response.
  • a patient's CD8 + T cells may be allowed to proliferate and destroy tumor cells.
  • administration of a monoclonal antibody targeted to by way of example, without limitation, PD-1 can lead to complete response and tumor remission.
  • the compounds described herein can be used in combination with one or more blocking antibodies targeted to an immune "checkpoint” molecule.
  • the present compounds provided herein can be used in combination with one or more blocking antibodies targeted to a molecule such as CTLA-4 or PD-1 .
  • the present compounds provided herein may be used in combination with an agent that blocks, reduces and/or inhibits PD-1 and PD-L1 or PD- L2 and/or the binding of PD-1 with PD-L1 or PD-L2 (by way of non-limiting example, one or more of nivolumab (ONO-4538/BMS-936558, MDX1 106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, Merck), pidilizumab (CT-011 , CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), MPDL3280A (ROCHE)).
  • an agent that blocks, reduces and/or inhibits PD-1 and PD-L1 or PD- L2 and/or the binding of PD-1 with PD-L1 or PD-L2
  • an agent that blocks, reduces and/or inhibits PD-1 and PD
  • the compounds provided herein may be used in combination with an agent that blocks, reduces and/or inhibits the activity of CTLA-4 and/or the binding of CTLA-4 with one or more receptors (e.g., CD80, CD86, AP2M1 , SHP-2, and PPP2R5A).
  • the immune-modulating agent is an antibody such as, by way of non- limitation, ipilimumab (MDX-010, M DX- 101 , Yervoy, BMS) and/or tremelimumab (Pfizer). Blocking antibodies against these molecules can be obtained from, for example, Bristol Myers Squibb (New York, NY), Merck (Kenilworth, NJ), Medlmmune (Gaithersburg, MD), and Pfizer (New York, NY).
  • the compounds provided herein can be used in combination with one or more blocking antibodies targeted to an immune "checkpoint” molecule such as for example, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160 (also referred to as BY55), CGEN-15049, CHK 1 and CHK2 kinases, A2aR, CEACAM (e.g., CEACAM-1 , CEACAM-3 and/or CEACAM-5), GITR, GITRL, galectin-9, CD244, CD160, TIGIT, SIRPa, ICOS, CD172a, and TMIGD2 and various B-7 family ligands (including, but are not limited to, B7-1 , B7-2, B7-DC, B7-H1 , B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7), LAG-3 and Tim-3.
  • an immune "checkpoint” molecule such as for example,
  • the subject is undergoing treatment with one or more immunotherapies.
  • the immunotherapy is an agent that modulates one or more PD-1 , PD-L1 , PD-L2, CTLA- 4, Tim-3, or LAG-3.
  • the method further comprises administering an agent that modulates one or more PD-1 , PD-L1 , PD-L2, CTLA-4, Tim-3, or LAG-3.
  • the agent that modulates PD-1 is an antibody or antibody format specific for PD-1.
  • the antibody or antibody format specific for PD-1 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.
  • the antibody or antibody format specific for PD-1 is selected from nivolumab, pembrolizumab, and pidilizumab.
  • the agent that modulates PD-L1 is an antibody or antibody format specific for PD-L1.
  • the antibody or antibody format specific for PD-L1 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.
  • the antibody or antibody format specific for PD-L1 is selected from atezolizumab, avelumab, durvalumab, and BMS-936559.
  • the agent that modulates PD-L2 is an antibody or antibody format specific for PD-L2.
  • the antibody or antibody format specific for PD-L2 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.
  • the agent that modulates CTLA-4 is an antibody or antibody format specific for CTLA-4.
  • the antibody or antibody format specific for CTLA-4 is selected from ipilimumab (YERVOY), tremelimumab, AGEN1884, and RG2077.
  • the disclosure provides a method for treating or preventing cancer, comprising administering a compound of Formula (I) or (II) as described herein.
  • the compounds of the present disclosure can be used in administration to a subject ⁇ e.g., a research animal or a mammal, such as a human, having a clinical condition such as cancer or an infection).
  • a subject e.g., a research animal or a mammal, such as a human, having a clinical condition such as cancer or an infection.
  • the compounds described herein can be administered to a subject for the treatment of cancer or infection.
  • this document provides methods for treating clinical conditions such as cancer or infection with the expression vectors provided herein.
  • the present disclosure pertains to cancers and/or tumors; for example, the treatment or prevention of cancers and/or tumors.
  • Cancers or tumors refer to an uncontrolled growth of cells and/or abnormal increased cell survival and/or inhibition of apoptosis which interferes with the normal functioning of the bodily organs and systems. Included are benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases. Also, included are cells having abnormal proliferation that is not impeded by the immune system ⁇ e.g., virus infected cells).
  • the cancer may be a primary cancer or a metastatic cancer.
  • the primary cancer may be an area of cancer cells at an originating site that becomes clinically detectable, and may be a primary tumor.
  • the metastatic cancer may be the spread of a disease from one organ or part to another non-adjacent organ or part.
  • the metastatic cancer may be caused by a cancer cell that acquires the ability to penetrate and infiltrate surrounding normal tissues in a local area, forming a new tumor, which may be a local metastasis.
  • the cancer may also be caused by a cancer cell that acquires the ability to penetrate the walls of lymphatic and/or blood vessels, after which the cancer cell is able to circulate through the bloodstream (thereby being a circulating tumor cell) to other sites and tissues in the body.
  • the cancer may be due to a process such as lymphatic or hematogeneous spread.
  • the cancer may also be caused by a tumor cell that comes to rest at another site, re-penetrates through the vessel or walls, continues to multiply, and eventually forms another clinically detectable tumor.
  • the cancer may be this new tumor, which may be a metastatic (or secondary) tumor.
  • the cancer may be caused by tumor cells that have metastasized, which may be a secondary or metastatic tumor.
  • the cells of the tumor may be like those in the original tumor.
  • the secondary tumor while present in the liver, is made up of abnormal breast or colon cells, not of abnormal liver cells.
  • the tumor in the liver may thus be a metastatic breast cancer or a metastatic colon cancer, not liver cancer.
  • the cancer may have an origin from any tissue.
  • the cancer may originate from melanoma, colon, breast, or prostate, and thus may be made up of cells that were originally skin, colon, breast, or prostate, respectively.
  • the cancer may also be a hematological malignancy, which may be lymphoma.
  • the cancer may invade a tissue such as liver, lung, bladder, or intestinal.
  • Representative cancers and/or tumors of the present invention include, but are not limited to, a basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer ⁇ e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx
  • the disclosure provides a method for treating or preventing an infection, comprising administering a compound of Formula (I) or Formula (II).
  • the compounds of the disclosure are used to treat one or more infections.
  • the infection is a microbial infection and/or chronic infection.
  • the infection is selected from Chagas disease, HIV/AIDS, tuberculosis, osteomyelitis, hepatitis B, hepatitis C, Epstein-Barr virus or parvovirus, T cell leukemia virus, bacterial overgrowth syndrome, fungal and parasitic infections.
  • the present compounds are used in methods of treating viral infections (including, for example, HIV and HCV), parasitic infections (including, for example, malaria), and bacterial infections.
  • the infections induce immunosuppression.
  • FI IV infections often result in immunosuppression in the infected subjects.
  • the treatment of such infections may involve, in various embodiments, modulating the immune system with the present compounds to favor immune stimulation over immune inhibition.
  • the present invention provides methods for treating infections that induce immunoactivation.
  • intestinal helminth infections have been associated with chronic immune activation.
  • the treatment of such infections may involve modulating the immune system with the present compounds to favor immune inhibition over immune stimulation.
  • the present disclosure provides methods of treating viral infections including, without limitation, acute or chronic viral infections, for example, of the respiratory tract, of papilloma virus infections, of herpes simplex virus (HSV) infection, of human immunodeficiency virus (HIV) infection, and of viral infection of internal organs such as infection with hepatitis viruses.
  • the viral infection is caused by a virus of family Flaviviridae.
  • the virus of family Flaviviridae is selected from Yellow Fever Virus, West Nile virus, Dengue virus, Japanese Encephalitis Virus, St. Louis Encephalitis Virus, and Hepatitis C Virus.
  • the viral infection is caused by a virus of family Picornaviridae, e.g., poliovirus, rhinovirus, coxsackievirus.
  • the viral infection is caused by a member of Orthomyxoviridae, e.g., an influenza virus.
  • the viral infection is caused by a member of Retroviridae, e.g., a lentivirus.
  • the viral infection is caused by a member of Paramyxoviridae, e.g., respiratory syncytial virus, a human parainfluenza virus, rubulavirus ⁇ e.g., mumps virus), measles virus, and human metapneumovirus.
  • the viral infection is caused by a member of Bunyaviridae, e.g., hantavirus. In other embodiments, the viral infection is caused by a member of Reoviridae, e.g., a rotavirus. [00103] In some embodiments, the method is in combination with an anti-infective agent.
  • the anti-infective agent is an anti-viral agent including, but not limited to, abacavir, acyclovir, adefovir, amprenavir, atazanavir, cidofovir, darunavir, delavirdine, didanosine, docosanol, efavirenz, elvitegravir, emtricitabine, enfuvirtide, etravirine, famciclovir, and foscarnet.
  • abacavir acyclovir
  • adefovir amprenavir
  • atazanavir cidofovir
  • darunavir delavirdine
  • didanosine didanosine
  • docosanol efavirenz
  • elvitegravir emtricitabine
  • enfuvirtide etravirine
  • famciclovir famciclovir
  • the anti-infective agent is an anti-bacterial agent selected from cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics (cipro, Levaquin, floxin, tequin, avelox, and norflox); tetracycline antibiotics (tetracycline, minocycline, oxytetracycline, and doxycycline); penicillin antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, and methicillin); monobactam antibiotics (aztreonam); and carbapenem antibiotics (ertapenem, doripenem, imipenem/cilastatin, and meropenem).
  • cephalosporin antibiotics cephalexin,
  • the anti-infectives include anti-malarial agents ⁇ e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline, artemether/lumefantrine, atovaquone/proguanil and sulfadoxine/pyrimethamine), metronidazole, tinidazole, ivermectin, pyrantel pamoate, and albendazole.
  • anti-malarial agents e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline, artemether/lumefantrine, atovaquone/proguanil and sulfadoxine/pyrimethamine
  • metronidazole tinidazole
  • ivermectin pyrantel pamoate
  • albendazole albendazole.
  • any compound and/or antibody or antibody format specific directed to immune checkpoint molecules used in methods of the present disclosure disclosed herein acts synergistically when co-administered with another agent and is administered at doses that are lower than the doses commonly employed when such agents are used as monotherapy.
  • the subject in need of a cancer treatment comprising any compound and/or antibody or antibody format specific directed to immune checkpoint molecules used in methods of the present disclosure, as disclosed herein, has been treated with, is contemporaneously treated with, or is subsequently treated with another anti-cancer therapy, as disclosed herein.
  • the anti-cancer therapy may comprise radiotherapy.
  • the anti-cancer therapy may include a synthetic polypeptide comprising at least one domain capable of binding an immune checkpoint molecule.
  • the immune checkpoint molecule is selected from PD-1 , PD-L1 , PD-L2, CTLA-4, Tim-3, or LAG-3.
  • the anti-cancer therapy may be surgery to excise the cancer, i.e., tumor.
  • the anti-cancer therapy may include administration of one more chemotherapeutic agents.
  • the disclosure provides for methods that further comprise administering an additional agent to a subject.
  • the disclosure pertains to co-administration and/or co-formulation.
  • the method increases a therapeutic window of the immune checkpoint immunotherapy. In some embodiments, the method elicits a potent immune response in less- immunogenic tumors. In some embodiments, the method converts a tumor with reduced inflammation (“cold tumor”) to a responsive, inflamed tumor (“hot tumor”).
  • cold tumor a tumor with reduced inflammation
  • hot tumor a responsive, inflamed tumor
  • the method makes the cancer responsive or more responsive to a combination therapy of the immune checkpoint immunotherapy and one or more chemotherapeutic agents and/or radiotherapy.
  • chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and CYTOXAN cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins ⁇ e.g., bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its ad
  • dynemicin including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, 6-diazo-5-oxo-L-norleucine
  • irinotecan Camptosar, CPT-11 (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (TYKERB); inhibitors of PKC-a, Raf, H-Ras, EGFR ⁇ e.g., erlotinib (Tarceva)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • the methods of treatment can further include the use
  • compositions containing any compound and/or antibody or antibody format specific directed to immune checkpoint molecules described herein, in combination with a physiologically and pharmaceutically acceptable carrier can include any of the well-known components useful for immunization.
  • the carrier can facilitate or enhance an immune response to an antigen administered in a vaccine.
  • the cell formulations can contain buffers to maintain a preferred pH range, salts or other components that present an antigen to an individual in a composition that stimulates an immune response to the antigen.
  • the physiologically acceptable carrier also can contain one or more adjuvants that enhance the immune response to an antigen.
  • Pharmaceutically acceptable carriers include, for example, pharmaceutically acceptable solvents, suspending agents, or any other pharmacologically inert vehicles for delivering compounds to a subject.
  • Pharmaceutically acceptable carriers can be liquid or solid, and can be selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, and other pertinent transport and chemical properties, when combined with one or more therapeutic compounds and any other components of a given pharmaceutical composition.
  • Typical pharmaceutically acceptable carriers include, without limitation: water, saline solution, binding agents ⁇ e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers ⁇ e.g., lactose or dextrose and other sugars, gelatin, or calcium sulfate), lubricants ⁇ e.g., starch, polyethylene glycol, or sodium acetate), disintegrates (e.g., starch or sodium starch glycolate), and wetting agents (e.g., sodium lauryl sulfate).
  • Compositions can be formulated for subcutaneous, intramuscular, or intradermal administration, or in any manner acceptable for immunization.
  • the compositions, e.g., pharmaceutical compositions, disclosed herein are resuspended in a saline buffer (including, without limitation TBS, PBS, and the like).
  • An adjuvant refers to a substance which, when added to an immunogenic agent nonspecifically enhances or potentiates an immune response to the agent in the recipient host upon exposure to the mixture.
  • Adjuvants can include, for example, oil-in-water emulsions, water-in oil emulsions, alum (aluminum salts), liposomes and microparticles, such as, polysytrene, starch, polyphosphazene and polylactide/polyglycosides.
  • the compounds disclosed herein are in the form of a pharmaceutically acceptable salt.
  • Adjuvants can also include, for example, squalene mixtures (SAF-I), muramyl peptide, saponin derivatives, mycobacterium cell wall preparations, monophosphoryl lipid A, mycolic acid derivatives, nonionic block copolymer surfactants, Quil A, cholera toxin B subunit, polyphosphazene and derivatives, and immunostimulating complexes (ISCOMs) such as those described by Takahashi et al., Nature 1990, 344:873-875.
  • SAF-I squalene mixtures
  • muramyl peptide saponin derivatives
  • saponin derivatives mycobacterium cell wall preparations
  • monophosphoryl lipid A mycolic acid derivatives
  • nonionic block copolymer surfactants nonionic block copolymer surfactants
  • Quil A cholera toxin B subunit
  • polyphosphazene and derivatives and immunostimulating complexes
  • IFA Incomplete Freund's Adjuvant
  • Various appropriate adjuvants are well known in the art (see, for example, Warren and Chedid, CRC Critical Reviews in Immunology 1988, 8:83; and Allison and Byars, in Vaccines: New Approaches to Immunological Problems, 1992, Ellis, ed., Butterworth-Heinemann, Boston).
  • Additional adjuvants include, for example, bacille Calmett-Guerin (BCG), DETOX (containing cell wall skeleton of Mycobacterium phlei (CWS) and monophosphoryl lipid A from Salmonella minnesota (MPL)), and the like (see, for example, Hoover et al., J Clin Oncol 1993, 11 :390; and Woodlock et al., J Immunother 1999, 22:251-259).
  • BCG Bacille Calmett-Guerin
  • DETOX containing cell wall skeleton of Mycobacterium phlei
  • MPL monophosphoryl lipid A from Salmonella minnesota
  • Routes of administration include, for example: intradermal, intratumoral, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin.
  • the administration is by intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or direct injection into a cancer tissue.
  • the compounds described herein can be administered to a subject one or more times ⁇ e.g., once, twice, two to four times, three to five times, five to eight times, six to ten times, eight to 12 times, or more than 12 times).
  • the compound as provided herein can be administered one or more times per day, one or more times per week, every other week, one or more times per month, once every two to three months, once every three to six months, or once every six to 12 months.
  • the compound can be administered over any suitable period of time, such as a period from about 1 day to about 12 months.
  • the period of administration can be from about 1 day to 90 days; from about 1 day to 60 days; from about 1 day to 30 days; from about 1 day to 20 days; from about 1 day to 10 days; from about 1 day to 7 days.
  • the period of administration can be from about 1 week to 50 weeks; from about 1 week to 50 weeks; from about 1 week to 40 weeks; from about 1 week to 30 weeks; from about 1 week to 24 weeks; from about 1 week to 20 weeks; from about 1 week to 16 weeks; from about 1 week to 12 weeks; from about 1 week to 8 weeks; from about 1 week to 4 weeks; from about 1 week to 3 weeks; from about 1 week to 2 weeks; from about 2 weeks to 3 weeks; from about 2 weeks to 4 weeks; from about 2 weeks to 6 weeks; from about 2 weeks to 8 weeks; from about 3 weeks to 8 weeks; from about 3 weeks to 12 weeks; or from about 4 weeks to 20 weeks.
  • the antibodies directed to immune checkpoint molecules and/or the compounds used in methods of the present invention are administered in the tumor microenvironment ⁇ e.g., cells, molecules, extracellular matrix and/or blood vessels that surround and/or feed a tumor cell, inclusive of, for example, tumor vasculature; tumor-infiltrating lymphocytes; fibroblast reticular cells; endothelial progenitor cells (EPC); cancer- associated fibroblasts; pericytes; other stromal cells; components of the extracellular matrix (ECM); dendritic cells; antigen presenting cells; T-cells; regulatory T cells; macrophages; neutrophils; and other immune cells located proximal to a tumor) or lymph node and/or targeted to the tumor microenvironment or lymph node.
  • the antibodies directed to immune checkpoint molecules and/or the compounds used in methods of the present invention are administered in the tumor microenvironment ⁇ e.g., cells, molecules, extracellular matrix and/or blood vessels that surround and/or feed a tumor cell,
  • Dosage forms suitable for parenteral administration include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions ⁇ e.g., lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art.
  • any antibody or antibody format specific directed to immune checkpoint molecules and/or compounds used in methods of the present disclosure (and/or additional agents) disclosed herein as well as the dosing schedule can depend on various parameters, including, but not limited to, the disease being treated, the subject's general health, and the administering physician's discretion.
  • Any antibody directed to immune checkpoint molecules and/or compounds used in methods of the present invention, disclosed herein, can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of an additional agent, to a subject in need thereof.
  • an additional agent e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks
  • an antibody or antibody format specific directed to immune checkpoint molecules and/or compounds used in methods of the present disclosure and an additional agent(s) are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 1 1 hours to 12 hours apart, 1 day apart, 2 days apart, 3 days apart, 4 days apart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeks apart, or 4 weeks apart.
  • any antibody or antibody format specific directed to immune checkpoint molecules and/or compounds used in methods of the present disclosure (and/or additional agents) disclosed herein can depend on several factors including the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the subject to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular subject may affect dosage used.
  • the exact individual dosages can be adjusted somewhat depending on a variety of factors, including the specific combination of the agents being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the particular disease being treated, the severity of the disorder, and the anatomical location of the disorder. Some variations in the dosage can be expected.
  • the dosage may be about 0.1 mg to about 250 mg per day, about 1 mg to about 20 mg per day, or about 3 mg to about 5 mg per day.
  • the dosage of any agent disclosed herein may be about 0.1 mg to about 1500 mg per day, or about 0.5 mg to about 10 mg per day, or about 0.5 mg to about 5 mg per day, or about 200 to about 1 ,200 mg per day ⁇ e.g., about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1 ,000 mg, about 1 ,100 mg, about 1 ,200 mg per day).
  • administration of the antibody or antibody format specific directed to immune checkpoint molecules and/or compounds used in methods of the present disclosure (and/or additional agents) disclosed herein is by parenteral injection at a dosage of about 0.1 mg to about 1500 mg per treatment, or about 0.5 mg to about 10 mg per treatment, or about 0.5 mg to about 5 mg per treatment, or about 200 to about 1 ,200 mg per treatment ⁇ e.g., about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1 ,000 mg, about 1 ,100 mg, about 1 ,200 mg per treatment).
  • a suitable dosage of the antibody or antibody format specific directed to immune checkpoint molecules and/or compounds used in methods of the present disclosure (and/or additional agents) is in a range of about 0.01 mg/kg to about 100 mg/kg of body weight or about 0.01 mg/kg to about 10 mg/kg of body weight of the subject, for example, about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 1.1 mg/kg, about 1 .2 mg/kg, about 1.3 mg/kg, about 1 .4 mg/kg, about 1.5 mg/
  • the compound of the disclosure is administered at a dose of 10 mg/kg/intraperitoneally twice a day.
  • Administration of any antibody or antibody format specific directed to immune checkpoint molecules and/or compounds used in methods of the present disclosure (and/or additional agents) disclosed herein can, independently, be one to four times daily or one to four times per month or one to six times per year or once every two, three, four or five years. Administration can be for the duration of one day or one month, two months, three months, six months, one year, two years, three years, and may even be for the life of the subject.
  • boosting doses of the compound as provided herein can be administered.
  • a boosting dose can be administered about 10 to 30 days, about 15 to 35 days, about 20 to 40 days, about 25 to 45 days, or about 30 to 50 days after a priming dose.
  • the methods provided herein can be used for controlling solid tumor growth ⁇ e.g., breast, prostate, melanoma, renal, colon, or cervical tumor growth) and/or metastasis.
  • the methods can include administering an effective amount of a compound as described herein to a subject in need thereof.
  • the subject is a mammal ⁇ e.g., a human).
  • the compounds and methods provided herein can be useful for stimulating an immune response against a tumor. Such immune response is useful in treating or alleviating a sign or symptom associated with the tumor.
  • treating is meant reducing, preventing, and/or reversing the symptoms in the individual to which a compound as described herein has been administered, as compared to the symptoms of an individual not being treated.
  • methods described herein are to be used in concomitance with continuous clinical evaluations by a skilled practitioner (physician or veterinarian) to determine subsequent therapy. Such evaluations will aid and inform in evaluating whether to increase, reduce, or continue a particular treatment dose, mode of administration, etc.
  • the methods provided herein can thus be used to treat a tumor, including, for example, a cancer.
  • the methods can be used, for example, to inhibit the growth of a tumor by preventing further tumor growth, by slowing tumor growth, or by causing tumor regression.
  • the methods can be used, for example, to treat a cancer.
  • the subject to which a compound is administered need not suffer from a specific traumatic state. Indeed, the compound described herein may be administered prophylactically, prior to development of symptoms ⁇ e.g., a patient in remission from cancer).
  • the terms "therapeutic” and “therapeutically,” and permutations of these terms, are used to encompass therapeutic, palliative, and prophylactic uses.
  • treating or alleviating the symptoms is meant reducing, preventing, and/or reversing the symptoms of the individual to which a therapeutically effective amount of a composition has been administered, as compared to the symptoms of an individual receiving no such administration.
  • the terms “effective amount” and “therapeutically effective amount” refer to an amount sufficient to provide the desired therapeutic ⁇ e.g., anti-cancer, anti-tumor, or anti-infection) effect in a subject (e.g., a human diagnosed as having cancer or an infection).
  • Anti-tumor and anti-cancer effects include, without limitation, modulation of tumor growth (e.g., tumor growth delay), tumor size, or metastasis, the reduction of toxicity and side effects associated with a particular anti-cancer agent, the amelioration or minimization of the clinical impairment or symptoms of cancer, extending the survival of the subject beyond that which would otherwise be expected in the absence of such treatment, and the prevention of tumor growth in an animal lacking tumor formation prior to administration, i.e., prophylactic administration.
  • administration of an effective amount of the compound can increase the activation or proliferation of tumor antigen specific T cells in a subject.
  • the activation or proliferation of tumor antigen specific T cells in the subject can be is increased by at least 10 percent (e.g., at least 25 percent, at least 50 percent, or at least 75 percent) as compared to the level of activation or proliferation of tumor antigen specific T cells in the subject prior to the administration.
  • Anti-infection effects include, for example, a reduction in the number of infective agents (e.g., viruses or bacteria).
  • infective agents e.g., viruses or bacteria.
  • administration of a compound as provided herein can stimulate the activation or proliferation of pathogenic antigen specific T cells in the subject.
  • an effective amount of a compound may be lowered or increased by fine tuning and/or by administering more than one dose.
  • This disclosure provides a method for tailoring the administration/treatment to the particular exigencies specific to a given mammal.
  • Therapeutically effective amounts can be determined by, for example, starting at relatively low amounts and using step-wise increments with concurrent evaluation of beneficial effects.
  • the methods provided herein thus can be used alone or in combination with other well-known tumor therapies, to treat a patient having a tumor.
  • One skilled in the art will readily understand advantageous uses of the compounds and methods provided herein, for example, in prolonging the life expectancy of a cancer patient and/or improving the quality of life of a cancer patient.
  • the subject and/or animal is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon.
  • the subject and/or animal is a non-mammal, such, for example, a zebrafish.
  • the subject and/or animal is a human.
  • the human is a pediatric human.
  • the human is an adult human.
  • the human is a geriatric human.
  • the human may be referred to as a patient.
  • the human has an age in a range of from about 0 months to about 6 months old, from about 6 to about 12 months old, from about 6 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old.
  • the subject is a non-human animal, and therefore the invention pertains to veterinary use.
  • the non-human animal is a household pet.
  • the non-human animal is a livestock animal.
  • the subject is predicted to be poorly responsive or non-responsive to the immune checkpoint immunotherapy based on expression of one or more of PD-1 , PD-L1 , or PD-L2, in a patient's biological specimen.
  • the subject is predicted to be poorly responsive or non-responsive to an agent that modulates one or more of PD-1 , PD-L1 , and PD-L2 based on low on expression of PD-1 , PD-L1 , and PD-L2 in a tumor specimen.
  • the subject is predicted to be poorly responsive or non-responsive to an agent that modulates one or more of PD-1 , PD-L1 , and PD-L2 tumor proportion score (TPS) of less than about 49% for PD-L1 staining.
  • TPS tumor proportion score
  • kits that can simplify the administration of the pharmaceutical compositions and/or compounds disclosed herein.
  • kits of the disclosure comprises any compound and/or antibody or antibody format specific directed to immune checkpoint molecules used in methods of the present disclosure and/or pharmaceutical composition disclosed herein in unit dosage form.
  • the unit dosage form is a container, such as a pre-filled syringe, which can be sterile, containing any agent disclosed herein and a pharmaceutically acceptable carrier, diluent, excipient, or vehicle.
  • the kit can further comprise a label or printed instructions instructing the use of any agent disclosed herein.
  • the kit may also include a lid speculum, topical anesthetic, and a cleaning agent for the administration location.
  • the kit can also further comprise one or more additional agent disclosed herein.
  • the kit comprises a container containing an effective amount of a composition of the invention and an effective amount of another composition, such those disclosed herein.
  • aspects of the present invention include use of a compound as disclosed herein in the manufacture of a medicament, e.g., a medicament for treatment of cancer and/or treatment of an infection. Any aspect or embodiment disclosed herein can be combined with any other aspect or embodiment as disclosed herein.
  • Example 1 Immunomodulatory Efects of PPP and Glycolytic Metabolism and their Impact on T Cell Function.
  • 6PGD fl0X/fl0X (6PGD +/+ ) and 6PGD fl0X/fl0X CD4-Cre (6PGD 7 -) mice were generated, which allows selective ablation of 6PGD only in T cells.
  • 6PGD-/- T cells After stimulation via TCR/CD3, 6PGD-/- T cells had significantly reduced levels of lactate but enhanced glucose uptake and elevated phosphoe-nolpyruvate (PEP) as well as altered levels of intermediates of the TCA cycle.
  • PPP phosphoe-nolpyruvate
  • 6PGD 7 - mice had markedly elevated numbers of TEFF cells that rapidly differentiated to central memory (TCM) in the absence of im-munization. 6PGD-/- T cells were able to contain infection with Listeria monocytogenes and reduce tumor burden more efficiently than 6PGD +/+ T cells. These results also showed that increased levels of citrate and acetyl-CoA generated from glucose in 6PGD 7 - T cells regulated H3K9/K27 acetylation, and event that may dictate the fate of 6PGD 7 - T cells.
  • CD4 + or CD8 + 6PGD 7 - T cells demonstrated significantly increased expression of CD69 and CD122 activation markers compared to 6PGD +/+ T cells in both spleen (Fig. 2D) and lymph nodes (Fig. 2E).
  • CD69 is one of the most characterized activation markers for T cells.
  • Enhanced CD122 (IL2Rb) expression is not only present on activated T cells but a key marker related to longevity and responsiveness to memory-driving cytokines such as IL-15. The same pattern of phenotype changes in T cells isolated from spleens and lymph nodes were observed. These results were interesting based on the current knowledge of how metabolic reprogramming affects CD8 + TEFF and TM differentiation. For these reasons, these studies focused on dissecting the effects of 6PGD on CD8+ T cell differentiation. 6PGD Deficiency in T Cells Induces a Memory Phenotype
  • 6PGD- T cells have a cell-intrinsic mechanism that alters their differentiation program in response to antigen exposure, resulting in the generation of TEFF and rapid differentiation to TCM phenotype.
  • T cells from OTI/6PGD 7 - and OTI control mice were used, which recognize Ova-derived peptide SIINFEKL (Ova257-264), and OVA-expressing EG7 cell line as a target.
  • OTI/6PGD 7 - CD8 + T cells showed higher CTL function (Fig. 4C).
  • Analysis of the activated CD8+ T cells for expression of genes with cytolytic function showed higher expression in cytotoxic profile genes including Granzyme family ⁇ Gzma, Gzmb, Gzmc, Gzmd, Gzme) and the cell death-inducing factor Fasi (Fig. 4D). Consistent with the increased production of IFN-g (Fig.
  • mRNA for IFN-g and the transcription factor Tbx21 (encoding for T-bet) were also elevated in 6PGD-/- cells as determined by qPCR (Fig. 4D) and intracellular staining (Fig. 4H).
  • Fig. 4E granzyme B
  • Fas/FasL Fig. 4F,G
  • 6-Aminonicotinamide (6-AN), A Small Molecule Inhibitor of 6PGD Phenocopies 6PGD ⁇ / - Results In Vitro
  • 6-AN a niacin analog that inhibits 6PGD was used. 6- AN has been previously used to sensitize tumors in-vivo to platinum drugs.
  • 6-AN can recapitulate the profile of T cells from 6PGD 7 - mice.
  • WT OTI T cells were stimulated with splenocytes and Ova257-264 peptides, in the presence of 6-AN, and examined their differentiation and function. Blocking of 6PGD in T cells resulted in significant increase of IFN-g production (Fig. 5A), enhanced expression of granzyme B (Fig. 5B) and enhanced CTL function as determined by co-culture of 6-AN-treated OTI cells with EG7 targets (Fig. 5D).
  • 6-AN-treated CD8 cells also displayed higher fatty acid uptake (Fig. 5C), a hallmark of TEFF cells.
  • 6PGD 7 - CD8+ T Cells have a Robust Gene Expression Signature of TEFF
  • 6PGD 7 - T cells their molecular profile after in-vitro stimulation with aCD3+aCD28 mAbs for 72 hours by QuantiGene Plex Assay (Affymetrix) was examined. Compared to similarly stimulated 6PGD +/+ T cells, 6PGD 7 - T cells were specifically enriched for the expression of genes encoding factors known to identify TEFF CD8 + T cells, including GZM family, Ifng, Ccr5, Ccr2, 1117a, Tbet, Zeb2, Klrgl and Stat3 (Fig. 6A). ZEB2 is critical for generation and expansion of terminally differentiated effector cells, in partnership with T-bet.
  • transcripts encoding key regulators of memory differentiation such as Tcf7 and Klf2 were not elevated.
  • Treg associated genes such as FoxP3 and Tgfbr3 were suppressed in 6PGD 7 - T cells (Fig. 6A).
  • 6PGD 7 T cells exhibited preferential elevation of several molecular hallmarks associated with TEFF cells.
  • chromatin accessibility of the 6PGD 7 - and 6PGD +/+ CD8 + T cells genome was analyzed by ATAC- Sequencing without any stimulation (Fig. 7).
  • Tbx21 (T-bet) is a strong driver of Th1 responses and Bhlhe40 works as a cofactor of T-bet for its function such as enhancing IFN-g production.
  • CELF2 splicing factor and is required for T- cell maturation and activation, and Runx3 supports differentiation into cytotoxic effector TEFF function.
  • TCR-mediated activation induces the expression of the glucose transporter Glutl , leading to internalization of glucose.
  • glucose transporter Glutl Once glucose is converted to glucose 6-phosphate, it has two fates, either it can be used via glycolysis or via PPP, which generates Rib-5P that is required for nucleotide synthesis, but also NADPH that is required for biosynthetic reactions and reducing power (Fig. 1A-H). Since, glucose usage correlates with IFN-g production, the ability of 6PGD 7 - T cells to uptake glucose after aCD3 + aCD28 mAbs stimulation was examined. It determined that 6PGD 7 - T cells had higher potency for glucose uptake as measured by 2-NBGD binding and Glutl expression (Fig. 8A-D).
  • Metabolomics analysis demonstrated that consistent with the lack of the 6PGD enzyme, there was an accumulation of 6-phosphoglucanate (6PG) in the 6PGD 7 - T cells, which increased following stimulation. In contrast no such increase of 6PG was observed in 6PGD +/+ T cells (Fig 9A). The functional consequences of increased 6PG and its degradation product gluconate in T cells are not yet known. Interestingly, 6PGD 7 - T altered the profile of glycolysis intermediates in response to stimulation with phosphoenolpyruvate (PEP) being higher and pyruvate lower compared to 6PGD +/+ cells.
  • PEP phosphoenolpyruvate
  • PEP that has been identified as a key factor to sustain NFAT signaling and anti-tumor effector function in both CD4 + and CD8+ T cells, may contribute to the improved effector and cytolytic function of the 6PGD 7 - T cells (Fig. 4A-I). Although activated 6PGD 7 - and 6PGD +/+ T cells contained comparable levels of malate and fumarate, 6PGD 7 - T cells had significantly higher levels of citrate compared to 6PGD +/+ control (Fig. 9). This metabolic profile with elevated citrate could suggest that in 6PGD 7 - T cells more pyruvate produced through enhanced glycolysis is shuttled into the TCA cycle.
  • the elevated SRC indicates that regardless of the enhanced features of activated TEFF, 6PGD 7 - T cells do not have a bioenergetic signature of activated TEFF cells which display a dramatic decline in SRC, but rather a bioenergetic signature of TM cells, which have elevated SRC.
  • Glucose-Pyruvate-Acetyl-CoA Axis Modulates Increased Histone Acetylation in 6PGCH- CD8 + T Cells
  • 6PGD 7 - T cells displayed elevated citrate (Fig. 9) but also elevated Acetyl-CoA (Fig. 11 D and E). 6PGD 7 T cells have a propensity to transition into TCM cells and recent studies have highlighted role of epigenetic mechanisms as being critical in the differentiation of T cells. Moreover, it is also recently demonstrated that glycolysis-derived Acetyl-CoA, promotes histone acetylation and open chromatin state. It was examined if a similar mechanism was operative in 6PGD 7 - T cells.
  • 6PGD 7 - had H3K9/K27 acetylation, which was inhibited by the HK2 inhibitor 2DG and by the ACLY inhibitor BMS303141 (Fig. 11 B and 11 C) indicating that glycolysis derived pyruvate leading to the generation of Acetyl-CoA was responsible for histone acetylation.
  • These events which are amplified in 6PGD 7 - CD8 T cells, may have a key role for the differentiation of effector cells or memory differentiation.
  • 6PGD 7 - cells were able to clear the Lm-Ova more effectively than control and Ag-specific T cells showed higher capacity in TNF-a production and expression of maturation markers (Fig. 12A).
  • adoptive transfer using T cells from OTI/6PGD 7 - and OTI/6PGD +/+ control mice followed by inoculation of Lm-Ova was performed. Recipients of OTI/6PGD 7 - cells, displayed higher rate of bacterial clearance compared to recipients of OTI/6PGD +/+ cells (Fig. 12B). Ag specific OTI/6PGD 7 - cells also showed higher IFN-g production (Fig. 12B).
  • 6PGD 7 - T Cells have Potent Anti-tumor Activity which Can be Recapitulated by and 6PGD Small Molecule Inhibitor 6-Aminonicotinamide (6-AN) In Vivo
  • T cell metabolic reprogramming is one of the key targets of cancer mediated immune dysfunction. Based on these results regarding the distinct metabolic properties of 6PGD 7 T cells, it was examined whether 6PGD 7 - T cells might maintain their superior effector function in the presence of tumor. To this end, OTI/6PGD +/+ and OTI/6PGD 7 - CD8 + T cells were adoptively transferred to mice bearing the OVA-expressing EG7 tumor. Compared to OTI/6PGD +/+ CD8 + T cells, OTI/6PGD 7 - CD8 + T cells were more efficient in reducing tumor growth (Fig. 13A) and expressed higher levels of granzyme B in the tumor environment (Fig. 13B).
  • 6PGD +/+ /OTI-CD8 + cells was pre-treated with 6-AN or vehicle control during in vitro culture with Ova peptide and adoptively transferred them to mice bearing Ova-expressing EG7 tumors. Strikingly, 6PGD +/+ /OTI-CD8 + pretreated with 6-AN abrogated tumor growth (Fig. 13C).
  • 6PGD 7 - TIL are Resistant to Metabolic Exhaustion and Tumor Microenvironment-Induced Mitochondrial Dysfunction
  • pmel-1 /6PGD 7 - and pmel- 1/6PGD +/+ T cells were transferred to tumor bearing hosts. Recipients of pmel-1/6PGD-/- T cells had significantly smaller tumors compared to recipients of pmel-1/6PGD +/+ T cells (Fig. 14B). Moreover, TIL from recipients of pmel-1/6PGD 7 - T cells had improved mitochondrial function compared to TIL from recipients of pmel-1/6PGD 7 - T cells as assessed by MitoT racker staining (Fig. 14B). This metabolic profile of 6PGD 7 - CD8 + TIL is consistent with resistance to tumor-mediated metabolic exhaustion and preserved anti-tumor function in the tumor microenvironment.
  • HSQC NMR analysis is employed to investigate the abundance of the 13C-3/13C-4-Glu and 13C-4-Glu-glutathione isotopomers in 6PGD 7 - CD8 + T and 6PGD +/+ CD8+ T cells, the abundance of 13C-4-Gln is assessed, which allow to identify 6PGD 7 - CD8 + T cells utilize glutamine based on the abundance of labeled products of 13C5, 15N2-Gln oxidation through the TCA cycle.
  • the PPP provides support for nucleotide biosynthesis, via the conversion of 6PG to ribose 5-phosphate which is subsequently converted to phosphoribosyl pyrophosphate (PRPP) followed by its conversion nucleotide synthesis pathway (Fig.
  • Results showed that 6PGD 7 - CD8 + T cells promoted the expression of genes involved in the fatty acid metabolism including Sterol O-acyltransferase 2, Acyl-CoA Oxidase, Aqp9, and Acs2 (Fig. 6A).
  • Flow cytometry based analysis using labeled fatty acids on 6PGD 7 - CD8+ T cells show enhanced fatty acid uptake with increased CD36 expression (Fig. 41) which is the hallmark of activated phenotype.
  • 6PGD 7 - CD8+ T selectively oxidize endogenous or exogenous FAs.
  • Results showed that 6PGD 7 CD8+ T cells contain higher levels of citrate (Fig. 9C) and Acetyl-CoA (Fig.
  • Glucose-derived Acetyl-CoA and citrate can be utilized for fatty acid and cholesterol biosynthesis (Fig. 1).
  • FAO is also a major source of Acetyl-CoA and FAO of glucose-derived lipids is a key metabolic program of TM cells. For these reasons, it is important to determine whether the functional differentiation of 6PGD-/- CD8+ T cells to effector might be related to altered lipid metabolism that might be promoted by increased glucose flux.
  • FTMS Full Transform Mass Spectrometry
  • 6PGD 7 - CD8 + T and 6PGD +/+ CD8 + T cells displayed an expression pattern of genes which can serve as hallmarks of effector cells. It was identified that signature genes of effector cell differentiation as well as genes encoding for metabolism enzymes were differentially expressed in 6PGD 7 - T cells compared to control. Detailed characterization of the gene signatures of 6PGD 7 - CD8 + T cells in order to identify the mechanisms for their distinct differentiation programs are performed. RNA-seq analysis that can provide a global assessment of differential gene expression that have been shown to be involved in regulating T cell effector or memory responses are used.
  • OTI+CD8+ cells from OTI/6PGD 7 - and OTI/6PGD +/+ mice are adoptively transferred into syngeneic recipients followed by inoculation of Lm-Ova to assess antigen-specific CD8+T cell responses (Fig. 11 B).
  • TEFF responses are assessed on days 4-8 as before.
  • memory cell differentiation the response for time course is analyzed at days 6, 10, 15 and after day 40 of i.v. injection. Based on these data, it is hypothesized that the switch to memory may happen at an enhanced rate and therefore these experiments are repeated few times to be able to catch differentiated cells at correct time window.
  • Antigen-specific T cells are analyzed with CD44+CD62L+ consistent with TCM, and CD44
  • 0W cells are also addressed, consistent with terminally differentiated short-lived effectors (SLEC), v/s KLRG lowCD127 hi 9 h phenotype, long-lived memory precursor cells (MPEC).
  • the functional properties of these antigen-specific memory cells are accessed for production of IFN-y by re-stimulating splenocytes ex vivo with cognate peptide antigen, SIINFEKL (Ova257-264).
  • SIINFEKL cognate peptide antigen
  • the function of memory cells in vivo is assessed by re-challenging the mice with a secondary infection.
  • Antigen specific CD44 + CD62L + are isolate consistent with TCM for molecular profiling for key gene markers related to TEFF and TCM.
  • CD8+ T cells that are found to have low-Dyiti and have memory gene signature have been associated with enhanced in vivo persistence and superior antitumor properties.
  • 6PGD 7 - CD8 + T cells have higher OCR, increased mitochondrial potential and substantial SRC in contrast to control (Fig. 10) combining characteristics of a CD8+ population with TEFF and TCM phenotype.
  • Example 3 To Determine How 6PGD Impacts the Differentiation of CD4+ T Cells and the Acquisition of Anti-tumor Functions.
  • a cardinal feature of the T cell immunity is the functional plasticity of na ' ive T cells to differentiate into lineage specific T effector functions with unique expression markers and metabolic signature.
  • Results support the hypothesis that 6PGD deficiency might affect the polarization program of CD4 + T cells, which have a critical role in mediating CD8 + function. These data justify the need for detailed characterization of metabolic program and gene signature of differentiation of na ' ive CD4 + T cells from 6PGD 7 - mice. For these studies T cells from 6PGD 7 - mice are used.
  • differentiated linages characterized by lineage-specific transcription factors (T-bet for Th1 cells, GATA3 for Th2 cells, Foxp3 and Runxl for Treg, RORyt and Runxl for Th17 cells) are evaluated.
  • Initial immune-phenotyping indicate enhanced TH1 , and TH17 cells within CD4+ population in 6PGD 7 - T cells with elevated expression pattern of T-bet and RoRyt.
  • Mitochondrial potential has a key role in the context of CD4 + T cell differentiation as they may drive CD4 + T enhanced sternness and improved functionality for immunotherapy.
  • Mitochondrial functions are characterized by accessing mitochondrial potential, mitochondrial DNA and markers of mitochondrial biogenesis in PGD 7 - TH subset from 6PGD 7 - and 6PGD +/+ in different culture polarizing conditions, ii) Differentiation of Na ' ive CD4+TFI subset Cells by pharmacological inhibition of 6PGD: Based on prior data, it is investigated whether 6-AN treatment of 6PGD +/+ CD4 + cells can phenocopy results observed in CD4+ T cells from 6PGD 7 - mice. Gene expression and cytokines are characterized to validate each subset.
  • IAA iodoacetate
  • GPDFI glyceraldehyde phosphate dehydrogenase
  • OXA oxalate
  • PK pyruvate kinase
  • DCA dichloroacetate
  • PDK mitochondrial pyruvate dehydrogenase kinase
  • PH pyruvate dehydrogenase
  • DCA is also known to inhibit the production of IL-17 and suppress expression of the Th17 transcription factor RORyt in cells cultured in Th17-skewing conditions is used.
  • Treg differentiation is modulated via differential usages of endogenous fatty acids
  • free fatty acids FLA are delivered during the in vitro polarizing conditions in 6PGD deficient naive CD4+ T to explore how FFA effects lineage differentiation in 6PGD deficient CD4+ T cells.
  • TRP-1 T CR transgenic mice is utilized to access how targeting 6PGD affects CD4+ anti tumor immunity.
  • TRP-1 -specific CD4 + T cells derived from TCR transgenic are transduced with control or 6PGD shRNA and adoptively transfer into B16 melanoma-bearing mice as recently described. Tumors are measured over 12 to 14 days and animal survival are determined. Separately, the expression of IFN- y, and IL17 in TRP-1 positive cells from tumors, spleen, and draining lymph nodes three days' post adoptive transfer are analyzed.
  • naive CD4+ T isolated from TRP-1 TCR transgenic mice cells with aCD8+aCD23 mAbs are stimulated in presence of 6-AN or a vehicle control for 4 days and assess polarization for either Th1 or Th17 by analyzing cytokine and transcription factor via intracellular staining.
  • Cells are adoptively transfer into mice bearing B16 melanoma tumors to test their antitumor function.
  • RORD small molecule agonist LYC-54143
  • Th 17 cells In context of cell based therapy, recent reports have highlighted the role of Th 17 cells in significantly eradicating large human and murine tumors better than bulk CD4+ T cells, Th1 or Th2 cells.
  • RORy activation with a small molecule agonist, LYC-54143 has been shown to be effective in generating anti-tumor immunity in syngeneic models.
  • the combination of RORy agonist with 6-AN in adoptive cell based therapy might result in improved efficacy in terms of significantly reduced tumor volumes.
  • naive CD4+ T isolated from TRP-1 T CR transgenic mice cells with a CD8+aCD23 mAbs in the presence of a) 6-AN alone, b) vehicle control, c) 6-AN plus RORy agonist, d) vehicle control plus RORy agonist for 4 to 6 days and adoptively transferred into mice bearing B16 melanoma tumors to test their anti-tumor function. Tumors are measured over 12 to 14 days and animal survival are determined. Separately, the expression of IFNy, and IL17 in Trp-1 positive cells from tumors, spleen, and draining lymph nodes three days' post adoptive transfer are also analyzed.
  • TEFF responses are critical for anti-tumor function in vivo.
  • the effectiveness of antitumor responses via adoptive T cell-based therapies has been associated with not only the effective cytolytic functions but also their ability to persist as long-term memory cells.
  • trafficking of CD8 + T cells to secondary lymph nodes and the ability of tumor-reactive cells to remain in activated state and avoiding exhaustion has been associated with successful immunotherapy.
  • Results showed that targeting 6PGD resulted in generation of potent TEFF cells and differentiation to TCM.
  • 6PGD 7 - TEFF cells significantly reduced expression of exhaustion markers. For these reasons, it is investigated whether 6PGD deficiency improves anti-tumor capabilities in vivo by preferentially enhancing the functions of the tumor-specific TEFF and TCM.
  • the glycolysis intermediate PEP has been implicated in enhancing T cell responses because it is necessary for maximal Ca2 + -NFAT 1 signaling. Since, increased levels of PEP along with low levels of pyruvate are observed, it is hypothesized that blockade of 6PGD activity might lead to the generation of potent effectors through regulation of this axis. To examine if 6PGD deficiency improves T cell effector function and anti-tumor properties through this mechanism, pmel-1/6PGD 7 - and pmel-1/6PGD +/+ T cells are used stimulated with cognate antigen gp100 and intracellular PEP levels using a fluorescence-based assay are assessed.
  • NFAT1 Nuclear v/s cytoplasmic distribution of NFAT1 are analyzed to access NFAT activity. NFAT activity are examined in TIL isolated from B16F10 tumors in recipients of adoptively transferred pmel-1/6PGD 7 - and pmel-1/6PGD +/+ T cells.
  • pmel-1/6PGD 7 - and pmel-1/6PGD +/+ T cells primed with gp100 peptide antigen for adoptive transfer to wild-type mice bearing B16F10 tumors are utilized.
  • Foxol functions are regulated by phosphorylation events, such that during TEFF differentiation, Foxol is sequestered and prevents expression of genes, selectin L ⁇ Sell ⁇ , which encodes CD62L), and Kruppel- like factor 2 (K/f2) that are important in homing to secondary lymphoid structures.
  • selectin L ⁇ Sell ⁇ which encodes CD62L
  • Kruppel- like factor 2 K/f2
  • Transcription factor T-bet as well as IFN-y for assessment of TEFF function is examined by intracellular staining as shown in Fig. 5.
  • TCM population by expression of transcription factors Tcf7 and Eomes and immune typing cells for CD44 l0W CD62L ,li 9 ,l CD122 ,li 9 ,l are also accessed.
  • Anti-tumor function in vivo is assessed by measuring tumor size and survival in these tumor bearing mice. Phosphorylation and intracellular localization of Foxol in tumor-specific pmel-1/6PGD 7 - and pmel-1/6PGD +/+ T cells using standard cell biology protocols is assessed.
  • 6PGD 7 - T cells not only display activated phenotype and elevated expression of PD-1 but lack expression of exhaustion markers Tim-3, LAG-3 and CTLA-4 (Fig. 18B-D and F-H). These findings strongly suggest that 6PGD 7 T cells has superior poly functionality as anti-tumor effectors due to the expression of lower number of exhaustion receptors.
  • tumor-reactive CD8+ TIL's are analyzed to assess the potency and longevity of 6PGD 7 - TIL's.
  • mitochondrial function is studied by accessing potential and Mitochondrial mass in pmel + CD8 + TILs from these tumors compared to non-draining lymph nodes.
  • 6PGD has a major effect on differentiation of CD8 + T cells and on their metabolic properties, how 6PGD affects the dynamics of tumor-specific T cells is investigated. These findings are critical to translational application of 6PGD inhibitors in adoptive cell based therapy.
  • mice are monitored for tumor growth and for dynamics of TCM response by assessing the presence of pmel+ T cells in lymphoid and non-lymphoid organs, in which TCM vs. TEFF cells reside differently. Gene expression profiles are examined by PCR based methods to identify transcripts that were differentially expressed.
  • 6-AN a small molecule inhibitor targeting 6PGD affects tumor specific T cell differentiation resulting in reduced tumor growth. It is examined whether pharmacologic intervention using, 6-AN, a small molecule inhibitor of 6PGD might recapitulate the effects of genetic 6PGD targeting on the metabolic and differentiation program of tumor-specific T cells.
  • the currently available small molecule 6PGD inhibitor has been previously tested for its potential application as an anti-cancer drug. The interest is to perform a short term ex vivo treatment of tumor-specific T cells with 6PGD inhibitor followed by adoptive transfer of these cells in vivo for cell-based cancer immunotherapy.
  • Results showed that 6PGD 7 - TEFF cells show increased expression of PD-1 but reduced expression of exhaustion markers (Fig. 18). Thus, it is highly likely that blocking the PD-1 : PD-L1 pathway concomitantly with 6PGD targeting might result in superior anti-tumor immunity.
  • two approaches are employed. First, B16F10 melanoma is implanted in the 6PGD 7 - and 6PGD +/+ control mice as in shown in Fig. 14A and tumor-bearing mice are treated with biwkeekly injections of anti-PD-1 blocking antibody based on standard protocols. Outcomes are monitored by assessing tumor size and metastasis.
  • pmel-1/6PGD +/+ T cells are used and during in vitro priming with cognate antigen gp100, and are incubated with 6-AN or vehicle control. Primed cells are transferred to tumor bearing mice, which are subsequently treated with biweekly injections of anti-PD-1 blocking antibody.
  • WT1 Wilms tumor antigen-1
  • 6-AN a vehicle control DMSO
  • 7- splecific CD8+ T cells from HLA-A * 0201 individuals are selected by tetramers and 6PGD are targeted with pharmacological inhibitor, 6-AN or a vehicle control DMSO.
  • Cells are cultured with dendritic cells pulsed with the HLA-A * 0201 -restricted WT1126-134 (RMFPNAPYL) epitope.
  • WT1- reactive T cells are assessed by analyze transcription factors associated with short-lived terminally differentiated TEFF cells.
  • Cells expressing CD45RO, CD62L, CD27, CD127, CD28, and CCR7 are examined, which identify an antigen-experienced but not terminally differentiated phenotype. These cells are known to be associated with anti-tumor and stem cell line properties of WT 1 -specific T cells generated with IL-21. Expression of markers such as CXCR3, CD122, and CD95 and that are known to correlate with a poorly differentiated long-lived memory subset in humans are analyzed.
  • CTL function is asessed by LDH release assay with HLA-A * 0201 , (TAP)-deficient T2 cells pulsed with WT 1 peptide and metabolic output and bioenergetics are examined, ii) Impact of 6PGD inhibition on anti-tumor function of WT1- specific human T cells in vivo.
  • TEP TAA-deficient T2 cells pulsed with WT 1 peptide and metabolic output and bioenergetics are examined
  • ii) Impact of 6PGD inhibition on anti-tumor function of WT1- specific human T cells in vivo To examine differential capacity of human tumor-specific CD8+ T cells, NOD/SCID mice bearing WT1 + BV173 leukemia are used. WT1+ human leukemia BV173 cells are inoculated intravenously in NOD/SCID. These mice are then given increasing doses of WT1 -specific T cells generated in vitro in the presence of 6-AN or vehicle control DMSO.
  • bone marrow from these mice is isolated for analysis of WT1-specific T cells (WT1-pentamer+/CD8+) and BV173 leukemia cells (human CD45- / HLA class I-/ CD8-) by flow cytometry.
  • WT1-specific T cells WT1-pentamer+/CD8+
  • BV173 leukemia cells human CD45- / HLA class I-/ CD8-
  • MDSCs derived in presence of DMSO from bone marrow are more suppressive than 6-AN derived as measured by their effect on T cell proliferation, indicating that 6-AN modulates MDSC, as shown in FIG. 19A-B and FIG. 20A-B.
  • Comparison of 6PGDf/f-FoxP3cre (FIG. 21 A) and 6PGDf/f- FoxP3WT (FIG. 21 B) in lung, pancreas and skin cells shows altered immunohistochemical expression. Similarly, alterations were observed in heart, intestine and liver cells (FIG. 22A-B).
  • FIG. 6B is a microarray image showing that the Th2 gene signature predominates in 6PGD-/- YFP+/+ T-regulatory cells (Tregs).
  • FIG. 6B shows that 6PGD-/- YFP+/+ Tregs express both TH1 and TH2 markers.
  • FIG. 6C shows a Real-Time PCR validation assay of the microarray experiment.
  • FIG. 23A-B are graphs of a Treg in vitro suppression assay showing that 6PGD-/- deficient T- regulatory cells lack in vitro suppressive activity.
  • Tregs YFP +
  • 6PGD fl/fl FoxP3 Cre mice were isolated from 6PGD fl/fl FoxP3 Cre mice and showed lower suppressive activity in the in vitro suppression assay.
  • FIG. 24A-C and FIG. 27A-B show that defiency is 6PGD in T regs abrogatge in vivo suppressive activity.
  • YFP + cells were isolated from 20 day old 6PGD +/+ FoxP3 Cre (WT) and 6PGD fl/fl FoxP3 Cre mice and FoxP3 mRNA levels were evaluated by real-time PCR.
  • Tregs (YFP + ) were isolated and cultured in vitro in the presence of IL-2 (700 lU/ml) and anti-CD3/anti-CD28 coated beads (Treg:beads ratio 1 :3) and cells number was assessed at 24 and 48 hour time points. Results are representative of three independent experiments.
  • FIG. 27A YFP + cells were isolated from 20 day old 6PGD +/+ FoxP3 Cre (WT) and 6PGD fl/fl FoxP3 Cre mice and FoxP3 mRNA levels were evaluated by real-time PCR.
  • Tregs (YFP + ) were isolated
  • FIG. 24A is a schematic showing Tregs (YFP + ) from WT and 6PGD fl/fl FoxP3 Cre mice and T effector (CD4 + CD45RB hi 9 h ) cells were isolated, mixed and adoptively transferred to Rag1 7 - mice. Colons were evaluated 45 days after adoptive transfer.
  • FIG. 24B shows representative hematoxylin and eosin staining of Rag1 7 - mice colon on day 45 after IBD induction via adoptive transfer. Rag1 7 - mice colon were evaluated for length and thickness on day 45 post IBD induction.
  • FIG. 24C is a immunohistochemical image of different Rag1 7 - mice colon that, were evaluated for length and thickness on day 45 post IBD induction.
  • FIG. 25A-D shows how 6-AN treatment drives lineage specific transcription factors.
  • FIG. 25A is a schematic showing how 6-AN treatment drives lineage specific transcription factors in Treg and Th2 cells.
  • FIG. 25B and FIG. 25C show isolated Tregs (YFP + ) from 6PGD fl/fl FoxP3 Cre mice having lower suppressive activity in a suppression assay.
  • FIG. 25D is a series of flow cytometry images showing isolated Tregs in the presence of 6-AN vs DMSO. These experiments demonstate that 6-AN treatment drives lineage specific transcription factors in Treg and Th2 cells.
  • FIG. 26A-M, FIG. 21A-B, and FIG. 22A-B shows deletion of 6PGD in Tregs results in early onset fatal autoimmune disorder.
  • Comparison of 6PGDf/f-FoxP3cre (FIG. 21 A) and 6PGDf/f-FoxP3WT (FIG. 21 B) in lung, pancreas and skin cells shows altered immunohistochemical expression.
  • alterations were observed in heart, intestine and liver cells (FIG. 22A-B).
  • FIG. 26B show expresion of 6PGD mRNA and protein in YFP + cells that were sorted from 6PGD +/+ FoxP3 Cre (WT) and 6PGD fl/fl FoxP3 Cre mice.
  • FIG. 26C is a representative image of 21 days old WT and 6PGD fl/fl FoxP3 Cre mice.
  • FIG. 26D is a representative image of lymphadenopathy in 6PGD fl/fl FoxP3 Cre compared to WT mice.
  • FIG. 26E shows absolute number of T cells per spleen and pLNs in WT and 6PGD fl/fl FoxP3 Cre mice. Results are representative of 12 mice per group.
  • FIG. 26F shows a survival curve of WT and 6PGD fl/fl FoxP3 Cre mice. Results are representative of 27 mice per group.
  • FIG. 26G show splenocytes from 19 days old WT and 6PGD fl/fl FoxP3 Cre mice that were harvested, and distribution of CD4 + vs CD8 + T cells were evaluated .
  • FIG. 26H shows that deletion of 6PGD induces enhanced effector phenotype (CD44 hi 9 h CD62L
  • FIG. 26I shows both CD4 + and CD8 + T cells that have elevated expression of CD69 activation marker in 6PGD fl/fl FoxP3 Cre compared to WT mice.
  • FIG. 26J shows splenocytes from 19 days old WT and 6PGD fl/fl FoxP3 Cre mice that were stimulated with PMA (50 ng/ml)/lonomycin (1 pg/ml) plus Golgiplug (1 mI/ml) for 4 hours and expression of IFN-g was evaluated by flow cytometry.
  • PMA 50 ng/ml
  • 6PGD fl/fl FoxP3 Cre mice were stimulated with PMA (50 ng/ml)/lonomycin (1 pg/ml) plus Golgiplug (1 mI/ml) for 4 hours and expression of IFN-g was evaluated by flow cytometry.
  • FIG. 26K shows granzyme B (top panel) and CD107a degranulation activation marker (bottom panel) expression was assessed on splenocytes of 19 days old WT and 6PGD fl/fl FoxP3 Cre mice by flow cytometry.
  • FIG. 26L and FIG. 26M shows serum collected from 20 days old WT and 6PGD fl/fl FoxP3 Cre mice and levels of serum antibodies (FIG. 26L), and IFN-g, IL-17A, IL-4 and IL-5 (FIG. 26M) was detected . Results are representative of 12 mice per group. These experiments demonstate that deletion of 6PGD in Tregs results in early onset fatal autoimmune disorder.

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Abstract

The present disclosure provides compounds and methods for modulating immune response, such as compounds that modulate 6-phosphogluconate dehydrogenase (6PGD).

Description

IMMUNOMODULATORY COMPOUNDS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 62/748,679, filed October 22, 2018, the contents of which are hereby incorporated by reference herein in their entirety.
FIELD OF THE DISCLOSURE
[0002] The disclosure is directed to compounds and methods for modulating immune response, such as compounds that modulate 6-phosphogluconate dehydrogenase (6PGD).
BACKGROUND
[0003] Cellular function is dependent on intracellular metabolism and energy consumption. Specifically, the pentose phosphate pathway (PPP) has been shown to play a crucial role in cancer cell growth and the inhibition of PPP key enzymes, including glucose-6-phosphate dehydrogenase (G6PD), strongly affects cancer cell proliferation in vitro, as well as in vivo. Alterations in metabolic reprogramming have also been shown impact the differentiation and function of immune cells and in particular T cells which serve as the main arms of immune responses. It is well established that in order to meet their bioenergetic demands, T effector (TEFF) cells use aerobic glycolysis leading to lactate production whereas memory (TM) cells switch to fatty acid oxidation (FAO). Inhibiting glycolysis or promoting FAO results in the generation of central memory (TCM) and stem cell memory (TSM) cells, which have improved longevity and anti-tumor function. However, it is not well understood how modulating the metabolism can regulate the activation and potency of cytolytic T cells. Understanding these events may provide improved approaches for efficacious T cell-based cancer immunotherapy by exploiting these metabolic based mechanisms. Thus, identifying metabolic checkpoints that reprogram glucose utilization may provide a means to modulate metabolic intermediates that play a vital role in cellular differentiation.
SUMMARY
[0004] The present disclosure provides novel immunomodulatory effects of Pentose Phosphate Pathway (PPP) and glycolytic metabolism and their impact on T cell function.
[0005] In one aspect, the disclosure provides a method for modulating an immune response, comprising administering a compound to a subject in need thereof that binds to or interacts with 6- phosphogluconate dehydrogenase (6PGD).
[0006] In one aspect, the disclosure provides a method for modulating an immune response, comprising administering a compound of Formula (I) or a pharmaceutically acceptable salt, hydrate or solvate thereof to a subject in need thereof:
Figure imgf000003_0001
wherein: R1, R2, R3 R4 and R5 each is independently selected from the group consisting of H, halo, (C1- C6)alkyl, (C1-C6)alkoxy, (C3-C7)cycloalkyl, -C(=0)0R6, -C(0)N(R6)2, -N(R¾ (C6-C10)aryl, and (C3- C10) heteroaryl, each alkyl, alkoxy, cycloalkyl, aryl and heteroaryl optionally substituted with one or more groups selected from hydroxyl, halo, and (C1-C6)alkyl; and each R6 is independently H or (C1-C6)alkyl.
[0007] In some embodiments, the R1, R2, R3 R4 and R5 each is independently selected from the group consisting of H, halo, (C1-C6)alkyl, (C1-C6)alkoxy, (C3-C7)cycloalkyl, -C(=0)0R6, -C(0)N(R6)2, and - N(R¾ each alkyl, alkoxy, and cycloalkyl optionally substituted with one or more groups selected from hydroxyl, halo, and (C1-C6)alkyl; and each R6 is independently H or (C1-C6)alkyl.
[0008] In some embodiments, the R1 is -N(R6)2; R2 is H or (C1-C6)alkyl; R3 is H or (C1-C6)alkyl; R4 is - C(0)N(R6)2; R5 is H or (C1-C6)alkyl; each R6 is independently H or (C1-C6)alkyl.
[0009] In some embodiments, the R1 is -N(R6)2; R2 is H; R3 is H; R4 is -C(0)N(R6)2; R5 is H; and each R6 is independently H or (C1-C6)alkyl.
[0010] In some embodiments, the compound is Formula (II) or a pharmaceutically acceptable salt, hydrate or solvate thereof.
Figure imgf000003_0002
(II).
[0011] In some embodiments, the compound binds to or interacts with 6PGD.
[0012] In some embodiments, the modulation of the immune response comprises stimulating an increase of cytotoxic T cells levels as compared to levels without administration of the compound.
[0013] In some embodiments, the modulation of the immune response comprises stimulating an increase of cytotoxic T cells activity as compared to activity without administration of the compound. [0014] In some embodiments, the modulation of the immune response comprises stimulating an increase in levels or activity of a granzyme.
[0015] In some embodiments, the granzyme is selected from granzyme A and granzyme B.
[0016] In some embodiments, the modulation of the immune response comprises stimulating an increase in levels or activity of an interferon as compared to levels without administration of the compound. In some embodiments, the interferon is selected from IFNa, IFN , and IFNy.
[0017] In some embodiments, the modulation of the immune response is within the tumor microenvironment. In some embodiments, the modulation of the immune response is a reduction or suppression of an immune inhibitory cell. In some embodiments, the modulation of the immune response is an increase or enhancing of an immune stimulatory cell. In some embodiments, the immune inhibitory cell is selected from myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), FoxP3+ T cells; tumor associated neutrophils (TANs), M2 macrophages, and tumor associated macrophages (TAMs). In some embodiments, the immune stimulatory cell is selected from T cells, cytotoxic T lymphocytes, T helper cells, natural killer (NK) cells, natural killer T (NKT) cells, B cells, and dendritic cells. In some embodiments, the modulation of the immune response increases a ratio of immune stimulatory cells to immune inhibitory cells. In some embodiments, the modulation of the immune response increases a ratio cytotoxic T cells (Tc) to regulatory T cell (Tregs).
[0018] In some embodiments, the modulation of the immune response comprises a reduction in checkpoint inhibition.
[0019] In one aspect, the disclosure provides a method for treating or preventing cancer, comprising administering a compound of Formula (I) to a subject in need thereof:
Figure imgf000004_0001
wherein: R1, R2, R3 R4 and R5 are defined as above.
[0020] In some embodiments, the subject is undergoing treatment with one or more immunotherapies.
[0021] In some embodiments, the immunotherapy is an agent that modulates one or more PD-1 , programmed death-ligand 1 (PD-L1 ), or programmed death-ligand 2 (PD-L2). In some embodiments, the method further comprises administering an agent that modulates one or more PD-1 , PD-L1 , PD-L2, CTLA-4, Tim-3, or LAG-3. In some embodiments, the administration is sequential or simultaneous. In some embodiments, the agent that modulates PD-1 is an antibody or antibody format specific for PD-1. In some embodiments, the antibody or antibody format specific for PD-1 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody. In some embodiments, the antibody or antibody format specific for PD-1 is selected from nivolumab, pembrolizumab, and pidilizumab.
[0022] In some embodiments, the agent that modulates PD-L1 is an antibody or antibody format specific for PD-L1. In some embodiments, the antibody or antibody format specific for PD-L1 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody. In some embodiments, the antibody or antibody format specific for PD-L1 is selected from atezolizumab, avelumab, durvalumab, and BMS-936559.
[0023] In some embodiments, the agent that modulates PD-L2 is an antibody or antibody format specific for PD-L2. In some embodiments, the antibody or antibody format specific for PD-L2 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.
[0024] In some embodiments, the agent that modulates CTLA-4 is an antibody or antibody format specific for CTLA-4. In some embodiments, the antibody or antibody format specific for CTLA-4 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen binding portion of an antibody. In some embodiments, the antibody or antibody format specific for CTLA- 4 is selected from ipilimumab (YERVOY), tremelimumab, AGEN1884, and RG2077.
[0025] In some embodiments, the administration is by intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or direct injection into cancer tissue. In some embodiments, the administration is intratumoral. In some embodiments, the subject is predicted to be poorly responsive or non-responsive to the immune checkpoint immunotherapy or has presented as poorly responsive or non-responsive to the immune checkpoint immunotherapy.
[0026] In some embodiments, the method reduces and/or mitigates one or more side effects of the immune checkpoint immunotherapy. In some embodiments, the side effect is selected from decreased appetite, rashes, fatigue, pneumonia, pleural effusion, pneumonitis, pyrexia, nausea, dyspnea, cough, constipation, diarrhea, immune-mediated pneumonitis, colitis, hepatitis, endocrinopathies, hypophysitis, iridocyclitis, and nephritis.
[0027] In some embodiments, the method reduces the dose of the immune checkpoint immunotherapy. In some embodiments, the method reduces number of administrations of the immune checkpoint immunotherapy.
[0028] In some embodiments, the method increases a therapeutic window of the immune checkpoint immunotherapy.
[0029] In some embodiments, the method elicits a potent immune response in less-immunogenic tumors.
[0030] In some embodiments, the method converts a tumor with reduced inflammation ("cold tumor”) to a responsive, inflamed tumor ("hot tumor”).
[0031] In some embodiments, the method makes the cancer responsive or more responsive to a combination therapy of the immune checkpoint immunotherapy and one or more chemotherapeutic agents and/or radiotherapy.
[0032] In some embodiments, the subject is predicted to be poorly responsive or non-responsive to the immune checkpoint immunotherapy based on expression of one or more of PD-1 , PD-L1 , or PD-L2, in a patient's biological specimen.
[0033] In some embodiments, the subject is predicted to be poorly responsive or non-responsive to an agent that modulates one or more of PD-1 , PD-L1 , and PD-L2 based on low on expression of PD-1 , PD- L1 , and PD-L2 in a tumor specimen.
[0034] In some embodiments, the subject is predicted to be poorly responsive or non-responsive to an agent that modulates one or more of PD-1 , PD-L1 , and PD-L2 tumor proportion score (TPS) of less than about 49% for PD-L1 staining.
[0035] In one aspect, the disclosure provides a method for treating or preventing an infection, comprising administering a compound of Formula (I) to a subject in need thereof:
Figure imgf000007_0001
wherein: R1, R2, R3 R4 and R5 as defined above.
[0036] In some embodiments, the infection is a microbial infection and/or chronic infection. In some embodiments, the infection is selected from Chagas disease, HIV/AIDS, tuberculosis, osteomyelitis, hepatitis B, hepatitis C, Epstein-Barr virus or parvovirus, T cell leukemia virus, bacterial overgrowth syndrome, fungal and parasitic infections. In some embodiments, the method is in combination with an anti-infective agent. In some embodiments, the anti-infective agent is an anti-viral agent including, but not limited to, abacavir, acyclovir, adefovir, amprenavir, atazanavir, cidofovir, darunavir, delavirdine, didanosine, docosanol, efavirenz, elvitegravir, emtricitabine, enfuvirtide, etravirine, famciclovir, and foscarnet.
[0037] In some embodiments, the anti-infective agent is an anti-bacterial agent selected from cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics (cipro, Levaquin, floxin, tequin, avelox, and norflox); tetracycline antibiotics (tetracycline, minocycline, oxytetracycline, and doxycycline); penicillin antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, and methicillin); monobactam antibiotics (aztreonam); and carbapenem antibiotics (ertapenem, doripenem, imipenem/cilastatin, and meropenem). In some embodiments, the anti-infectives include anti-malarial agents (e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline, artemether/lumefantrine, atovaquone/proguanil and sulfadoxine/pyrimethamine), metronidazole, tinidazole, ivermectin, pyrantel pamoate, and albendazole.
[0038] In one aspect, the disclosure provides a method of making an immunomodulatory cancer treatment, comprising: (a) identifying an immunomodulatory anti-cancer agent by: (i) determining whether the agent binds to or interacts with 6-phosphogluconate dehydrogenase (6PGD); and (ii) classifying the agent as immunomodulatory based on an ability to bind to or interact with 6PGD; and (b) formulating the agent for cancer treatment.
[0039] In some embodiments, the compound binds to or interacts with 6PGD.
[0040] In some embodiments, the agent stimulates an increase in levels or activity of a granzyme. In some embodiments, the granzyme is selected from granzyme A and granzyme B. [0041] In some embodiments, the agent stimulates an increase in levels or activity of an interferon as compared to levels without administration of the compound. In some embodiments, the interferon is selected from IFNa, IFN , and IFNy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] FIG. 1 A-H is an illustrative schematic representation of the metabolic pathway with X representing 6PGD inhibition. Metabolic analysis confirming increased metabolite 6-Phosphogluconate (FIG. 1 C) accumulation in 6-PGD cd4-Cre mine when compared to Glucose (FIG. 1 B), Phosphoenolpyruvate (PEP) (FIG. 1 D), Pyruvate (FIG. 1 E), and Lactate (FIG. 1 F). FIG. 1G and 1 FI show an increase in OCR in 6PGD7- compared to 6PGD+/+ treatments.
[0043] FIG. 2A-E are a series of flow cytometry images showing that 6PGD deficiency induces an activated T cell phenotype in thymus (FIG. 2A), spleen (FIG. 2B and 2D), and lymph nodes (FIG. 2C and 2E).
[0044] FIG. 3A-B are a pair of flow cytometry images showing that 6PGD deficiency in T cells induces a memory phenotype in 20 weeks old (FIG. 3A) and 18 weeks old (FIG. 3B).
[0045] FIG. 4A-I show that 6PGD7- CD8+ T cells display enhanced TEFF function. IFN-g production as determined by intracellular staining and by ELISA of culture supernatants are shown in Fig. 4A and B. Compared to OTI/6PGD+/+ control, OTI/6PGD7- CD8+ T cells showed higher CTL function (Fig. 4C). Analysis of the activated CD8+ T cells for expression of genes with cytolytic function showed higher expression in cytotoxic profile genes including Granzyme family ( Gzma , Gzmb, Gzmc, Gzmd, Gzme) and the cell death-inducing factor Fasl (Fig. 4D). mRNA for IFN-g and the transcription factor Tbx21 (encoding for T-bet) were also elevated in 6PGD7- cells as determined by qPCR (Fig. 4D) and intracellular staining (Fig. 4H). Higher expression of granzyme B (Fig. 4E) and Fas/FasL (Fig. 4F and 4G) on CD8+ 6PGD7- T cells, which are consistent with their enhanced cytolytic function.
[0046] FIG. 5A-C show that 6-Aminonicotinamide (6-AN), a small molecule inhibitor of 6PGD phenocopies 6PGD7- results in vitro. Blocking of 6PGD in T cells resulted in significant increase of IFN-y production (Fig. 5A), enhanced expression of granzyme B (Fig. 5B) and enhanced CTL function as determined by co-culture of 6-AN-treated OTI cells with EG7 targets (Fig. 5D). 6-AN-treated CD8 cells also displayed higher fatty acid uptake (Fig. 5C), a hallmark of TEFF cells.
[0047] FIG. 6A is a microarray image showing that 6PGD7- CD8+ T cells have a robust gene expression signature of TEFF. FIG. 6B is a microarray image showing that the Th2 gene signature predominates in 6PGD7- YFP+/+ T-regulatory cells (Tregs). FIG. 6C shows a Real-Time PCR validation assay. [0048] FIG. 7 is a microarray image showing chromatin accessibility of the 6PGD7- and 6PGD+/+ CD8+ T cells genome by ATAC-Sequencing without any stimulation.
[0049] FIG. 8A-D are a series of histograms and bar graphs showing altered utilization of glucose in 6PGD7- T cells. FIG. 8A and 8C show glucose uptake and FIG. 8B and 8D shows Glutl expression.
[0050] FIG. 9A-J show that deficiency in 6PGD7-T cells reprograms metabolic circuits. PGD7- T altered the profile of glycolysis intermediates in response to stimulation with phosphoenolpyruvate (PEP) (FIG. 9D) being higher and pyruvate (FIG. 9F) lower compared to 6PGD+/+ cells. Although activated 6PGD7- and 6PGD+/+ T cells contained comparable levels of malate (FIG. 9G) and fumarate (FIG. 91 ), 6PGD7- T cells had significantly higher levels of citrate compared to 6PGD+/+ control.
[0051] FIG. 10A-F show that 6PGD7-T cells demonstrate higher mitochondrial activity that supports anti tumor function. Analysis of mitochondrial function demonstrated that CD8+ T cells from 6PGD7- mice develop significantly higher mitochondrial mass, membrane potential (DYiti) and production of reactive oxygen species (ROS) compared to 6PGD+/+ cells (Fig 10A-C). 6PGD7 T cells displayed higher oxygen consumption rate (OCR) at base line, a robust increase of mitochondrial metabolism in response to mitostress and elevated spare respiratory capacity (SRC) (Fig. 10D-F).
[0052] FIG. 11A-E shows that Glucose-Pyruvate-Acetyl-CoA axis modulates increased histone acetylation in 6PGD7 CD8+ T cells. Elevated Acetyl-CoA levels are shown in Fig. 11 D and E. 6PGD7 had H3K9/K27 acetylation, which was inhibited by the HK2 inhibitor 2DG and by the ACLY inhibitor BMS303141 (Fig. 11B and 11C).
[0053] FIG. 12A-B show the effective response of 6PGD7- CD8+ T cells to Listeria m. infection in vivo. 6PGD7- cells were able to clear the Lm-Ova more effectively than control and Ag-specific T cells showed higher capacity in TNF-a production and expression of maturation markers (Fig. 12A). Recipients of OTI/6PGD7- cells, displayed higher rate of bacterial clearance compared to recipients of OTI/6PGD+/+ cells (Fig. 12B) and Ag specific OTI/6PGD7- cells also showed higher IFN-g production.
[0054] FIG. 13A-C shows that 6PGD7 T cells have potent anti-tumor activity which can be recapitulated by and 6PGD small molecule inhibitor 6-Aminonicotinamide (6-AN) in vivo. Compared to OTI/6PGD+/+ CD8+ T cells, OTI/6PGD7- CD8+ T cells were more efficient in reducing tumor growth (Fig. 13A) and expressed higher levels of granzyme B in the tumor environment (Fig. 13B).
[0055] FIG. 14A-B shows 6PGD7- TIL are resistant to metabolic exhaustion and tumor microenvironment-induced mitochondrial dysfunction. 6PGD7- mice had significantly smaller tumors compared to 6PGD+/+ control group (Fig. 14A) and Recipients of pmel-1/6PGD7- T cells had significantly smaller tumors compared to recipients of pmel-1/6PGD+/+ T cells (Fig. 14B). [0056] FIG. 15A-D shows how 6PGD deficiency alters differentiation of naive CD4+TH subset Cells. IFNgamma expression was significantly elevated in 6PGD7- compared to control CD4+ T cells (Fig. 15A). increased T-bet expression in 6PGD7- CD4+ T cells is shown in Fig. 15B. an increased number of IL-17A+ RORyt+ CD4+ T cells in 6PGD7- mice compared to control (Fig. 15C and D).
[0057] FIG. 16 shows a high number of 6PGD7- T mice developed rectal prolapse between 4 to 5 months of age, strongly indicative of colitis.
[0058] FIG. 17 shows iodoacetate (IAA), which targets glyceraldehyde phosphate dehydrogenase (GAPDFI) and oxalate (OXA) which targets pyruvate kinase (PK).
[0059] FIG. 18A-H are a series of flow cytometry images showing that 6PGD7- T cells not only display activated phenotype and elevated expression of PD-1 but lack expression of exhaustion markers in the Spleen for PD-1 (Fig. 18A), Tim-3 (Fig. 18B), LAG-3 (Fig. 18C) and CTLA-4 (Fig. 18D) and in the Lypm node, for PD-1 (Fig. 18E), Tim-3 (Fig. 18F), LAG-3 (Fig. 18G) and CTLA-4 (Fig. 18H).
[0060] FIG. 19A-B are a series of flow cytometry images showing Bone Marrow Myeloid Derived Stem Cells (MDSCs) in the presence of 6AN vs DMSO.
[0061] FIG. 20A-B are a series of histograms showing bone marrow derived MDSCs suppression assay following 6AN and DMSO treatment. MDSCs derived in presence of DMSO from bone marrow are more suppressive than 6-AN derived as measured by their effect on T cell proliferation, indicating that 6-AN reduces suppressive activity of MDSCs.
[0062] FIG. 21A-B are a series of immunohistochemical images comparing 6PGDf/f-FoxP3cre (FIG. 21 A) and 6PGDf/f-FoxP3WT (FIG. 21 B) in lung, pancreas and skin cells.
[0063] FIG. 22A-B are a series of immunohistochemical images comparing 6PGDf/f-FoxP3cre (FIG. 22A) and 6PGDf/f-FoxP3WT (FIG. 22B) in heart, intestine and liver cells.
[0064] FIG. 23A-B are graphs of a Treg in vitro suppression assay showing that 6PGD7- deficient T- regulatory cells lack in vitro suppressive activity.
[0065] FIG. 24A-D shows how 6PGD7- deficient T-regulatory cells abrogate in vivo suppressive activity. FIG. 24A shows a schematic of the Rag17- Colitis model. FIG. 24B shows a immunohistochemical image of hematoxylin and eosin staining of Rag17- mice. FIG. 24C shows the colon of Rag17- mice under different conditions.
[0066] FIG. 25A-D shows how 6-AN treatment drives lineage specific transcription factors. FIG. 25A is a schematic showing how 6-AN treatment drives lineage specific transcription factors in Treg and Th2 cells. FIG. 25B and FIG. 25C show isolated Tregs (YFP+) from 6PGDfl/fl FoxP3Cre mice having lower suppressive activity in a suppression assay. FIG. 25D is a series of flow cytometry images showing isolated Tregs in the presence of 6-AN vs DMSO.
[0067] FIG. 26A-M shows deletion of 6PGD in Tregs results in early onset fatal autoimmune disorder. FIG. 26A and FIG. 26B show expresion of 6PGD mRNA and protein in YFP+ cells that were sorted from 6PGD+/+ FoxP3Cre (WT) and 6PGDfl/fl FoxP3Cre mice. FIG. 26C is a representative image of 21 days old WT and 6PGDfl/fl FoxP3Cre mice. FIG. 26D is a representative image of lymphadenopathy in 6PGDfl/fl FoxP3Cre compared to WT mice. FIG. 26E shows absolute number of T cells per spleen and pLNs in WT and 6PGDfl/fl FoxP3Cre mice. Results are representative of 12 mice per group. FIG. 26F shows a survival curve of WT and 6PGDfl/fl FoxP3Cre mice. Results are representative of 27 mice per group. FIG. 26G show splenocytes from 19 days old WT and 6PGDfl/fl FoxP3Cre mice that were harvested, and distribution of CD4+ vs CD8+ T cells were evaluated. FIG. 26H shows that deletion of 6PGD induces enhanced effector phenotype (CD44hi9h CD62L|0W) both in CD4+ T cells (top panel) and CD8+ T cells (bottom panel) in 19 days old 6PGDfl/fl FoxP3Cre compared to WT mice. FIG. 26I shows both CD4+ and CD8+ T cells that have elevated expression of CD69 activation marker in 6PGDfl/fl FoxP3Cre compared to WT mice. FIG. 26J shows splenocytes from 19 days old WT and 6PGDfl/fl FoxP3Cre mice that were stimulated with PMA (50 ng/ml)/lonomycin (1 pg/ml) plus Golgiplug (1 mI/ml) for 4 hours and expression of IFN-g was evaluated by flow cytometry. FIG. 26K shows granzyme B (top panel) and CD107a degranulation activation marker (bottom panel) expression was assessed on splenocytes of 19 days old WT and 6PGDfl/fl FoxP3Cre mice by flow cytometry. FIG. 26L and FIG. 26M shows serum collected from 20 days old WT and 6PGDfl/fl FoxP3Cre mice and levels of serum antibodies (FIG. 26L), and IFN-g, IL-17A, IL-4 and IL-5 (FIG. 26M) was detected . Results are representative of 12 mice per group.
[0068] FIG. 27A shows YFP+ cells that were isolated from 20 day old 6PGD+/+ FoxP3Cre (WT) and 6PGDfl/fl FoxP3Cre mice. FoxP3 mRNA levels were evaluated by real-time PCR. FIG. 27B shows isolated Tregs (YFP+) that were cultured in vitro in the presence of IL-2 (700 lU/ml) and anti-CD3/anti-CD28 coated beads (Treg:beads ratio 1 :3), and cell numbers were assessed at 24 and 48 hour time points. Results are representative of three independent experiments.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0069] The present disclosure is based at least in part, on the surprising discovery that compounds that inhibit 6 Phosphogluconate dehydrogenase (6PGD) in a manner similar to 6-Aminonicotinamide (6AN) are able to activate T cell that can be used in cellular therapy. Moreover, immune modulation of T cells results in enhanced effector phenotype and function, with especial effect on PD-1 /PD-L1 pathway. These findings have potential clinical applicability as pharmacologic inhibition of 6PGD with a small molecule inhibitor recapitulates the metabolic, immunologic and functional features of 6PGD deficient T cells.
[0070] In one aspect, the disclosure provides a method for modulating an immune response, comprising administering a compound of Formula (I) or a pharmaceutically acceptable salt, hydrate or solvate thereof to a subject in need thereof:
Figure imgf000012_0001
wherein: R1, R2, R3 R4 and R5 as defined above.
[0071] In some embodiments, the compound is Formula (II) or a pharmaceutically acceptable salt, hydrate or solvate thereof:
Figure imgf000012_0002
(II).
[0072] In one aspect, the disclosure provides a method for treating or preventing cancer, comprising administering a compound of Formula (I) to a subject in need thereof:
Figure imgf000012_0003
wherein: R1, R2, R3 R4 and R5 as defined above.
[0073] In one aspect, the disclosure provides a method for treating or preventing an infection, comprising administering a compound of Formula (I) to a subject in need thereof:
Figure imgf000013_0001
[0074] wherein: R1, R2, R3 R4 and R5 as defined above.
[0075] In one aspect, the disclosure provides a method of making an immunomodulatory cancer treatment, comprising: (a) identifying an immunomodulatory anti-cancer agent by: (i) determining whether the agent binds to or interacts with 6-phosphogluconate dehydrogenase (6PGD); and (ii) classifying the agent as immunomodulatory based on an ability to bind to or interact with 6PGD; and (b) formulating the agent for cancer treatment.
[0076] Cellular metabolism has a key role in T cell differentiation. The success of antitumor responses via adoptive T cell-based therapies not only requires effective cytolytic function but also the ability of T cells to persist as long-term memory cells. Recent data suggest that metabolic reprogramming drives the divergence and regulates T effector (TEFF) and memory (TM) cell differentiation and can be critical to effective immune therapy. Glycolysis and PPP are two key metabolic routes that utilize glucose to fulfill the bioenergetics and biomass requirements of a cell. Although recent studies suggest that accumulation of certain glycolytic intermediates can drive TEFF, there is very sparse knowledge about how alteration in glucose utilization between glycolysis and PPP impacts T cell differentiation. Understanding these events provide an ideal approach to improve the efficacy of T cell-based cancer immunotherapy by exploiting the plasticity of metabolic re-wiring of glucose metabolism. PPP consists of two branches, (i) the largely irreversible NADPH-producing oxidative branch starting from glucose-6-phosphate (G6P) that produces ribulose-5-phosphate (Rib-5P) or (ii) the reversible non-oxidative branch that converts glyceraldehyde-3- phosphate (GAP) and fructose-6-phosphate (F6P) to Rib-5P and Xyl-5-P. Blocking PPP enhances glucose utilization resulting in robust increase in mitochondrial potential that overrides tumor microenvironment-induced exhaustion. 6PGD is upregulated in human cancers and inactivation of 6PGD in tumor cells results in decreased tumorigenesis. Upregulation of 6PGD activity is a mechanism of tumor resistance. There is a potential clinical applicability for a small molecule inhibitor such as the compounds described herein which can recapitulate the metabolic and functional features of 6PGD deficiency resulting in potent antigen-specific CD4+ and CD8+ cells.
Compounds of the disclosure
[0077] 6-PGD small molecule inhibitors include one or more compounds that inhibit one or more proteins upstream of 6-PGD in the PPP, such as a G6PD inhibitor, a 6-phosphogluconolactonase; one or more compounds that inhibit one or more proteins downstream of 6-PGD in the PPP. An exemplary 6-PGD small molecule inhibitor further includes glucose 1 ,6-diphosphate.
[0078] 6-PGD antagonists include one or more agents or compounds that directly or indirectly inhibit 6- PGD gene expression, protein expression, or enzymatic activity. Exemplary 6-PGD antagonists include an, an anti- 6-PGD antibody, and small molecule inhibitors. Additional 6-PGD antagonists can be identified by any useful method, such as by inhibiting or activating one or more proteins upstream of 6- PGD in the PPP that results in 6-PGD inhibition.
[0079] In one aspect, the disclosure provides a method for modulating an immune response, comprising administering a compound of Formula (I) or a pharmaceutically acceptable salt, hydrate or solvate thereof to a subject in need thereof:
Figure imgf000014_0001
wherein: R1, R2, R3 R4 and R5 each is independently selected from the group consisting of H, halo, (C1- C6)alkyl, (C1 -C6)alkoxy, (C3-C7)cycloalkyl, -C(=0)0R6, -C(0)N(R6)2, -N(R¾ (C6-C10)aryl, and (C3- C10) heteroaryl, each alkyl, alkoxy, cycloalkyl, aryl and heteroaryl optionally substituted with one or more groups selected from hydroxyl, halo, and (C1 -C6)alkyl; each R6 is independently H or (C1-C6)alkyl.
[0080] In some embodiments, the compound is Formula (II) or a pharmaceutically acceptable salt, hydrate or solvate thereof.
[0081] In some embodiments, the compound binds to or interacts with 6PGD. In some embodiments, the binds to or interacts with 6PGD comprises Polydatin (3,4',5-trihydroxystilbene-3- -d-glucoside; trans- resveratrol 3- -mono-D-glucoside; piceid), Physcion (1 ,8-Dihydroxy-3-methoxy-6-methylanthraquinone, Emodin-3-methyl ether).
[0082] In some embodiments, the modulation of the immune response comprises stimulating an increase in levels or activity of an interferon as compared to levels without administration of the compound.
[0083] In some embodiments, the interferon is selected from IFNa, IFN , and IFNy.
[0084] In some embodiments, the modulation of the immune response is within the tumor microenvironment. [0085] In some embodiments, the modulation of the immune response is a reduction or suppression of an immune inhibitory cell. In some embodiments, the modulation of the immune response is an increase or enhancing of an immune stimulatory cell. In some embodiments, the immune inhibitory cell is selected from MDSCs, Tregs, FoxP3+ T cells; TANs, M2 macrophages, and TAMs. In some embodiments, the immune stimulatory cell is selected from T cells, cytotoxic T lymphocytes, T helper cells, NK cells, NKT cells, B cells, and dendritic cells. In some embodiments, the modulation of the immune response increases a ratio of immune stimulatory cells to immune inhibitory cells. In some embodiments, the modulation of the immune response increases a ratio cytotoxic T cells (Tc) to Tregs.
Checkpoint Blockade / Blockage of Tumor Immunosuppression
[0086] Some human tumors can be eliminated by a patient's immune system. For example, administration of a monoclonal antibody targeted to an immune "checkpoint” molecule can lead to complete response and tumor remission. A mode of action of such antibodies is through inhibition of an immune regulatory molecule that the tumors have co-opted as protection from an anti-tumor immune response. By inhibiting these "checkpoint” molecules {e.g., with an antagonistic antibody), a patient's CD8+ T cells may be allowed to proliferate and destroy tumor cells. For example, administration of a monoclonal antibody targeted to by way of example, without limitation, PD-1 can lead to complete response and tumor remission. The mode of action of such antibodies is through inhibition of PD-1 that the tumors have co-opted as protection from an anti-tumor immune response. By inhibiting these "checkpoint” molecules {e.g., with an antagonistic antibody), a patient's CD8+ T cells may be allowed to proliferate and destroy tumor cells.
[0087] The compounds described herein can be used in combination with one or more blocking antibodies targeted to an immune "checkpoint” molecule. For instance, in some embodiments, the present compounds provided herein can be used in combination with one or more blocking antibodies targeted to a molecule such as CTLA-4 or PD-1 . For example, the present compounds provided herein may be used in combination with an agent that blocks, reduces and/or inhibits PD-1 and PD-L1 or PD- L2 and/or the binding of PD-1 with PD-L1 or PD-L2 (by way of non-limiting example, one or more of nivolumab (ONO-4538/BMS-936558, MDX1 106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, Merck), pidilizumab (CT-011 , CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), MPDL3280A (ROCHE)). In an embodiment, the compounds provided herein may be used in combination with an agent that blocks, reduces and/or inhibits the activity of CTLA-4 and/or the binding of CTLA-4 with one or more receptors (e.g., CD80, CD86, AP2M1 , SHP-2, and PPP2R5A). For instance, in some embodiments, the immune-modulating agent is an antibody such as, by way of non- limitation, ipilimumab (MDX-010, M DX- 101 , Yervoy, BMS) and/or tremelimumab (Pfizer). Blocking antibodies against these molecules can be obtained from, for example, Bristol Myers Squibb (New York, NY), Merck (Kenilworth, NJ), Medlmmune (Gaithersburg, MD), and Pfizer (New York, NY).
[0088] Further, the compounds provided herein can be used in combination with one or more blocking antibodies targeted to an immune "checkpoint” molecule such as for example, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160 (also referred to as BY55), CGEN-15049, CHK 1 and CHK2 kinases, A2aR, CEACAM (e.g., CEACAM-1 , CEACAM-3 and/or CEACAM-5), GITR, GITRL, galectin-9, CD244, CD160, TIGIT, SIRPa, ICOS, CD172a, and TMIGD2 and various B-7 family ligands (including, but are not limited to, B7-1 , B7-2, B7-DC, B7-H1 , B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7), LAG-3 and Tim-3.
In some embodiments, the subject is undergoing treatment with one or more immunotherapies. In some embodiments, the immunotherapy is an agent that modulates one or more PD-1 , PD-L1 , PD-L2, CTLA- 4, Tim-3, or LAG-3.
In some embodiments, the method further comprises administering an agent that modulates one or more PD-1 , PD-L1 , PD-L2, CTLA-4, Tim-3, or LAG-3.
[0089] In some embodiments, the agent that modulates PD-1 is an antibody or antibody format specific for PD-1. In some embodiments, the antibody or antibody format specific for PD-1 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody. In some embodiments, the antibody or antibody format specific for PD-1 is selected from nivolumab, pembrolizumab, and pidilizumab.
[0090] In some embodiments, the agent that modulates PD-L1 is an antibody or antibody format specific for PD-L1. In some embodiments, the antibody or antibody format specific for PD-L1 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody. In some embodiments, the antibody or antibody format specific for PD-L1 is selected from atezolizumab, avelumab, durvalumab, and BMS-936559.
[0091] In some embodiments, the agent that modulates PD-L2 is an antibody or antibody format specific for PD-L2. In some embodiments, the antibody or antibody format specific for PD-L2 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.
[0092] In some embodiments, the agent that modulates CTLA-4 is an antibody or antibody format specific for CTLA-4. In some embodiments, the antibody or antibody format specific for CTLA-4 is selected from ipilimumab (YERVOY), tremelimumab, AGEN1884, and RG2077.
Method of Treatment
[0093] In one aspect, the disclosure provides a method for treating or preventing cancer, comprising administering a compound of Formula (I) or (II) as described herein.
[0094] The compounds of the present disclosure can be used in administration to a subject {e.g., a research animal or a mammal, such as a human, having a clinical condition such as cancer or an infection). For example, the compounds described herein can be administered to a subject for the treatment of cancer or infection. Thus, this document provides methods for treating clinical conditions such as cancer or infection with the expression vectors provided herein.
[0095] In some embodiments, the present disclosure pertains to cancers and/or tumors; for example, the treatment or prevention of cancers and/or tumors. Cancers or tumors refer to an uncontrolled growth of cells and/or abnormal increased cell survival and/or inhibition of apoptosis which interferes with the normal functioning of the bodily organs and systems. Included are benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases. Also, included are cells having abnormal proliferation that is not impeded by the immune system {e.g., virus infected cells). The cancer may be a primary cancer or a metastatic cancer. The primary cancer may be an area of cancer cells at an originating site that becomes clinically detectable, and may be a primary tumor. In contrast, the metastatic cancer may be the spread of a disease from one organ or part to another non-adjacent organ or part. The metastatic cancer may be caused by a cancer cell that acquires the ability to penetrate and infiltrate surrounding normal tissues in a local area, forming a new tumor, which may be a local metastasis. The cancer may also be caused by a cancer cell that acquires the ability to penetrate the walls of lymphatic and/or blood vessels, after which the cancer cell is able to circulate through the bloodstream (thereby being a circulating tumor cell) to other sites and tissues in the body. The cancer may be due to a process such as lymphatic or hematogeneous spread. The cancer may also be caused by a tumor cell that comes to rest at another site, re-penetrates through the vessel or walls, continues to multiply, and eventually forms another clinically detectable tumor. The cancer may be this new tumor, which may be a metastatic (or secondary) tumor. [0096] The cancer may be caused by tumor cells that have metastasized, which may be a secondary or metastatic tumor. The cells of the tumor may be like those in the original tumor. As an example, if a breast cancer or colon cancer metastasizes to the liver, the secondary tumor, while present in the liver, is made up of abnormal breast or colon cells, not of abnormal liver cells. The tumor in the liver may thus be a metastatic breast cancer or a metastatic colon cancer, not liver cancer.
[0097] The cancer may have an origin from any tissue. The cancer may originate from melanoma, colon, breast, or prostate, and thus may be made up of cells that were originally skin, colon, breast, or prostate, respectively. The cancer may also be a hematological malignancy, which may be lymphoma. The cancer may invade a tissue such as liver, lung, bladder, or intestinal.
[0098] Representative cancers and/or tumors of the present invention include, but are not limited to, a basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer {e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.
Infections [0099] In one aspect, the disclosure provides a method for treating or preventing an infection, comprising administering a compound of Formula (I) or Formula (II).
[00100] In some embodiments the compounds of the disclosure are used to treat one or more infections. In some embodiments, the infection is a microbial infection and/or chronic infection. In some embodiments, the infection is selected from Chagas disease, HIV/AIDS, tuberculosis, osteomyelitis, hepatitis B, hepatitis C, Epstein-Barr virus or parvovirus, T cell leukemia virus, bacterial overgrowth syndrome, fungal and parasitic infections.
[00101] In some embodiments, the present compounds are used in methods of treating viral infections (including, for example, HIV and HCV), parasitic infections (including, for example, malaria), and bacterial infections. In various embodiments, the infections induce immunosuppression. For example, FI IV infections often result in immunosuppression in the infected subjects. Accordingly, as described elsewhere herein, the treatment of such infections may involve, in various embodiments, modulating the immune system with the present compounds to favor immune stimulation over immune inhibition. Alternatively, the present invention provides methods for treating infections that induce immunoactivation. For example, intestinal helminth infections have been associated with chronic immune activation. In these embodiments, the treatment of such infections may involve modulating the immune system with the present compounds to favor immune inhibition over immune stimulation.
[00102] In some embodiments, the present disclosure provides methods of treating viral infections including, without limitation, acute or chronic viral infections, for example, of the respiratory tract, of papilloma virus infections, of herpes simplex virus (HSV) infection, of human immunodeficiency virus (HIV) infection, and of viral infection of internal organs such as infection with hepatitis viruses. In some embodiments, the viral infection is caused by a virus of family Flaviviridae. In some embodiments, the virus of family Flaviviridae is selected from Yellow Fever Virus, West Nile virus, Dengue virus, Japanese Encephalitis Virus, St. Louis Encephalitis Virus, and Hepatitis C Virus. In other embodiments, the viral infection is caused by a virus of family Picornaviridae, e.g., poliovirus, rhinovirus, coxsackievirus. In other embodiments, the viral infection is caused by a member of Orthomyxoviridae, e.g., an influenza virus. In other embodiments, the viral infection is caused by a member of Retroviridae, e.g., a lentivirus. In other embodiments, the viral infection is caused by a member of Paramyxoviridae, e.g., respiratory syncytial virus, a human parainfluenza virus, rubulavirus {e.g., mumps virus), measles virus, and human metapneumovirus. In other embodiments, the viral infection is caused by a member of Bunyaviridae, e.g., hantavirus. In other embodiments, the viral infection is caused by a member of Reoviridae, e.g., a rotavirus. [00103] In some embodiments, the method is in combination with an anti-infective agent. In some embodiments, the anti-infective agent is an anti-viral agent including, but not limited to, abacavir, acyclovir, adefovir, amprenavir, atazanavir, cidofovir, darunavir, delavirdine, didanosine, docosanol, efavirenz, elvitegravir, emtricitabine, enfuvirtide, etravirine, famciclovir, and foscarnet.
[00104] In some embodiments, the anti-infective agent is an anti-bacterial agent selected from cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics (cipro, Levaquin, floxin, tequin, avelox, and norflox); tetracycline antibiotics (tetracycline, minocycline, oxytetracycline, and doxycycline); penicillin antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, and methicillin); monobactam antibiotics (aztreonam); and carbapenem antibiotics (ertapenem, doripenem, imipenem/cilastatin, and meropenem). In some embodiments, the anti-infectives include anti-malarial agents {e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline, artemether/lumefantrine, atovaquone/proguanil and sulfadoxine/pyrimethamine), metronidazole, tinidazole, ivermectin, pyrantel pamoate, and albendazole.
Combination Therapies
[00105] In some embodiments, any compound and/or antibody or antibody format specific directed to immune checkpoint molecules used in methods of the present disclosure disclosed herein acts synergistically when co-administered with another agent and is administered at doses that are lower than the doses commonly employed when such agents are used as monotherapy.
[00106] In some embodiments, the subject in need of a cancer treatment comprising any compound and/or antibody or antibody format specific directed to immune checkpoint molecules used in methods of the present disclosure, as disclosed herein, has been treated with, is contemporaneously treated with, or is subsequently treated with another anti-cancer therapy, as disclosed herein. In some embodiments, the anti-cancer therapy may comprise radiotherapy. In some embodiments, the anti-cancer therapy may include a synthetic polypeptide comprising at least one domain capable of binding an immune checkpoint molecule. In embodiments, the immune checkpoint molecule is selected from PD-1 , PD-L1 , PD-L2, CTLA-4, Tim-3, or LAG-3. In some embodiments, the anti-cancer therapy may be surgery to excise the cancer, i.e., tumor. In some embodiments, the anti-cancer therapy may include administration of one more chemotherapeutic agents. In some embodiments, the disclosure provides for methods that further comprise administering an additional agent to a subject. In some embodiments, the disclosure pertains to co-administration and/or co-formulation.
[00107] In some embodiments, the method increases a therapeutic window of the immune checkpoint immunotherapy. In some embodiments, the method elicits a potent immune response in less- immunogenic tumors. In some embodiments, the method converts a tumor with reduced inflammation ("cold tumor”) to a responsive, inflamed tumor ("hot tumor”).
[00108] In some embodiments, the method makes the cancer responsive or more responsive to a combination therapy of the immune checkpoint immunotherapy and one or more chemotherapeutic agents and/or radiotherapy.
[00109] In some embodiments, inclusive of, without limitation, cancer applications, the present invention pertains to chemotherapeutic agents as additional agents. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and CYTOXAN cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins {e.g., bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins {e.g., cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB 1 -TM1 ); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem. Inti. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxu ridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as minoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2- ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylamine; trichothecenes {e.g., T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), and TAXOTERE doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE. vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (TYKERB); inhibitors of PKC-a, Raf, H-Ras, EGFR {e.g., erlotinib (Tarceva)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above. In addition, the methods of treatment can further include the use of radiation. In addition, the methods of treatment can further include the use of photodynamic therapy.
Pharmaceutical composition
[00110] This present disclosure therefore also provides compositions containing any compound and/or antibody or antibody format specific directed to immune checkpoint molecules described herein, in combination with a physiologically and pharmaceutically acceptable carrier. The physiologically and pharmaceutically acceptable carrier can include any of the well-known components useful for immunization. The carrier can facilitate or enhance an immune response to an antigen administered in a vaccine. The cell formulations can contain buffers to maintain a preferred pH range, salts or other components that present an antigen to an individual in a composition that stimulates an immune response to the antigen. The physiologically acceptable carrier also can contain one or more adjuvants that enhance the immune response to an antigen. Pharmaceutically acceptable carriers include, for example, pharmaceutically acceptable solvents, suspending agents, or any other pharmacologically inert vehicles for delivering compounds to a subject. Pharmaceutically acceptable carriers can be liquid or solid, and can be selected with the planned manner of administration in mind so as to provide for the desired bulk, consistency, and other pertinent transport and chemical properties, when combined with one or more therapeutic compounds and any other components of a given pharmaceutical composition. Typical pharmaceutically acceptable carriers include, without limitation: water, saline solution, binding agents {e.g., polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers {e.g., lactose or dextrose and other sugars, gelatin, or calcium sulfate), lubricants {e.g., starch, polyethylene glycol, or sodium acetate), disintegrates (e.g., starch or sodium starch glycolate), and wetting agents (e.g., sodium lauryl sulfate). Compositions can be formulated for subcutaneous, intramuscular, or intradermal administration, or in any manner acceptable for immunization. In some embodiments, the compositions, e.g., pharmaceutical compositions, disclosed herein are resuspended in a saline buffer (including, without limitation TBS, PBS, and the like).
[00111] An adjuvant refers to a substance which, when added to an immunogenic agent nonspecifically enhances or potentiates an immune response to the agent in the recipient host upon exposure to the mixture. Adjuvants can include, for example, oil-in-water emulsions, water-in oil emulsions, alum (aluminum salts), liposomes and microparticles, such as, polysytrene, starch, polyphosphazene and polylactide/polyglycosides.
[00112] In some embodiments, the compounds disclosed herein are in the form of a pharmaceutically acceptable salt.
[00113] Adjuvants can also include, for example, squalene mixtures (SAF-I), muramyl peptide, saponin derivatives, mycobacterium cell wall preparations, monophosphoryl lipid A, mycolic acid derivatives, nonionic block copolymer surfactants, Quil A, cholera toxin B subunit, polyphosphazene and derivatives, and immunostimulating complexes (ISCOMs) such as those described by Takahashi et al., Nature 1990, 344:873-875. For veterinary use and for production of antibodies in animals, mitogenic components of Freund's adjuvant (both complete and incomplete) can be used. In humans, Incomplete Freund's Adjuvant (IFA) is a useful adjuvant. Various appropriate adjuvants are well known in the art (see, for example, Warren and Chedid, CRC Critical Reviews in Immunology 1988, 8:83; and Allison and Byars, in Vaccines: New Approaches to Immunological Problems, 1992, Ellis, ed., Butterworth-Heinemann, Boston). Additional adjuvants include, for example, bacille Calmett-Guerin (BCG), DETOX (containing cell wall skeleton of Mycobacterium phlei (CWS) and monophosphoryl lipid A from Salmonella minnesota (MPL)), and the like (see, for example, Hoover et al., J Clin Oncol 1993, 11 :390; and Woodlock et al., J Immunother 1999, 22:251-259).
Administration, Dosing, and Treatment Regimens
[00114] Routes of administration include, for example: intradermal, intratumoral, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin. In some embodiments, the administration is by intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or direct injection into a cancer tissue.
[00115] In some embodiments, the compounds described herein can be administered to a subject one or more times {e.g., once, twice, two to four times, three to five times, five to eight times, six to ten times, eight to 12 times, or more than 12 times). The compound as provided herein can be administered one or more times per day, one or more times per week, every other week, one or more times per month, once every two to three months, once every three to six months, or once every six to 12 months. The compound can be administered over any suitable period of time, such as a period from about 1 day to about 12 months. In some embodiments, for example, the period of administration can be from about 1 day to 90 days; from about 1 day to 60 days; from about 1 day to 30 days; from about 1 day to 20 days; from about 1 day to 10 days; from about 1 day to 7 days. In some embodiments, the period of administration can be from about 1 week to 50 weeks; from about 1 week to 50 weeks; from about 1 week to 40 weeks; from about 1 week to 30 weeks; from about 1 week to 24 weeks; from about 1 week to 20 weeks; from about 1 week to 16 weeks; from about 1 week to 12 weeks; from about 1 week to 8 weeks; from about 1 week to 4 weeks; from about 1 week to 3 weeks; from about 1 week to 2 weeks; from about 2 weeks to 3 weeks; from about 2 weeks to 4 weeks; from about 2 weeks to 6 weeks; from about 2 weeks to 8 weeks; from about 3 weeks to 8 weeks; from about 3 weeks to 12 weeks; or from about 4 weeks to 20 weeks.
[00116] In specific embodiments, it may be desirable to administer locally to the area in need of treatment. In embodiments, for instance in the treatment of cancer, the antibodies directed to immune checkpoint molecules and/or the compounds used in methods of the present invention (and/or additional agents) are administered in the tumor microenvironment {e.g., cells, molecules, extracellular matrix and/or blood vessels that surround and/or feed a tumor cell, inclusive of, for example, tumor vasculature; tumor-infiltrating lymphocytes; fibroblast reticular cells; endothelial progenitor cells (EPC); cancer- associated fibroblasts; pericytes; other stromal cells; components of the extracellular matrix (ECM); dendritic cells; antigen presenting cells; T-cells; regulatory T cells; macrophages; neutrophils; and other immune cells located proximal to a tumor) or lymph node and/or targeted to the tumor microenvironment or lymph node. In embodiments, for instance in the treatment of cancer, the antibodies directed to immune checkpoint molecules and/or the compounds used in methods of the present disclosure (and/or additional agents) are administered intratumorally.
[00117] Dosage forms suitable for parenteral administration {e.g., intravenous, intramuscular, intraperitoneal, subcutaneous and intra-articular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions {e.g., lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art.
[00118] The dosage of any antibody or antibody format specific directed to immune checkpoint molecules and/or compounds used in methods of the present disclosure (and/or additional agents) disclosed herein as well as the dosing schedule can depend on various parameters, including, but not limited to, the disease being treated, the subject's general health, and the administering physician's discretion. Any antibody directed to immune checkpoint molecules and/or compounds used in methods of the present invention, disclosed herein, can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of an additional agent, to a subject in need thereof.
[00119] In embodiments, an antibody or antibody format specific directed to immune checkpoint molecules and/or compounds used in methods of the present disclosure and an additional agent(s) are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 1 1 hours to 12 hours apart, 1 day apart, 2 days apart, 3 days apart, 4 days apart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeks apart, or 4 weeks apart. [00120] The dosage of any antibody or antibody format specific directed to immune checkpoint molecules and/or compounds used in methods of the present disclosure (and/or additional agents) disclosed herein can depend on several factors including the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the subject to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular subject may affect dosage used. Furthermore, the exact individual dosages can be adjusted somewhat depending on a variety of factors, including the specific combination of the agents being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the particular disease being treated, the severity of the disorder, and the anatomical location of the disorder. Some variations in the dosage can be expected.
[00121] For administration of any antibody or antibody format specific directed to immune checkpoint molecules and/or compounds used in methods of the present disclosure (and/or additional agents) disclosed herein by parenteral injection, the dosage may be about 0.1 mg to about 250 mg per day, about 1 mg to about 20 mg per day, or about 3 mg to about 5 mg per day. Generally, when orally or parenterally administered, the dosage of any agent disclosed herein may be about 0.1 mg to about 1500 mg per day, or about 0.5 mg to about 10 mg per day, or about 0.5 mg to about 5 mg per day, or about 200 to about 1 ,200 mg per day {e.g., about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1 ,000 mg, about 1 ,100 mg, about 1 ,200 mg per day).
[00122] In some embodiments, administration of the antibody or antibody format specific directed to immune checkpoint molecules and/or compounds used in methods of the present disclosure (and/or additional agents) disclosed herein is by parenteral injection at a dosage of about 0.1 mg to about 1500 mg per treatment, or about 0.5 mg to about 10 mg per treatment, or about 0.5 mg to about 5 mg per treatment, or about 200 to about 1 ,200 mg per treatment {e.g., about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1 ,000 mg, about 1 ,100 mg, about 1 ,200 mg per treatment).
[00123] In embodiments, a suitable dosage of the antibody or antibody format specific directed to immune checkpoint molecules and/or compounds used in methods of the present disclosure (and/or additional agents) is in a range of about 0.01 mg/kg to about 100 mg/kg of body weight or about 0.01 mg/kg to about 10 mg/kg of body weight of the subject, for example, about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 1.1 mg/kg, about 1 .2 mg/kg, about 1.3 mg/kg, about 1 .4 mg/kg, about 1.5 mg/kg, about 1.6 mg/kg, about 1.7 mg/kg, about 1.8 mg/kg, 1 .9 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg body weight, inclusive of all values and ranges therebetween.
[00124] In some embodiments, the compound of the disclosure is administered at a dose of 10 mg/kg/intraperitoneally twice a day.
[00125] Administration of any antibody or antibody format specific directed to immune checkpoint molecules and/or compounds used in methods of the present disclosure (and/or additional agents) disclosed herein can, independently, be one to four times daily or one to four times per month or one to six times per year or once every two, three, four or five years. Administration can be for the duration of one day or one month, two months, three months, six months, one year, two years, three years, and may even be for the life of the subject.
[00126] In some embodiments, after an initial dose (sometimes referred to as a "priming” dose) of a compound has been administered and a maximal antigen-specific immune response has been achieved, one or more boosting doses of the compound as provided herein can be administered. For example, a boosting dose can be administered about 10 to 30 days, about 15 to 35 days, about 20 to 40 days, about 25 to 45 days, or about 30 to 50 days after a priming dose.
[00127] In some embodiments, the methods provided herein can be used for controlling solid tumor growth {e.g., breast, prostate, melanoma, renal, colon, or cervical tumor growth) and/or metastasis. The methods can include administering an effective amount of a compound as described herein to a subject in need thereof. In some embodiments, the subject is a mammal {e.g., a human).
[00128] The compounds and methods provided herein can be useful for stimulating an immune response against a tumor. Such immune response is useful in treating or alleviating a sign or symptom associated with the tumor. As used herein, by "treating” is meant reducing, preventing, and/or reversing the symptoms in the individual to which a compound as described herein has been administered, as compared to the symptoms of an individual not being treated. A practitioner will appreciate that the methods described herein are to be used in concomitance with continuous clinical evaluations by a skilled practitioner (physician or veterinarian) to determine subsequent therapy. Such evaluations will aid and inform in evaluating whether to increase, reduce, or continue a particular treatment dose, mode of administration, etc. [00129] The methods provided herein can thus be used to treat a tumor, including, for example, a cancer. The methods can be used, for example, to inhibit the growth of a tumor by preventing further tumor growth, by slowing tumor growth, or by causing tumor regression. Thus, the methods can be used, for example, to treat a cancer. It will be understood that the subject to which a compound is administered need not suffer from a specific traumatic state. Indeed, the compound described herein may be administered prophylactically, prior to development of symptoms {e.g., a patient in remission from cancer). The terms "therapeutic” and "therapeutically,” and permutations of these terms, are used to encompass therapeutic, palliative, and prophylactic uses. Thus, as used herein, by "treating or alleviating the symptoms” is meant reducing, preventing, and/or reversing the symptoms of the individual to which a therapeutically effective amount of a composition has been administered, as compared to the symptoms of an individual receiving no such administration.
[00130] As used herein, the terms "effective amount” and "therapeutically effective amount” refer to an amount sufficient to provide the desired therapeutic {e.g., anti-cancer, anti-tumor, or anti-infection) effect in a subject (e.g., a human diagnosed as having cancer or an infection). Anti-tumor and anti-cancer effects include, without limitation, modulation of tumor growth (e.g., tumor growth delay), tumor size, or metastasis, the reduction of toxicity and side effects associated with a particular anti-cancer agent, the amelioration or minimization of the clinical impairment or symptoms of cancer, extending the survival of the subject beyond that which would otherwise be expected in the absence of such treatment, and the prevention of tumor growth in an animal lacking tumor formation prior to administration, i.e., prophylactic administration. In some embodiments, administration of an effective amount of the compound can increase the activation or proliferation of tumor antigen specific T cells in a subject. For example, the activation or proliferation of tumor antigen specific T cells in the subject can be is increased by at least 10 percent (e.g., at least 25 percent, at least 50 percent, or at least 75 percent) as compared to the level of activation or proliferation of tumor antigen specific T cells in the subject prior to the administration.
[00131] Anti-infection effects include, for example, a reduction in the number of infective agents (e.g., viruses or bacteria). When the clinical condition in the subject to be treated is an infection, administration of a compound as provided herein can stimulate the activation or proliferation of pathogenic antigen specific T cells in the subject.
[00132] One of skill will appreciate that an effective amount of a compound may be lowered or increased by fine tuning and/or by administering more than one dose. This disclosure provides a method for tailoring the administration/treatment to the particular exigencies specific to a given mammal. Therapeutically effective amounts can be determined by, for example, starting at relatively low amounts and using step-wise increments with concurrent evaluation of beneficial effects. The methods provided herein thus can be used alone or in combination with other well-known tumor therapies, to treat a patient having a tumor. One skilled in the art will readily understand advantageous uses of the compounds and methods provided herein, for example, in prolonging the life expectancy of a cancer patient and/or improving the quality of life of a cancer patient.
Subjects and/or Animals
[00133] In embodiments, the subject and/or animal is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon. In embodiments, the subject and/or animal is a non-mammal, such, for example, a zebrafish. In embodiments, the subject and/or animal is a human. In embodiments, the human is a pediatric human. In embodiments, the human is an adult human. In embodiments, the human is a geriatric human. In embodiments, the human may be referred to as a patient.
[00134] In certain embodiments, the human has an age in a range of from about 0 months to about 6 months old, from about 6 to about 12 months old, from about 6 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old.
[00135] In embodiments, the subject is a non-human animal, and therefore the invention pertains to veterinary use. In a specific embodiment, the non-human animal is a household pet. In another specific embodiment, the non-human animal is a livestock animal.
[00136] In some embodiments, the subject is predicted to be poorly responsive or non-responsive to the immune checkpoint immunotherapy based on expression of one or more of PD-1 , PD-L1 , or PD-L2, in a patient's biological specimen. In some embodiments, the subject is predicted to be poorly responsive or non-responsive to an agent that modulates one or more of PD-1 , PD-L1 , and PD-L2 based on low on expression of PD-1 , PD-L1 , and PD-L2 in a tumor specimen. In some embodiments, the subject is predicted to be poorly responsive or non-responsive to an agent that modulates one or more of PD-1 , PD-L1 , and PD-L2 tumor proportion score (TPS) of less than about 49% for PD-L1 staining. Kits and Medicaments
[00137] Aspects of the present disclosure provide kits that can simplify the administration of the pharmaceutical compositions and/or compounds disclosed herein.
[00138] An illustrative kit of the disclosure comprises any compound and/or antibody or antibody format specific directed to immune checkpoint molecules used in methods of the present disclosure and/or pharmaceutical composition disclosed herein in unit dosage form. In embodiments, the unit dosage form is a container, such as a pre-filled syringe, which can be sterile, containing any agent disclosed herein and a pharmaceutically acceptable carrier, diluent, excipient, or vehicle. The kit can further comprise a label or printed instructions instructing the use of any agent disclosed herein. The kit may also include a lid speculum, topical anesthetic, and a cleaning agent for the administration location. The kit can also further comprise one or more additional agent disclosed herein. In embodiments, the kit comprises a container containing an effective amount of a composition of the invention and an effective amount of another composition, such those disclosed herein.
[00139] Aspects of the present invention include use of a compound as disclosed herein in the manufacture of a medicament, e.g., a medicament for treatment of cancer and/or treatment of an infection. Any aspect or embodiment disclosed herein can be combined with any other aspect or embodiment as disclosed herein.
EXAMPLES
[00140] In order that the invention disclosed herein may be more efficiently understood, examples are provided below. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the invention in any manner.
Example 1: Immunomodulatory Efects of PPP and Glycolytic Metabolism and their Impact on T Cell Function.
[00141] These studies are based on outcomes of targeting PPP on T cell fate and function. It was found that genetic targeting of 6PGD in T cells reprogrammed glucose utilization leading to altered accumulation of metabolic intermediates and their utilization in the TCA cycle. The metabolic and molecular changes induced by 6PGD targeting were driving differentiation of CD8+ and CD4+ T cells resulting in elevated number of TEFF and generation of hybrid TH1/TH17 respectively. These findings support the hypothesis that identifying checkpoints that can reprogram the interplay between metabolic pathways might regulate cellular differentiation. These studies investigate for the first time how alteration in metabolic diversion of glucose between glycolysis and PPP, as a result of 6PGD deficiency, affects CD8+ T cell function but also drives CD4+ T cells towards Th1/Th17 hybrid phenotype. These studies also highlight that blocking PPP enhances glucose utilization resulting in robust increase in mitochondrial potential that overrides tumor microenvironment-induced exhaustion. These studies also have potential clinical applicability as they demonstrate that a small molecule inhibitor can recapitulate the metabolic and functional features of 6PGD deficiency resulting in potent antigen-specific CD4+ and CD8+ cells. This approach has significant implications for cell-based immunotherapies.
Metabolite Accumulation and Altered Glucose Utilization May Regulate T Cell Differentiation
[00142] To study how targeting PPP -that might alter cells' ability to metabolize glucose- would impact the differen-tiation and function of T cells, 6PGDfl0X/fl0X(6PGD+/+) and 6PGDfl0X/fl0XCD4-Cre (6PGD7-) mice were generated, which allows selective ablation of 6PGD only in T cells. After stimulation via TCR/CD3, 6PGD-/- T cells had significantly reduced levels of lactate but enhanced glucose uptake and elevated phosphoe-nolpyruvate (PEP) as well as altered levels of intermediates of the TCA cycle. 6PGD7- mice had markedly elevated numbers of TEFF cells that rapidly differentiated to central memory (TCM) in the absence of im-munization. 6PGD-/- T cells were able to contain infection with Listeria monocytogenes and reduce tumor burden more efficiently than 6PGD+/+ T cells. These results also showed that increased levels of citrate and acetyl-CoA generated from glucose in 6PGD7- T cells regulated H3K9/K27 acetylation, and event that may dictate the fate of 6PGD7- T cells. Not only 6PGD targeting affected CD8+ T cells but also repro-grammed the differentiation of CD4+T cells resulting in the generation of a Th 1 /Th 17 hybrid population, previously identified to have potent anti-tumor function. Thus, the scientific premise of these studies was to dissect how targeting of 6PGD affects the differentiation and function of T cells. Understanding these events provide an ideal approach to improve the efficacy of T cell-based therapies.
6PGD Deficiency Induces An Activated T Cell Phenotype
[00143] The importance of PPP in T cells function is not clearly understood. To determine how targeting of key enzyme (6PGD) within this pathway would impact on the properties and functions of T cells 6PGDfl/fl mice were crossed with mice expressing Cre recombinase under the control of the CD4 promoter, which allows for deletion in CD4+/CD8+ double positive thymocyte stage and in the subsequent single positive CD4+ and CD8+ T cells. Assessment of CD4+and CD8+T cell populations in thymus, lymph nodes, and spleen showed that there was significant difference between 6PGDfl/fl/CD4-Cre (thereafter named 6PGD7-) and 6PGDfl/fl (thereafter named 6PGD+/+) mice (Fig. 2A-E). Although thymic differentiation was comparable, both CD4+ and CD8+ T cells from 6PGD7- spleens and lymph nodes were reduced compared to littermate 6PGD+/+mice, but demonstrated an altered immune phenotype, characterized by elevated CD44hi,CD62Ll0W and KLRG1 hi,CD127low (IL-7Ra) cells (Fig. 2D and 2E). Moreover, CD4+ or CD8+ 6PGD7- T cells demonstrated significantly increased expression of CD69 and CD122 activation markers compared to 6PGD+/+ T cells in both spleen (Fig. 2D) and lymph nodes (Fig. 2E). CD69 is one of the most characterized activation markers for T cells. Enhanced CD122 (IL2Rb) expression is not only present on activated T cells but a key marker related to longevity and responsiveness to memory-driving cytokines such as IL-15. The same pattern of phenotype changes in T cells isolated from spleens and lymph nodes were observed. These results were intriguing based on the current knowledge of how metabolic reprogramming affects CD8+ TEFF and TM differentiation. For these reasons, these studies focused on dissecting the effects of 6PGD on CD8+ T cell differentiation. 6PGD Deficiency in T Cells Induces a Memory Phenotype
[00144] It was observed that 6PGD7- TEFF cells differentiated into TCM at a rate significantly faster compared to 6PGD+/+ T cells without immunization in vivo (Fig. 3A-B). Deficiency of 6PGD generated CD44+CD62L|0W T cells, consistent with TEFF phenotype in 6-8 weeks old mice (Fig. 3A). At 20 weeks of age, there was a significant increase in CD44+CD62L+ T cells consistent with TCM (Fig. 3B) compared to littermate control mice. Because this response is driven by self-antigens, this finding suggests that 6PGD- T cells have a cell-intrinsic mechanism that alters their differentiation program in response to antigen exposure, resulting in the generation of TEFF and rapid differentiation to TCM phenotype.
6PGD7- CD8+ T Cells Display Enhanced TEFF Function
[00145] To determine whether 6PGD7- CD8+ cells could develop enhanced effector function after stimulation in vitro , IFN-g was examined after in vitro culture with aCD3+aCD28 mAbs. Elevated IFN-y production was observed as determined by intracellular staining and by ELISA of culture supernatants (Fig. 4A and B). To examine whether 6PGD ablation would alter the cytolytic function of CD8+ T cells, 6PGD7- and 6PGD+/+ control mice was crossed with OTI TCR transgenic mice. T cells from OTI/6PGD7- and OTI control mice were used, which recognize Ova-derived peptide SIINFEKL (Ova257-264), and OVA-expressing EG7 cell line as a target. Compared to OTI/6PGD+/+ control, OTI/6PGD7- CD8+ T cells showed higher CTL function (Fig. 4C). Analysis of the activated CD8+ T cells for expression of genes with cytolytic function showed higher expression in cytotoxic profile genes including Granzyme family {Gzma, Gzmb, Gzmc, Gzmd, Gzme) and the cell death-inducing factor Fasi (Fig. 4D). Consistent with the increased production of IFN-g (Fig. 4A and 4B), mRNA for IFN-g and the transcription factor Tbx21 (encoding for T-bet) were also elevated in 6PGD-/- cells as determined by qPCR (Fig. 4D) and intracellular staining (Fig. 4H). Higher expression of granzyme B (Fig. 4E) and Fas/FasL (Fig. 4F,G) on CD8+6PGD7- T cells was confirmed, which are consistent with their enhanced cytolytic function. Because the Fas/FasL pathway is also a key regulator of activation induced cell death (AICD) during T cell responses, this finding accounts for the reduced T cell numbers in 6PGD7- mice (Fig. 2B and C).
6-Aminonicotinamide (6-AN), A Small Molecule Inhibitor of 6PGD Phenocopies 6PGD~/- Results In Vitro [00146] The lower numbers of mature CD4+ and CD8+ T cells in the secondary lymphoid organs of 6PG7- mouse might raise the question whether the changes observed in 6PGD7- T cells are mediated through cell-intrinsic mechanisms or might be secondary to lymphopenia-driven homeostatic expansion of autoreactive T cells. To address this question, 6-AN, a niacin analog that inhibits 6PGD was used. 6- AN has been previously used to sensitize tumors in-vivo to platinum drugs. To investigate whether 6-AN can recapitulate the profile of T cells from 6PGD7- mice, the effects of 6-AN on purified T cells cultured in-vitro was tested. WT OTI T cells were stimulated with splenocytes and Ova257-264 peptides, in the presence of 6-AN, and examined their differentiation and function. Blocking of 6PGD in T cells resulted in significant increase of IFN-g production (Fig. 5A), enhanced expression of granzyme B (Fig. 5B) and enhanced CTL function as determined by co-culture of 6-AN-treated OTI cells with EG7 targets (Fig. 5D). 6-AN-treated CD8 cells also displayed higher fatty acid uptake (Fig. 5C), a hallmark of TEFF cells. These changes induced by 6-AN in vitro , are consistent with the findings in 6PGD7 T cells (Fig. 4A, C, E, I) and provide evidence that the altered properties of T cells in 6PGD7- mice are mediated by cell intrinsic mechanisms induced by abrogated 6PGD function.
6PGD7- CD8+ T Cells have a Robust Gene Expression Signature of TEFF
[00147] To further investigate the properties of 6PGD7- T cells, their molecular profile after in-vitro stimulation with aCD3+aCD28 mAbs for 72 hours by QuantiGene Plex Assay (Affymetrix) was examined. Compared to similarly stimulated 6PGD+/+ T cells, 6PGD7- T cells were specifically enriched for the expression of genes encoding factors known to identify TEFF CD8+ T cells, including GZM family, Ifng, Ccr5, Ccr2, 1117a, Tbet, Zeb2, Klrgl and Stat3 (Fig. 6A). ZEB2 is critical for generation and expansion of terminally differentiated effector cells, in partnership with T-bet. In contrast, transcripts encoding key regulators of memory differentiation such as Tcf7 and Klf2 were not elevated. Treg associated genes such as FoxP3 and Tgfbr3 were suppressed in 6PGD7- T cells (Fig. 6A). Thus, at this early time after stimulation 6PGD7 T cells exhibited preferential elevation of several molecular hallmarks associated with TEFF cells. To investigate whether the changes in 6PGD7- T cells metabolism lead to epigenetic changes, chromatin accessibility of the 6PGD7- and 6PGD+/+ CD8+ T cells genome was analyzed by ATAC- Sequencing without any stimulation (Fig. 7). Genes encoding for transcription factors involved in Th1 and effector functions including Tbx21, Bhlhe40, and Runx3 showed significantly open chromatin region whereas chromatins of regulatory transcription factors including FoxP1 and Smad4 were highly closed in 6PGD7-T cells (Fig. 7). Tbx21 (T-bet) is a strong driver of Th1 responses and Bhlhe40 works as a cofactor of T-bet for its function such as enhancing IFN-g production. CELF2 splicing factor and is required for T- cell maturation and activation, and Runx3 supports differentiation into cytotoxic effector TEFF function. Moreover, it is known that mature naive CD8+T cells lacking the transcription factor Foxpl gained effector phenotype and function and proliferated directly in response to IL-7 in vitro. Cxcr4 is known to be repressed in differentiated CD8+T cells. These results demonstrate that blocking 6PGD, changes cell metabolism, and induces epigenetic alterations in T cells, which govern their differentiation fate.
Altered Utilization of Glucose in 6PGD7- T cells
[00148] In T cells, TCR-mediated activation induces the expression of the glucose transporter Glutl , leading to internalization of glucose. Once glucose is converted to glucose 6-phosphate, it has two fates, either it can be used via glycolysis or via PPP, which generates Rib-5P that is required for nucleotide synthesis, but also NADPH that is required for biosynthetic reactions and reducing power (Fig. 1A-H). Since, glucose usage correlates with IFN-g production, the ability of 6PGD7- T cells to uptake glucose after aCD3+aCD28 mAbs stimulation was examined. It determined that 6PGD7- T cells had higher potency for glucose uptake as measured by 2-NBGD binding and Glutl expression (Fig. 8A-D).
Deficiency in 6PGD- T Cells Reprograms Metabolic Circuits
[00149] Metabolomics analysis demonstrated that consistent with the lack of the 6PGD enzyme, there was an accumulation of 6-phosphoglucanate (6PG) in the 6PGD7- T cells, which increased following stimulation. In contrast no such increase of 6PG was observed in 6PGD+/+ T cells (Fig 9A). The functional consequences of increased 6PG and its degradation product gluconate in T cells are not yet known. Interestingly, 6PGD7- T altered the profile of glycolysis intermediates in response to stimulation with phosphoenolpyruvate (PEP) being higher and pyruvate lower compared to 6PGD+/+ cells. PEP that has been identified as a key factor to sustain NFAT signaling and anti-tumor effector function in both CD4+ and CD8+ T cells, may contribute to the improved effector and cytolytic function of the 6PGD7- T cells (Fig. 4A-I). Although activated 6PGD7- and 6PGD+/+ T cells contained comparable levels of malate and fumarate, 6PGD7- T cells had significantly higher levels of citrate compared to 6PGD+/+ control (Fig. 9). This metabolic profile with elevated citrate could suggest that in 6PGD7- T cells more pyruvate produced through enhanced glycolysis is shuttled into the TCA cycle. This would be consistent the higher PEP in 6PGD7- T cells (indicating more active glycolysis) the lower pyruvate and lactate levels indicating less pyruvate to lactate conversion. Together with observed T cell phenotype, the only interpretation of these findings, is that pyruvate enters the TCA cycle resulting in higher citrate levels in stimulated 6PGD7- compared to control T cells. The enhanced citrate production might be responsible for the elevated acetyl- coA that leads to histone acetylation, but also to elevated FA synthesis, which is required for memory cell differentiation. These preliminary results justify thorough tracer studies in order to identify how altered glucose utilization by blocking 6PGD function results in such significant changes in TEFF and TM differentiation.
6PGD7- T Cells Demonstrate Higher Mitochondrial Activity that Supports Anti-tumor Function
[00150] Analysis of mitochondrial function demonstrated that CD8+ T cells from 6PGD7- mice develop significantly higher mitochondrial mass, membrane potential (DYiti) and production of reactive oxygen species (ROS) compared to 6PGD+/+ cells (Fig 10A-C). The elevated ROS levels have an indispensable role in TEFF cell function after exposure to antigen. Consistent with an enhanced mitochondrial metabolism and function, these bioenergetics studies also showed that 6PGD7- T cells displayed higher oxygen consumption rate (OCR) at base line, a robust increase of mitochondrial metabolism in response to mitostress and elevated spare respiratory capacity (SRC) (Fig. 10D-F), an indicator of mitochondrial reserve required to function under conditions of stress. Importantly, the elevated SRC indicates that regardless of the enhanced features of activated TEFF, 6PGD7- T cells do not have a bioenergetic signature of activated TEFF cells which display a dramatic decline in SRC, but rather a bioenergetic signature of TM cells, which have elevated SRC.
Glucose-Pyruvate-Acetyl-CoA Axis Modulates Increased Histone Acetylation in 6PGCH- CD8+ T Cells
[00151] In the metabolite analysis, it was observed that after stimulation, 6PGD7- T cells displayed elevated citrate (Fig. 9) but also elevated Acetyl-CoA (Fig. 11 D and E). 6PGD7 T cells have a propensity to transition into TCM cells and recent studies have highlighted role of epigenetic mechanisms as being critical in the differentiation of T cells. Moreover, it is also recently demonstrated that glycolysis-derived Acetyl-CoA, promotes histone acetylation and open chromatin state. It was examined if a similar mechanism was operative in 6PGD7- T cells. 6PGD7- had H3K9/K27 acetylation, which was inhibited by the HK2 inhibitor 2DG and by the ACLY inhibitor BMS303141 (Fig. 11 B and 11 C) indicating that glycolysis derived pyruvate leading to the generation of Acetyl-CoA was responsible for histone acetylation. These events, which are amplified in 6PGD7- CD8 T cells, may have a key role for the differentiation of effector cells or memory differentiation.
Effective Response of 6PGD- CD8+ T Cells to Listeria m. Infection In Vivo
[00152] To examine the role of 6PGD during Ag-specific stimulation in-vivo, a well-established model was used, which allows assessment of CD8+ TEFF and TM cell differentiation and function during primary infection and re-challenge with L. monocytogenes ( Lm ). Lm-specific TEFF CD8+ T cells produce IFN-y, which is essential for defense against this pathogen. By using Lm engineered to express ovalbumin {Lm- Ova), Ag-specific CD8+T cells, identified by using an H-2Kb tetramer containing the Ova-derived peptide SIINFEKL (Ova257-264), are assessed in the spleen and peak on day 4 after i.v. injection. 6PGD7- cells were able to clear the Lm-Ova more effectively than control and Ag-specific T cells showed higher capacity in TNF-a production and expression of maturation markers (Fig. 12A). To eliminate the possibility of cell extrinsic factor might have altered responses of Ag-specific 6PGD7- CD8+ cells in 6PGDfl/fl-CD4-Cre mice, adoptive transfer using T cells from OTI/6PGD7- and OTI/6PGD+/+ control mice followed by inoculation of Lm-Ova was performed. Recipients of OTI/6PGD7- cells, displayed higher rate of bacterial clearance compared to recipients of OTI/6PGD+/+ cells (Fig. 12B). Ag specific OTI/6PGD7- cells also showed higher IFN-g production (Fig. 12B).
6PGD7- T Cells have Potent Anti-tumor Activity which Can be Recapitulated by and 6PGD Small Molecule Inhibitor 6-Aminonicotinamide (6-AN) In Vivo
[00153] T cell metabolic reprogramming is one of the key targets of cancer mediated immune dysfunction. Based on these results regarding the distinct metabolic properties of 6PGD7 T cells, it was examined whether 6PGD7- T cells might maintain their superior effector function in the presence of tumor. To this end, OTI/6PGD+/+ and OTI/6PGD7- CD8+ T cells were adoptively transferred to mice bearing the OVA-expressing EG7 tumor. Compared to OTI/6PGD+/+ CD8+ T cells, OTI/6PGD7- CD8+ T cells were more efficient in reducing tumor growth (Fig. 13A) and expressed higher levels of granzyme B in the tumor environment (Fig. 13B). To examine whether these effects of OTI/6PGD7- T cells would be recapitulated and persist in vivo after ex vivo 6-AN treatment, 6PGD+/+/OTI-CD8+ cells was pre-treated with 6-AN or vehicle control during in vitro culture with Ova peptide and adoptively transferred them to mice bearing Ova-expressing EG7 tumors. Strikingly, 6PGD+/+/OTI-CD8+ pretreated with 6-AN abrogated tumor growth (Fig. 13C).
6PGD7- TIL are Resistant to Metabolic Exhaustion and Tumor Microenvironment-Induced Mitochondrial Dysfunction
[00154] To examine if the enhanced anti-tumor function of 6PGD7- T cells was also observed in the presence of a true tumor antigen, B16 mouse melanoma in 6PGD7- and 6PGD+/+ mice and monitored tumor growth was inoculated. During the 2 week monitoring, 6PGD7-mice had significantly smaller tumors compared to 6PGD+/+ control group (Fig. 14A) (p=0.0065). To explore how the properties of tumor- specific T cells were affected, 6PGD7- mice was crossed with pmel-1 T CR transgenic mice, which express a transgenic TCR specific for gp100 expressed on B16 murine melanoma (H2-Db). According to established methods, after ex vivo priming with cognate antigen gp100, pmel-1 /6PGD7- and pmel- 1/6PGD+/+ T cells were transferred to tumor bearing hosts. Recipients of pmel-1/6PGD-/- T cells had significantly smaller tumors compared to recipients of pmel-1/6PGD+/+ T cells (Fig. 14B). Moreover, TIL from recipients of pmel-1/6PGD7- T cells had improved mitochondrial function compared to TIL from recipients of pmel-1/6PGD7- T cells as assessed by MitoT racker staining (Fig. 14B). This metabolic profile of 6PGD7- CD8+TIL is consistent with resistance to tumor-mediated metabolic exhaustion and preserved anti-tumor function in the tumor microenvironment.
[00155] These results provide evidence that blockade of pentose phosphate pathway at the 6PGD step in T cells reprograms metabolism and gene expression resulting in differentiation of T cells with functional features that support potent anti-tumor immunity.
Example 2
[00156] To determine how 6PGD deficiency regulates the differentiation of CD8+ effector and memory T cells. Data indicates that in absence of 6PGD, CD8+ T cells acquire a TEFF phenotype with altered metabolic utilization program for glucose. We observed induced TEFF phenotype by specific cell markers along with enhanced effector functions including in vivo functional assay for listeria and tumor clearance. It was also found that not only 6PGD7- CD8+ T differentiate to potent TEFF cells but also have a propensity to shift towards T CM. These data justify the need for detailed characterization of metabolic program, gene network and epigenetic signatures that induce 6PGD7- CD8+ T cells to effector and memory T cells. These studies elucidate the molecular mechanisms involved in their distinct functions and properties observed in 6PGD7- CD8+ T cells. Within these studies, T cells from pmel-1 and OTI TCR transgenic mice is used to investigate metabolic changes during antigen encounter.
Global Metabolic Analysis of 6PGD-/- CD8+ T
[00157] Metabolic reprogramming of tumor reactive antigen T cells, by global metabolite analysis, which provides an extensive and unbiased characterization of the cell's metabolic state and can identify altered utilization of various classes of nutrients is examined. These analyses provide unpredictable findings as it explores the global network of metabolites in multiple connected pathways, na'ive pmel- 1/6PGD7 and pmel-1/6PGD+/+ as well as OTI/6PGD7- and OTI/6PGD+/+ T cells are used and after stimulation with cognate peptide antigens (gp100 and SIINFEKL [Ova257-264], respectively), the metabolite profiles in these purified T cells are assessed by GC-MS.
Altered Utilization of Glucose and Glutamine by Tracer Analysis.
[00158] Based the studies from metabolomics analysis, activated 6PGD7- CD8+ T cells demonstrate an altered glucose utilization program. In order to get better understanding of how 6-PGD deficiency alters the flow of glucose carbon to rewire the metabolism, stable isotope-resolved metabolomic (SIRM) analysis of control or 6PGD7- TEFF cells subjected to treatment with 13C6-glucose (Glc) and 13C5.15N2- glutamine (Gin) tracer are performed, amounts of total and 13C-enriched TCA (Krebs) cycle metabolites (malate, aspartate citrate, cis-aconitate, a-KG and fumarate) derived from either 13C-Glc or 13C-Gln are assessed, which can be used in the TCA cycle. Since data shows increased PEP, decreased pyruvate and reduced TCA metabolites in 6PGD7- T cells, it is important to follow how other intermediate glycolytic metabolites in tumor reactive 6PGD7- CD8+ T cells are altered compared to 6PGD+/+ CD8+ T cells using tracer analysis. It is clarified, whether TCA cycle is either broken or cycles rapidly to metabolize TCA metabolites; hence less accumulation in TCA metabolites is observed. This is critical, as it was observed that 6PGD deficient T cells have increased acetyl Co-A and citrate that may impact the glycolytic- lipogenic pathway. Using tracer analysis, it is deciphered whether increase in PEP is generated from 2- phospgo-glycerate (2PG) step of glycolysis or via phosphoenol pyruvate carboxykinase (PCK1) from TCA cycle (Fig. 1). altered flux through the Krebs cycle is examined by assessing 13C4-isotopologues of citrate, fumarate and malate derived during the first turn of the cycle. HSQC NMR analysis is employed to investigate the abundance of the 13C-3/13C-4-Glu and 13C-4-Glu-glutathione isotopomers in 6PGD7- CD8+ T and 6PGD+/+ CD8+ T cells, the abundance of 13C-4-Gln is assessed, which allow to identify 6PGD7- CD8+ T cells utilize glutamine based on the abundance of labeled products of 13C5, 15N2-Gln oxidation through the TCA cycle. The PPP provides support for nucleotide biosynthesis, via the conversion of 6PG to ribose 5-phosphate which is subsequently converted to phosphoribosyl pyrophosphate (PRPP) followed by its conversion nucleotide synthesis pathway (Fig. 1A-H). As mentioned in the results (Fig . 2A-E), the numbers of CD8+ T cells in the spleen and lymph node of 6PGD- A mice are reduced compared with their littermate control. The lower T cell numbers might be due to the elevated expression of the Fas: FasL pathway but may also be due to the reduced levels of ribose phosphate that is required for nucleotide synthesis. These racer studies elucidate how nucleotide synthesis is regulated and altered during 6PGD deficiency.
Lipidomic Analysis of 6PGD CD8+ T
[00159] Results showed that 6PGD7- CD8+ T cells promoted the expression of genes involved in the fatty acid metabolism including Sterol O-acyltransferase 2, Acyl-CoA Oxidase, Aqp9, and Acs2 (Fig. 6A). Flow cytometry based analysis using labeled fatty acids on 6PGD7- CD8+ T cells show enhanced fatty acid uptake with increased CD36 expression (Fig. 41) which is the hallmark of activated phenotype. However, it is unknown whether 6PGD7- CD8+ T selectively oxidize endogenous or exogenous FAs. Results showed that 6PGD7 CD8+ T cells contain higher levels of citrate (Fig. 9C) and Acetyl-CoA (Fig. 11 D). Glucose-derived Acetyl-CoA and citrate can be utilized for fatty acid and cholesterol biosynthesis (Fig. 1). FAO is also a major source of Acetyl-CoA and FAO of glucose-derived lipids is a key metabolic program of TM cells. For these reasons, it is important to determine whether the functional differentiation of 6PGD-/- CD8+ T cells to effector might be related to altered lipid metabolism that might be promoted by increased glucose flux. To identify the lipid profile of 6PGD7- CD8+T and 6PGD+/+ CD8+ T cells static metabolite analysis are performed by FTMS (Fourier Transform Mass Spectrometry). Metabolite tracing with either 13C6-Glc, 13C5,15N2-Gln or 13C8-octanoate are performed to determine whether FAs utilized for FAO are synthesized endogenously or acquired from extracellular sources. It is found that 6PGD7- CD8+ T selectively promotes utilization of FAs from cell intrinsic sources, it is investigated whether mechanisms of lipolysis are involved. Results showing diminished histone acetylation in the presence of 2DG suggests that in this system, glucose has an important role in the generation of Acetyl-CoA (Fig. 11). These results do not necessarily rule out generation of Acetyl-CoA from FAO derived lipids. Therefore, role of Fatty Acid derived Acetyl-CoA is explored to address alteration in lipid metabolism. Gene Expression
[00160] Based on the initial immune-phenotyping it is examined whether 6PGD7- CD8+ T and 6PGD+/+ CD8+ T cells displayed an expression pattern of genes which can serve as hallmarks of effector cells. It was identified that signature genes of effector cell differentiation as well as genes encoding for metabolism enzymes were differentially expressed in 6PGD7- T cells compared to control. Detailed characterization of the gene signatures of 6PGD7- CD8+ T cells in order to identify the mechanisms for their distinct differentiation programs are performed. RNA-seq analysis that can provide a global assessment of differential gene expression that have been shown to be involved in regulating T cell effector or memory responses are used. Differential expression of genes identified by this approach by qPCR are validated, whether some of these differentially expressed genes alter the differentiation of CD8+ T cells by either targeting them for deletion using retroviral/lentivirus based shRNA or overexpression systems for in vitro and in vivo functional studies are investigated.
[00161] To investigate the link between metabolic changes and differentiation of 6PGD deficient T cells. Results in Fig. 3A-B suggest that 6PGD7- T cells have a cell-intrinsic mechanism that alters their differentiation program in response to antigen exposure, resulting in the generation of TEFF and rapid differentiation to TCM phenotype. This initial observation justifies further mechanistic characterization. For this purpose, the well-established mouse model, such as L. monocytogenes (Lm)-Ova is used, which allows assessment of TCM cell differentiation and function during primary infection and re-challenge. OTI+CD8+ cells from OTI/6PGD7- and OTI/6PGD+/+ mice are adoptively transferred into syngeneic recipients followed by inoculation of Lm-Ova to assess antigen-specific CD8+T cell responses (Fig. 11 B). TEFF responses are assessed on days 4-8 as before. For memory cell differentiation, the response for time course is analyzed at days 6, 10, 15 and after day 40 of i.v. injection. Based on these data, it is hypothesized that the switch to memory may happen at an enhanced rate and therefore these experiments are repeated few times to be able to catch differentiated cells at correct time window. Antigen-specific T cells are analyzed with CD44+CD62L+ consistent with TCM, and CD44|0WCD62L+ for TSCM populations. KLRG1 +CD127|0W cells are also addressed, consistent with terminally differentiated short-lived effectors (SLEC), v/s KLRG lowCD127hi9h phenotype, long-lived memory precursor cells (MPEC). The functional properties of these antigen-specific memory cells are accessed for production of IFN-y by re-stimulating splenocytes ex vivo with cognate peptide antigen, SIINFEKL (Ova257-264). In parallel, the function of memory cells in vivo is assessed by re-challenging the mice with a secondary infection. Antigen specific CD44+CD62L+ are isolate consistent with TCM for molecular profiling for key gene markers related to TEFF and TCM.
Mitochondrial Potential Dictates Effector and Memory Cells
[00162] CD8+ T cells that are found to have low-Dyiti and have memory gene signature have been associated with enhanced in vivo persistence and superior antitumor properties. Results indicated that metabolic reprogramming mediated by targeting 6PGD may have direct implications on mitochondrial metabolism of CD8+ T cells. The findings that 6PGD7- CD8+ T cells have higher OCR, increased mitochondrial potential and substantial SRC in contrast to control (Fig. 10) combining characteristics of a CD8+ population with TEFF and TCM phenotype. To address how 6PGD deficiency may regulate mitochondrial function and how this regulation is different in 6PGD7' TEFF or TCM, simultaneous uptake of 2-NBDG and Mitotracker-FM in TEFF or TCM isolated from tumors after adoptive transfer of pmel-1/6PGD- Aand pmel-1/6PGD+/+ T cells are examined. Mitochondrial DNA, markers of mitochondrial biogenesis are examined. Electron microscopy analysis are performed to assess the morphologic structure of mitochondria which is linked to their function.
[00163] To investigate whether histone acetylation plays a role in the differentiation of 6PGD7- CD8+ T cells: Recent literature points to a role of pyruvate to acetyl Acetyl-CoA step in epigenetic regulation of gene expression via histone modification in hematopoietic stem cells. Results showed 6PGD7- T cells have increased Acetyl-CoA and that glycolysis-derived pyruvate is required for H3K9/K27 acetylation (Fig. 11 C). These findings provide a strong rationale that similar mechanisms might be responsible for the altered differentiation of 6PGD7- T cells. Epigenetic mechanisms are critical components in the specification of distinct T-cell subsets. Flow histone acetylation modulates gene expression during differentiation is still an area of active investigation. Because these studies provided evidence that PGD- T cells had increased Acetyl-CoA and H3K9/K27 acetylation (Fig. 11 D, 11 E and 11 B), chromatin immunoprecipitation is used as detailed in with an antibody for H3K9/K27 coupled with parallel sequencing (ChIP-Seq) to map genome-wide acetylation-based histone modifications. pmel-1/ 6PGD7-T cells and pmel-1/ 6PGD+/+ T cells stimulated in vitro are used. Promoters and genes related to cell differentiation are examined and these findings are validated in conjunction with the RNA-seq analysis of pmel-1/ 6PGD7- T cells and pmel-1/ 6PGD+/+ TILs
Example 3: To Determine How 6PGD Impacts the Differentiation of CD4+ T Cells and the Acquisition of Anti-tumor Functions.
[00164] A cardinal feature of the T cell immunity is the functional plasticity of na'ive T cells to differentiate into lineage specific T effector functions with unique expression markers and metabolic signature. Results support the hypothesis that 6PGD deficiency might affect the polarization program of CD4+T cells, which have a critical role in mediating CD8+ function. These data justify the need for detailed characterization of metabolic program and gene signature of differentiation of na'ive CD4+ T cells from 6PGD7- mice. For these studies T cells from 6PGD7- mice are used. Investigate how 6PGD deficiency alters differentiation of na'ive CD4+TFI subset Cells, i) Differentiation of Naive CD4+TFI subset Cells from 6PGD7- T mice: 6PGD deficiency not only resulted in differentiation of CD8+ T cells, but also impacted CD4+ T cells. It was observed the IFN-y expression was significantly elevated in 6PGD7- compared to control CD4+ T cells (Fig. 15A). Since, T-bet directly activates transcription of the IFN-g gene and can regulate Th1 phenotype, increased T-bet expression in 6PGD7- CD4+ T cells was assessed and confirmed (Fig. 15B). Moreover, it was observed that a high number of 6PGD7 T mice developed rectal prolapse between 4 to 5 months of age, strongly indicative of colitis (Fig. 16). Because Th17 cells are the most potent mediators of colitis, it was examined whether 6PGD7- mice might have elevated Th17 cells. An increased number of IL-17A+ RORyt+CD4+ T cells in 6PGD7- mice compared to control was observed (Fig. 15C and D). With these significant, how 6PGD targeting affects the differentiation and polarization program of CD4+ TH subsets specifically Th1 , Th2, Th17, and Treg is examined. First, how 6PGD ablation affects the differentiation of these Th subsets in vivo using standard methodology of flow cytometry for the assessment of relevant markers is examined. Second, whether the metabolic changes induced by 6PGD deficiency alter the polarization capacity of T helper cells during culture in vitro is examined. The studies are divided into two parts; i) neutral condition i.e. stimulation of na'ive CD4+TFI cells from 6PGD7- and 6PGD+/+ T mice with aCD3+aCD28 mAbs in vitro in standard culture media and analyze different subsets, ii) polarizing conditions i.e. analysis of properties for cultured na'ive CD4+ T cells from 6PGD7 T mice and 6PGD+/+ T mice in established polarizing culture condition for Th1 , Th2, Th17, and Treg. As lineage polarization in neutral culture conditions might not allow for detection of differences, hence polarization culture conditions might help to magnify the changes. The functional properties of these cells by assessing IFN-y, IL-4 or IL-17 producers by intracellular staining in flow cytometry is investigated. To investigate whether inhibition of 6PGD induces an altered CD4+ polarization program, differentiated linages characterized by lineage-specific transcription factors (T-bet for Th1 cells, GATA3 for Th2 cells, Foxp3 and Runxl for Treg, RORyt and Runxl for Th17 cells) are evaluated. Initial immune-phenotyping indicate enhanced TH1 , and TH17 cells within CD4+ population in 6PGD7- T cells with elevated expression pattern of T-bet and RoRyt. Differentiation and Mitochondrial potential. Mitochondrial potential has a key role in the context of CD4+T cell differentiation as they may drive CD4+ T enhanced sternness and improved functionality for immunotherapy. Mitochondrial functions are characterized by accessing mitochondrial potential, mitochondrial DNA and markers of mitochondrial biogenesis in PGD7- TH subset from 6PGD7- and 6PGD+/+ in different culture polarizing conditions, ii) Differentiation of Na'ive CD4+TFI subset Cells by pharmacological inhibition of 6PGD: Based on prior data, it is investigated whether 6-AN treatment of 6PGD+/+ CD4+ cells can phenocopy results observed in CD4+ T cells from 6PGD7- mice. Gene expression and cytokines are characterized to validate each subset.
Modulation of Metabolic Reprogramming of the TH Subset by 6PGD Deficiency.
[00165] To investigate how metabolic changes mediated by 6PGD deficiency induce differentiation, metabolite tracer analysis for glucose, glutamine and lipids as detailed in SA1c-d in PGD7- TH subset from 6 PGD7- and 6PGD+/+ CD4+ T cells in different culture polarizing conditions is performed. Mechanistic understanding of how metabolic changes mediated by 6PGD deficiency induce differentiation. Th1 , Th2, and Th17 cells are highly glycolytic in nature in contrast to Treg, which are reliant on lipid oxidation for their differentiation. To investigate how metabolic changes, as a result of 6PGD deficiency alter regulation of lineage commitment, the in vitro lineage culture conditions for 6PGD deficient na'ive CD4+ T are perturbed by variety of metabolic pharmacologic inhibitors. In these studies, Biochemical assays such as glucose uptake, lipid oxidation as previously described as well as mitochondrial potential and functional assay for specific lineage commitment are used to measure. Of particular interest are selective effects of each of these steps of glycolysis on Th17 polarization because previous studies have indicated that enhanced glycolytic machinery is actively regulated to direct the differentiation of TH17 and Treg cells. For example, iodoacetate (IAA), which targets glyceraldehyde phosphate dehydrogenase (GAPDFI) and oxalate (OXA) which targets pyruvate kinase (PK) are used (Fig. 17). These have confirmed specificity and previously gave informative results in T cells. Moreover, Pyruvate dehydrogenase (PDH), that converts pyruvate to Acetyl-CoA is a key bifurcation point between T cell glycolytic and oxidative metabolism and plays important role in Th1 , Th17, and Treg differentiation. To target PDH, dichloroacetate (DCA) that blocks the inhibitory effect of mitochondrial pyruvate dehydrogenase kinase (PDK) on pyruvate dehydrogenase (PDH) and promotes conversion of pyruvate to Acetyl-CoA. DCA is also known to inhibit the production of IL-17 and suppress expression of the Th17 transcription factor RORyt in cells cultured in Th17-skewing conditions is used. Since, Treg differentiation is modulated via differential usages of endogenous fatty acids, free fatty acids (FFA) are delivered during the in vitro polarizing conditions in 6PGD deficient naive CD4+ T to explore how FFA effects lineage differentiation in 6PGD deficient CD4+ T cells. These studies are designed to test these effects at the beginning or during the polarization process. Therefore, either the inhibitors are added before starting in vitro differentiation, or after the in vitro differentiation is almost completed. These studies help in deciphering how 6PGD7- reprograms metabolic circuits and whether metabolic intervention can flip-flop the lineage depending on the time of treatment. These results also provide insight into how to exploit metabolic intervention for increased efficacy of immunotherapy.
Characterization of 6PGD Deficient CD4+ T Cells that Reduces Tumor Growth In Vivo.
[00166] After characterizing of CD4+ lineage differentiation and predominant metabolic pathways on CD4+ 6PGD7- cells, these subsets functional activity within tumor condition are checked. There are several studies showing that CD4+ T helper cells are capable of protecting the host against tumor challenge and may even mediate tumor regression on their own. Data shows that deficiency of 6PGD alters CD4+ T cells such that these cells phenocopy hybrid Th1/17 population (Fig.15). This hybrid population of cells has‘anti-tumor effector' function of Th1 cells and‘sternness characteristics' of Th 17 cells. These strong findings justify the hypothesis that 6PGD7- CD4+ T cells might play a key role in anti tumor immunity. TRP-1 T CR transgenic mice is utilized to access how targeting 6PGD affects CD4+ anti tumor immunity. TRP-1 -specific CD4+ T cells derived from TCR transgenic are transduced with control or 6PGD shRNA and adoptively transfer into B16 melanoma-bearing mice as recently described. Tumors are measured over 12 to 14 days and animal survival are determined. Separately, the expression of IFN- y, and IL17 in TRP-1 positive cells from tumors, spleen, and draining lymph nodes three days' post adoptive transfer are analyzed. Alternatively, naive CD4+ T isolated from TRP-1 TCR transgenic mice cells with aCD8+aCD23 mAbs are stimulated in presence of 6-AN or a vehicle control for 4 days and assess polarization for either Th1 or Th17 by analyzing cytokine and transcription factor via intracellular staining. Cells are adoptively transfer into mice bearing B16 melanoma tumors to test their antitumor function. [00167] Combination with RORD small molecule agonist (LYC-54143) to enhance Th17/Th1 profile of 6PGD7- CD4+ T cells for improved tumor outcomes. In context of cell based therapy, recent reports have highlighted the role of Th 17 cells in significantly eradicating large human and murine tumors better than bulk CD4+ T cells, Th1 or Th2 cells. RORy activation with a small molecule agonist, LYC-54143 has been shown to be effective in generating anti-tumor immunity in syngeneic models. The combination of RORy agonist with 6-AN in adoptive cell based therapy might result in improved efficacy in terms of significantly reduced tumor volumes. For these studies, naive CD4+ T isolated from TRP-1 T CR transgenic mice cells with a CD8+aCD23 mAbs in the presence of a) 6-AN alone, b) vehicle control, c) 6-AN plus RORy agonist, d) vehicle control plus RORy agonist for 4 to 6 days and adoptively transferred into mice bearing B16 melanoma tumors to test their anti-tumor function. Tumors are measured over 12 to 14 days and animal survival are determined. Separately, the expression of IFNy, and IL17 in Trp-1 positive cells from tumors, spleen, and draining lymph nodes three days' post adoptive transfer are also analyzed.
[00168] To examine how modulation of 6PGD expression and activity regulates the anti-tumor function of CD8+ T cells: TEFF responses are critical for anti-tumor function in vivo. The effectiveness of antitumor responses via adoptive T cell-based therapies has been associated with not only the effective cytolytic functions but also their ability to persist as long-term memory cells. Moreover, trafficking of CD8+ T cells to secondary lymph nodes and the ability of tumor-reactive cells to remain in activated state and avoiding exhaustion has been associated with successful immunotherapy. Results showed that targeting 6PGD resulted in generation of potent TEFF cells and differentiation to TCM. Moreover, 6PGD7- TEFF cells significantly reduced expression of exhaustion markers. For these reasons, it is investigated whether 6PGD deficiency improves anti-tumor capabilities in vivo by preferentially enhancing the functions of the tumor-specific TEFF and TCM.
[00169] To investigate how metabolite changes as a result of deficiency of 6PGD enhances generation of anti-tumor TEFF cells. Results showed that 6PGD7- CD8+ T cells were more potent TEFF as accessed by various surface markers (Fig. 2) and produce higher levels of IFN-y and able to clear Lm Ova (Fig. 12) as well as tumors more effectively than their wild type counterparts (Fig. 13 and 14). These metabolite studies showed that in the absence of 6PGD activity, T cells have elevated phosphoenolpyruvate (PEP) (Fig. 9), and other intermediates of glycolysis and decreased pyruvate (Fig. 9). The glycolysis intermediate PEP has been implicated in enhancing T cell responses because it is necessary for maximal Ca2+-NFAT 1 signaling. Since, increased levels of PEP along with low levels of pyruvate are observed, it is hypothesized that blockade of 6PGD activity might lead to the generation of potent effectors through regulation of this axis. To examine if 6PGD deficiency improves T cell effector function and anti-tumor properties through this mechanism, pmel-1/6PGD7- and pmel-1/6PGD+/+ T cells are used stimulated with cognate antigen gp100 and intracellular PEP levels using a fluorescence-based assay are assessed. Nuclear v/s cytoplasmic distribution of NFAT1 are analyzed to access NFAT activity. NFAT activity are examined in TIL isolated from B16F10 tumors in recipients of adoptively transferred pmel-1/6PGD7- and pmel-1/6PGD+/+ T cells.
Investigate Properties of 6PGD Deficient Tumor-Specific T Cells In Vivo.
[00170] pmel-1/6PGD7- and pmel-1/6PGD+/+ T cells primed with gp100 peptide antigen for adoptive transfer to wild-type mice bearing B16F10 tumors are utilized. Based on the data that 6PGD commit to TEFF differentiation and recent studies have highlighted the role of that Foxol in modulating TEFF differentiation and homing properties CD8+ T cells, it is investigated whether Foxol function is affected in 6PGD deficient TIL and plays a role in trafficking to intra-tumoral sites and lymphoid organs. Foxol functions are regulated by phosphorylation events, such that during TEFF differentiation, Foxol is sequestered and prevents expression of genes, selectin L {Sell·, which encodes CD62L), and Kruppel- like factor 2 (K/f2) that are important in homing to secondary lymphoid structures. Hence, immune- phenotype tumor infiltrating cells from pmel-1/6PGD7- and pmel-1/6PGD+/+ T cells after gating on pmel-1 with V13 positive cells using activated TEFF cell markers such as CD127, CD44, KLRG and CD62L are examined. Transcription factor T-bet as well as IFN-y for assessment of TEFF function is examined by intracellular staining as shown in Fig. 5. TCM population by expression of transcription factors Tcf7 and Eomes and immune typing cells for CD44l0WCD62L,li9,lCD122,li9,l are also accessed. Anti-tumor function in vivo is assessed by measuring tumor size and survival in these tumor bearing mice. Phosphorylation and intracellular localization of Foxol in tumor-specific pmel-1/6PGD7- and pmel-1/6PGD+/+ T cells using standard cell biology protocols is assessed. Results show that 6PGD7- T cells not only display activated phenotype and elevated expression of PD-1 but lack expression of exhaustion markers Tim-3, LAG-3 and CTLA-4 (Fig. 18B-D and F-H). These findings strongly suggest that 6PGD7 T cells has superior poly functionality as anti-tumor effectors due to the expression of lower number of exhaustion receptors. To this end tumor-reactive CD8+ TIL's are analyzed to assess the potency and longevity of 6PGD7- TIL's. As detailed in Fig. 14, mitochondrial function is studied by accessing potential and Mitochondrial mass in pmel+ CD8+ TILs from these tumors compared to non-draining lymph nodes.
[00171] Does Preferential Differentiation of 6PGD- T Cells to TM Tesult in Prolonged Anti-Tumor Immunity? Preliminary experiments to test effector function were carried out with pmel-1 were performed with tumors inoculated in naive host without any prior recurring tumor challenge (Fig. 14). Given the striking preliminary data, (Fig. 2) suggesting that 6PGD7- T cells preferentially transition 6PGD7 TCM, it is investigated whether 6PGD7- T cells can provide prolonged anti-tumor immunity by generating and maintaining TCM cells that are able to respond to tumor re-challenge. Because 6PGD has a major effect on differentiation of CD8+ T cells and on their metabolic properties, how 6PGD affects the dynamics of tumor-specific T cells is investigated. These findings are critical to translational application of 6PGD inhibitors in adoptive cell based therapy. A large cohort of congenic C57BL6 CD45. Mice are used in which B16F10 is inoculated followed by adoptive transfer of pmel-1/6PGD7- and pmel-1/6PGD+/+ T cells. Following adoptive transfer, mice that have eradicated the tumors are identified and re-challenged them with B16F10. Mice are monitored for tumor growth and for dynamics of TCM response by assessing the presence of pmel+ T cells in lymphoid and non-lymphoid organs, in which TCM vs. TEFF cells reside differently. Gene expression profiles are examined by PCR based methods to identify transcripts that were differentially expressed.
[00172] To determine if 6-AN, a small molecule inhibitor targeting 6PGD affects tumor specific T cell differentiation resulting in reduced tumor growth. It is examined whether pharmacologic intervention using, 6-AN, a small molecule inhibitor of 6PGD might recapitulate the effects of genetic 6PGD targeting on the metabolic and differentiation program of tumor-specific T cells. The currently available small molecule 6PGD inhibitor has been previously tested for its potential application as an anti-cancer drug. The interest is to perform a short term ex vivo treatment of tumor-specific T cells with 6PGD inhibitor followed by adoptive transfer of these cells in vivo for cell-based cancer immunotherapy. Based on the preliminary data, short term treatment (4 days) of CD8+ T cells with 6-AN in presence of IL2 and aCD3+aCD28 mAbs results in enhanced increased in expression of IFN-y and GranzB (Fig. 5) and significantly reduces established EG7-Ova tumors (Fig. 13B). pmel-1/6PGD+/+ splenocytes are stimulated with cognate antigen gp100 and IL-2 in the presence of 6-AN or vehicle control DMSO. The goal is to assess the effect of treatment on the activation and differentiation program of tumor-specific T cells and on their anti-tumor properties in vivo using pmel-1 transgenic mice. Expression of perforin and granzyme B, are examined and the activation markers CD44, CD25, and CD62. Tumor infiltrating cells are isolated and characterized. Their anti-tumor function in vivo is assessed by tumor size and survival.
Combination of 6PGD Blockade with PD-1 Checkpoint pathway to Improve Immunotherapy.
[00173] Results showed that 6PGD7- TEFF cells show increased expression of PD-1 but reduced expression of exhaustion markers (Fig. 18). Thus, it is highly likely that blocking the PD-1 : PD-L1 pathway concomitantly with 6PGD targeting might result in superior anti-tumor immunity. To address this issue, two approaches are employed. First, B16F10 melanoma is implanted in the 6PGD7- and 6PGD+/+ control mice as in shown in Fig. 14A and tumor-bearing mice are treated with biwkeekly injections of anti-PD-1 blocking antibody based on standard protocols. Outcomes are monitored by assessing tumor size and metastasis. As a second approach, pmel-1/6PGD+/+ T cells are used and during in vitro priming with cognate antigen gp100, and are incubated with 6-AN or vehicle control. Primed cells are transferred to tumor bearing mice, which are subsequently treated with biweekly injections of anti-PD-1 blocking antibody.
[00174] To investigate effects of targeting 6-PDG on tumor-specific human T cells, i) Pharmacological intervention with 6-AN for in vitro characterization: Wilms tumor antigen-1 (WT1) is zinc finger transcription factor is expressed in various cancers and correlates with cancer aggressiveness. WT1- splecific CD8+ T cells from HLA-A*0201 individuals are selected by tetramers and 6PGD are targeted with pharmacological inhibitor, 6-AN or a vehicle control DMSO. Cells are cultured with dendritic cells pulsed with the HLA-A*0201 -restricted WT1126-134 (RMFPNAPYL) epitope. Differentiation of WT1- reactive T cells are assessed by analyze transcription factors associated with short-lived terminally differentiated TEFF cells. Cells expressing CD45RO, CD62L, CD27, CD127, CD28, and CCR7 are examined, which identify an antigen-experienced but not terminally differentiated phenotype. These cells are known to be associated with anti-tumor and stem cell line properties of WT 1 -specific T cells generated with IL-21. Expression of markers such as CXCR3, CD122, and CD95 and that are known to correlate with a poorly differentiated long-lived memory subset in humans are analyzed. CTL function is asessed by LDH release assay with HLA-A*0201 , (TAP)-deficient T2 cells pulsed with WT 1 peptide and metabolic output and bioenergetics are examined, ii) Impact of 6PGD inhibition on anti-tumor function of WT1- specific human T cells in vivo. To examine differential capacity of human tumor-specific CD8+ T cells, NOD/SCID mice bearing WT1 + BV173 leukemia are used. WT1+ human leukemia BV173 cells are inoculated intravenously in NOD/SCID. These mice are then given increasing doses of WT1 -specific T cells generated in vitro in the presence of 6-AN or vehicle control DMSO. At 4 and 5 weeks after transfer, bone marrow from these mice is isolated for analysis of WT1-specific T cells (WT1-pentamer+/CD8+) and BV173 leukemia cells (human CD45- / HLA class I-/ CD8-) by flow cytometry.
[00175] 6-AN Reduces Suppressive Activity ofMDSCs
[00176] MDSCs derived in presence of DMSO from bone marrow are more suppressive than 6-AN derived as measured by their effect on T cell proliferation, indicating that 6-AN modulates MDSC, as shown in FIG. 19A-B and FIG. 20A-B. Comparison of 6PGDf/f-FoxP3cre (FIG. 21 A) and 6PGDf/f- FoxP3WT (FIG. 21 B) in lung, pancreas and skin cells shows altered immunohistochemical expression. Similarly, alterations were observed in heart, intestine and liver cells (FIG. 22A-B).
[00177] 6-AN Blocks Treg Function and Drives Tregs toward the Th2 Phenotype [00178] The experiments of this example demonstrate that 6-AN blocks Treg function and drives Tregs toward the Th2 Phenotype. FIG. 6B is a microarray image showing that the Th2 gene signature predominates in 6PGD-/- YFP+/+ T-regulatory cells (Tregs). FIG. 6B shows that 6PGD-/- YFP+/+ Tregs express both TH1 and TH2 markers. FIG. 6C shows a Real-Time PCR validation assay of the microarray experiment.
[00179] FIG. 23A-B are graphs of a Treg in vitro suppression assay showing that 6PGD-/- deficient T- regulatory cells lack in vitro suppressive activity. In Fig. 23A, Tregs (YFP+) were isolated from 6PGDfl/fl FoxP3Cre mice and showed lower suppressive activity in the in vitro suppression assay. Representative histogram of CFSE dilution pattern (Treg:Teff = 1 :1 ratio) (Fig. 23A) and bar graph analysis of serial ratios of Treg and Teff used in the in vitro suppression assay (Fig. 23B) are shown. Results are from three independent experiments.
[00180] FIG. 24A-C and FIG. 27A-B show that defiency is 6PGD in T regs abrogatge in vivo suppressive activity. In FIG. 27A, YFP+ cells were isolated from 20 day old 6PGD+/+ FoxP3Cre (WT) and 6PGDfl/fl FoxP3Cre mice and FoxP3 mRNA levels were evaluated by real-time PCR. In FIG. 27B, Tregs (YFP+) were isolated and cultured in vitro in the presence of IL-2 (700 lU/ml) and anti-CD3/anti-CD28 coated beads (Treg:beads ratio 1 :3) and cells number was assessed at 24 and 48 hour time points. Results are representative of three independent experiments. FIG. 24A is a schematic showing Tregs (YFP+) from WT and 6PGDfl/fl FoxP3Cre mice and T effector (CD4+CD45RBhi9h) cells were isolated, mixed and adoptively transferred to Rag17- mice. Colons were evaluated 45 days after adoptive transfer. FIG. 24B shows representative hematoxylin and eosin staining of Rag17- mice colon on day 45 after IBD induction via adoptive transfer. Rag17- mice colon were evaluated for length and thickness on day 45 post IBD induction. FIG. 24C is a immunohistochemical image of different Rag17- mice colon that, were evaluated for length and thickness on day 45 post IBD induction. Representative hematoxylin and eosin staining of Rag 17- mice colon on day 45 after IBD induction via adoptive transfer. These experiments demonstrate that defiency of 6PGD in Tregs abrogatge in vivo suppressive activity.
[00181] FIG. 25A-D shows how 6-AN treatment drives lineage specific transcription factors. FIG. 25A is a schematic showing how 6-AN treatment drives lineage specific transcription factors in Treg and Th2 cells. FIG. 25B and FIG. 25C show isolated Tregs (YFP+) from 6PGDfl/fl FoxP3Cre mice having lower suppressive activity in a suppression assay. FIG. 25D is a series of flow cytometry images showing isolated Tregs in the presence of 6-AN vs DMSO. These experiments demonstate that 6-AN treatment drives lineage specific transcription factors in Treg and Th2 cells.
[00182] FIG. 26A-M, FIG. 21A-B, and FIG. 22A-B shows deletion of 6PGD in Tregs results in early onset fatal autoimmune disorder. Comparison of 6PGDf/f-FoxP3cre (FIG. 21 A) and 6PGDf/f-FoxP3WT (FIG. 21 B) in lung, pancreas and skin cells shows altered immunohistochemical expression. Similarly, alterations were observed in heart, intestine and liver cells (FIG. 22A-B). FIG. 26A and FIG. 26B show expresion of 6PGD mRNA and protein in YFP+ cells that were sorted from 6PGD+/+ FoxP3Cre (WT) and 6PGDfl/fl FoxP3Cre mice. FIG. 26C is a representative image of 21 days old WT and 6PGDfl/fl FoxP3Cre mice. FIG. 26D is a representative image of lymphadenopathy in 6PGDfl/fl FoxP3Cre compared to WT mice. FIG. 26E shows absolute number of T cells per spleen and pLNs in WT and 6PGDfl/fl FoxP3Cre mice. Results are representative of 12 mice per group. FIG. 26F shows a survival curve of WT and 6PGDfl/fl FoxP3Cre mice. Results are representative of 27 mice per group. FIG. 26G show splenocytes from 19 days old WT and 6PGDfl/fl FoxP3Cre mice that were harvested, and distribution of CD4+ vs CD8+ T cells were evaluated . FIG. 26H shows that deletion of 6PGD induces enhanced effector phenotype (CD44hi9h CD62L|0W) both in CD4+ T cells (top panel) and CD8+ T cells (bottom panel) in 19 days old 6PGDfl/fl FoxP3Cre compared to WT mice. FIG. 26I shows both CD4+ and CD8+ T cells that have elevated expression of CD69 activation marker in 6PGDfl/fl FoxP3Cre compared to WT mice. FIG. 26J shows splenocytes from 19 days old WT and 6PGDfl/fl FoxP3Cre mice that were stimulated with PMA (50 ng/ml)/lonomycin (1 pg/ml) plus Golgiplug (1 mI/ml) for 4 hours and expression of IFN-g was evaluated by flow cytometry. FIG. 26K shows granzyme B (top panel) and CD107a degranulation activation marker (bottom panel) expression was assessed on splenocytes of 19 days old WT and 6PGDfl/fl FoxP3Cre mice by flow cytometry. FIG. 26L and FIG. 26M shows serum collected from 20 days old WT and 6PGDfl/fl FoxP3Cre mice and levels of serum antibodies (FIG. 26L), and IFN-g, IL-17A, IL-4 and IL-5 (FIG. 26M) was detected . Results are representative of 12 mice per group. These experiments demonstate that deletion of 6PGD in Tregs results in early onset fatal autoimmune disorder.
Statistical Considerations
[00183] For the in vivo mouse studies described in this application, statistical analysis is performed by student's t-test or Wilcoxon-Mann-Whitney test. Power calculations are two-sided at the significance level of 0.05. To achieve at least 80% power, at least eight mice per each test group are studied for each experimental question and the experiment are repeated three times. For gene expression and metabolism studies statistical analysis are performed by the specialized facilities involved.
OTHER EMBODIMENTS
[00184] It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
INCORPORATION BY REFERENCE
[00185] All patents and publications referenced herein are hereby incorporated by reference in their entireties. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. As used herein, all headings are simply for organization and are not intended to limit the disclosure in anyway.

Claims

CLAIMS What is claimed is:
1. A method for modulating an immune response, comprising administering a compound of Formula (I) or a pharmaceutically acceptable salt, hydrate or solvate thereof to a subject in need thereof:
Figure imgf000051_0001
wherein: R1, R2, R3 R4 and R5 each is independently selected from the group consisting of H, halo, (C1- C6)alkyl, (C1-C6)alkoxy, (C3-C7)cycloalkyl, -C(=0)0R6, -C(0)N(R6)2, -N(R6)2, (C6-C10)aryl, and (C3- C10) heteroaryl, each alkyl, alkoxy, cycloalkyl, aryl and heteroaryl optionally substituted with one or more groups selected from hydroxyl, halo, and (C1-C6)alkyl; and each R6 is independently H or (C1-C6)alkyl.
2. The method of claim 1 , wherein R1, R2, R3 R4 and R5 each is independently selected from the group consisting of H, halo, (C1-C6)alkyl, (C1-C6)alkoxy, (C3-C7)cycloalkyl, -C(=0)0R6, -C(0)N(R6)2, and -N(R6)2, each alkyl, alkoxy, and cycloalkyl optionally substituted with one or more groups selected from hydroxyl, halo, and (C1-C6)alkyl; and each R6 is independently H or (C1-C6)alkyl.
3. The method of claim 1 , wherein R1 is -N(R6)2; R2 is H or (C1-C6)alkyl; R3 is H or (C1-C6)alkyl; R4 is -C(0)N(R6)2; R5 is H or (C1-C6)alkyl; and each R6 is independently H or (C1-C6)alkyl.
4. The method of claim 1 , wherein R1 is -N(R6)2; R2 is H; R3 is H; R4 is -C(0)N(R6)2; R5 is H; and each R6 is independently H or (C1-C6)alkyl.
5. The method of claim 1 , wherein the compound is Formula (II) or a pharmaceutically acceptable salt, hydrate or solvate thereof:
Figure imgf000051_0002
6. The method of claim 1 , wherein the compound binds to or interacts with 6-phosphogluconate dehydrogenase (6PGD).
7. The method of any of the above claims, wherein the modulation of the immune response comprises stimulating an increase of cytotoxic T cells levels as compared to levels without administration of the compound.
8. The method of any of the above claims, wherein the modulation of the immune response comprises stimulating an increase of cytotoxic T cells activity as compared to activity without administration of the compound.
9. The method of any of the above claims, wherein the modulation of the immune response comprises stimulating an increase in levels or activity of a granzyme.
10. The method of claim 9, wherein the granzyme is selected from granzyme A and granzyme B.
11. The method of any of the above claims, wherein the modulation of the immune response comprises stimulating an increase in levels or activity of an interferon as compared to levels without administration of the compound.
12. The method of claim 11 , wherein the interferon is selected from IFNa, IFN , and IFNy.
13. The method of any of the above claims, wherein the modulation of the immune response is within the tumor microenvironment.
14. The method of any of the above claims, wherein the modulation of the immune response is a reduction or suppression of an immune inhibitory cell.
15. The method of any of the above claims, wherein the modulation of the immune response is an increase or enhancing of an immune stimulatory cell.
16. The method of claim 14, wherein the immune inhibitory cell is selected from myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), FoxP3+ T cells; tumor associated neutrophils (TANs), M2 macrophages, and tumor associated macrophages (TAMs).
17. The method of claim 15, wherein the immune stimulatory cell is selected from T cells, cytotoxic T lymphocytes, T helper cells, natural killer (NK) cells, natural killer T (NKT) cells, B cells, and dendritic cells.
18. The method of any one of claims 14-17, wherein the modulation of the immune response increases a ratio of immune stimulatory cells to immune inhibitory cells.
19. The method of any one of claims 14-17, wherein the modulation of the immune response increases a ratio of cytotoxic T cells (Tc) to regulatory T cell (Tregs).
20. The method of any one of claims 14-17, wherein the modulation of the immune response comprises a reduction in checkpoint inhibition.
21. A method for treating or preventing cancer, comprising administering a compound of Formula (I) to a subject in need thereof:
Figure imgf000053_0001
wherein: R1, R2, R3 R4 and R5 each is independently selected from the group consisting of H, halo, (C1- C6)alkyl, (C1-C6)alkoxy, (C3-C7)cycloalkyl, -C(=0)0R6, -C(0)N(R6)2, -N(R6)2, (C6-C10)aryl, and (C3- C10) heteroaryl, each alkyl, alkoxy, cycloalkyl, aryl and heteroaryl optionally substituted with one or more groups selected from hydroxyl, halo, and (C1-C6)alkyl; and each R6 is independently H or (C1-C6)alkyl.
22. The method of claim 21 , wherein R1, R2, R3 R4 and R5 each is independently selected from the group consisting of H, halo, (C1-C6)alkyl, (C1-C6)alkoxy, (C3-C7)cycloalkyl, -C(=0)0R6, -C(0)N(R6)2, and - N(R6)2, each alkyl, alkoxy, and cycloalkyl optionally substituted with one or more groups selected from hydroxyl, halo, and (C1-C6)alkyl; and each R6 is independently H or (C1-C6)alkyl.
23. The method of claim 21 , wherein R1 is -N(R6)2; R2 is H or (C1-C6)alkyl; R3 is H or (C1-C6)alkyl; R4 is -C(0)N(R6)2; R5 is H or (C1-C6)alkyl; and each R6 is independently H or (C1-C6)alkyl.
24. The method of claim 21 , wherein R1 is -N(R6)2; R2 is H; R3 is H; R4 is -C(0)N(R6)2; R5 is H; and each R6 is independently H or (C1-C6)alkyl.
25. The method of claim 21 , wherein the compound is Formula (II) or a pharmaceutically acceptable salt, hydrate or solvate thereof:
Figure imgf000054_0001
(II).
26. The method of claim 21 , wherein the compound binds to or interacts with 6-phosphogluconate dehydrogenase (6PGD).
27. The method of any one of claims 21-26, wherein the modulation of the immune response comprises stimulating an increase of cytotoxic T cells levels as compared to levels without administration of the compound.
28. The method of any one of claims 21-27, wherein the modulation of the immune response comprises stimulating an increase of cytotoxic T cells activity as compared to activity without administration of the compound.
29. The method of any one of claims 21-28, wherein the modulation of the immune response comprises stimulating an increase in levels or activity of a granzyme.
30. The method of claim 29, wherein the granzyme is selected from granzyme A and granzyme B.
31. The method of any one of claims 21-30, wherein the modulation of the immune response comprises stimulating an increase in levels or activity of an interferon as compared to levels without administration of the compound.
32. The method of claim 31 , wherein the interferon is selected from IFNa, IFN , and IFNy.
33. The method of any one of claims 21-32, wherein the modulation of the immune response is within the tumor microenvironment.
34. The method of any one of claims 21 -33, wherein the modulation of the immune response is a reduction or suppression of an immune inhibitory cell.
35. The method of any one of claims 21 -34, wherein the modulation of the immune response is an increase or enhancing of an immune stimulatory cell.
36. The method of claim 34, wherein the immune inhibitory cell is selected from myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), FoxP3+ T cells; tumor associated neutrophils (TANs), M2 macrophages, and tumor associated macrophages (TAMs).
37. The method of claim 35, wherein the immune stimulatory cell is selected from T cells, cytotoxic T lymphocytes, T helper cells, natural killer (NK) cells, natural killer T (NKT) cells, B cells, and dendritic cells.
38. The method of any one of claims 34-37, wherein the modulation of the immune response increases a ratio of immune stimulatory cells to immune inhibitory cells.
39. The method of any one of claims 34-37, wherein the modulation of the immune response increases a ratio of cytotoxic T cells (Tc) to regulatory T cell (Tregs).
40. The method of any one of claims 34-37, wherein the modulation of the immune response comprises a reduction in checkpoint inhibition.
41. The method of any one of claims 21-40, wherein the subject is undergoing treatment with one or more immunotherapies.
42. The method of claim 41 , wherein the immunotherapy is an agent that modulates one or more PD-1 , programmed death-ligand 1 (PD-L1), or programmed death-ligand 2 (PD-L2).
43. The method of any one of claims 21-42, wherein the method further comprises administering an agent that modulates one or more PD-1 , PD-L1 , PD-L2, CTLA-4, Tim-3, or LAG-3.
44. The method of claim 43, wherein the administration is sequential or simultaneous.
45. The method of claim 43, wherein the agent that modulates PD-1 is an antibody or antibody format specific for PD-1.
46. The method of claim 45, wherein the antibody or antibody format specific for PD-1 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.
47. The method of claim 45, wherein the antibody or antibody format specific for PD-1 is selected from nivolumab, pembrolizumab, and pidilizumab.
48. The method of claim 43, wherein the agent that modulates PD-L1 is an antibody or antibody format specific for PD-L1.
49. The method of claim 48, wherein the antibody or antibody format specific for PD-L1 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.
50. The method of claim 48, wherein the antibody or antibody format specific for PD-L1 is selected from atezolizumab, avelumab, durvalumab, and BMS-936559.
51. The method of claim 43, wherein the agent that modulates PD-L2 is an antibody or antibody format specific for PD-L2.
52. The method of claim 51 , wherein the antibody or antibody format specific for PD-L2 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.
53. The method of claim 43, wherein the agent that modulates CTLA-4 is an antibody or antibody format specific for CTLA-4.
54. The method of claim 53, wherein the antibody or antibody format specific for CTLA-4 is selected from ipilimumab (YERVOY), tremelimumab, AGEN1884, and RG2077.
55. The method of any one of claims 43-54, wherein the administration is by intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or direct injection into cancer tissue.
56. The method of claim 55, wherein the administration is intratumoral.
57. The method of any one of claims 41-56, wherein the subject is predicted to be poorly responsive or non-responsive to the immune checkpoint immunotherapy or has presented as poorly responsive or non- responsive to the immune checkpoint immunotherapy.
58. The method of any one of claims 41-56, wherein the method reduces and/or mitigates one or more side effects of the immune checkpoint immunotherapy.
59. The method of claim 56, wherein the side effect is selected from decreased appetite, rashes, fatigue, pneumonia, pleural effusion, pneumonitis, pyrexia, nausea, dyspnea, cough, constipation, diarrhea, immune-mediated pneumonitis, colitis, hepatitis, endocrinopathies, hypophysitis, iridocyclitis, and nephritis.
60. The method of any one of claims 41-59, wherein the method reduces the dose of the immune checkpoint immunotherapy.
61. The method of any one of claims 41-60, wherein the method reduces number of administrations of the immune checkpoint immunotherapy.
62. The method of any one of claims 41-61 , wherein the method increases a therapeutic window of the immune checkpoint immunotherapy.
63. The method of any one of claims 41-62, wherein the method elicits a potent immune response in less-immunogenic tumors.
64. The method of any one of claims 41-63, wherein the method converts a tumor with reduced inflammation ("cold tumor”) to a responsive, inflamed tumor ("hot tumor”).
65. The method of any one of claims 41-64, wherein the method makes the cancer responsive or more responsive to a combination therapy of the immune checkpoint immunotherapy and one or more chemotherapeutic agents and/or radiotherapy.
66. The method of 65, wherein the chemotherapeutic agent is selected from one or more of daunorubicin, doxorubicin, epirubicin, idarubicin, adriamycin, vincristine, carmustine, cisplatin, 5-fluorouracil, tamoxifen, prodasone, sandostatine, mitomycin C, foscarnet, paclitaxel, docetaxel, gemcitabine, fludarabine, carboplatin, leucovorin, tamoxifen, goserelin, ketoconazole, leuprolide flutamide, vinblastine, vindesine, vinorelbine, camptothecin, topotecan, irinotecan hydrochloride, etoposide, mitoxantrone, teniposide, amsacrine, merbarone, piroxantrone hydrochloride, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine (Ara-C), trimetrexate, acivicin, alanosine, pyrazofurin, pentostatin, 5-azacitidine, 5-azacitidine, 5-Aza-5-Aza-2'-deoxycytidine, adenosine arabinoside (Ara-A), cladribine, ftorafur, LIFT (combination of uracil and florafur), 5-fluoro-2'-deoxyuridine, 5-fluorouridine, 5'-deoxy-5-fluorouridine, hydroxyurea, dihyd rolenchiorambucil, tiazofurin, oxaliplatin, melphalan, thiotepa, busulfan, chlorambucil, plicamycin, dacarbazine, ifosfamide phosphate, cyclophosphamide, pipobroman, 4-ipomeanol, dihydrolenperone, spiromustine, geldenamycin, cytochalasins, depsipeptide, 4'-cyano-3-(4-(e.g., ZOLADEX) and 4'-cyano- 3-(4-fluorophenylsulphonyl)-2-hydroxy-3-methyl-3'-(trifluorometh- yl)propionanilide.
67. The method of any one of claims 41 -66, wherein the subject is predicted to be poorly responsive or non-responsive to the immune checkpoint immunotherapy based on expression of one or more of PD-1 , PD-L1 , or PD-L2, in a subject's biological specimen.
68. The method of any one of claims 41 -67, wherein the subject is predicted to be poorly responsive or non-responsive to an agent that modulates one or more of PD-1 , PD-L1 , and PD-L2 based on low on expression of PD-1 , PD-L1 , and PD-L2 in a tumor specimen.
69. The method of any one of claims 41 -68, wherein the subject is predicted to be poorly responsive or non-responsive to an agent that modulates one or more of PD-1 , PD-L1 , and PD-L2 tumor proportion score (TPS) of less than about 49% for PD-L1 staining.
70. The method of any one of claims 21-69, wherein the cancer is selected from one or more of basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (e.g. that associated with brain tumors), and Meigs' syndrome.
71. A method for treating or preventing an infection, comprising administering a compound of Formula (I) to a subject in need thereof:
Figure imgf000059_0001
wherein: R1, R2, R3 R4 and R5 each is independently selected from the group consisting of H, halo, (C1- C6)alkyl, (C1-C6)alkoxy, (C3-C7)cycloalkyl, -C(=0)0R6, -C(0)N(R6)2, -N(R6)2, (C6-C10)aryl, and (C3- C10) heteroaryl, each alkyl, alkoxy, cycloalkyl, aryl and heteroaryl optionally substituted with one or more groups selected from hydroxyl, halo, and (C1-C6)alkyl; and each R6 is independently H or (C1-C6)alkyl.
72. The method of claim 71 , wherein R1, R2, R3 R4 and R5 each is independently selected from the group consisting of H, halo, (C1-C6)alkyl, (C1-C6)alkoxy, (C3-C7)cycloalkyl, -C(=0)0R6, -C(0)N(R6)2, and - N(R6)2, each alkyl, alkoxy, and cycloalkyl optionally substituted with one or more groups selected from hydroxyl, halo, and (C1-C6)alkyl; and each R6 is independently H or (C1-C6)alkyl.
73. The method of claim 71 , wherein R1 is -N(R6)2; R2 is H or (C1-C6)alkyl; R3 is H or (C1-C6)alkyl; R4 is -C(0)N(R6)2; R5 is H or (C1-C6)alkyl; and each R6 is independently H or (C1-C6)alkyl.
74. The method of claim 71 , wherein R1 is -N(R6)2; R2 is H; R3 is H; R4 is -C(0)N(R6)2; R5 is H; and each R6 is independently H or (C1-C6)alkyl.
75. The method of claim 71 , wherein the compound is Formula (II) or a pharmaceutically acceptable salt, hydrate or solvate thereof:
Figure imgf000059_0002
(II).
76. The method of claim 71 , wherein the compound binds to or interacts with 6-phosphogluconate dehydrogenase (6PGD).
77. The method of any one of claims 71-76, wherein the modulation of the immune response comprises stimulating an increase of cytotoxic T cells levels as compared to levels without administration of the compound.
78. The method of any one of claims 71-77, wherein the modulation of the immune response comprises stimulating an increase of cytotoxic T cells activity as compared to activity without administration of the compound.
79. The method of any one of claims 71-78, wherein the modulation of the immune response comprises stimulating an increase in levels or activity of a granzyme.
80. The method of claim 79, wherein the granzyme is selected from granzyme A and granzyme B.
81. The method of any one of claims 71-80, wherein the modulation of the immune response comprises stimulating an increase in levels or activity of an interferon as compared to levels without administration of the compound.
82. The method of claim 81 , wherein the interferon is selected from IFNa, IFN , and IFNy.
83. The method of any one of claims 71 -82, wherein the modulation of the immune response is within the tumor microenvironment.
84. The method of any one of claims 71-83, wherein the modulation of the immune response is a reduction or suppression of an immune inhibitory cell.
85. The method of any one of claims 71-83, wherein the modulation of the immune response is an increase or enhancing of an immune stimulatory cell.
86. The method of claim 84, wherein the immune inhibitory cell is selected from myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), FoxP3+ T cells; tumor associated neutrophils (TANs), M2 macrophages, and tumor associated macrophages (TAMs).
87. The method of claim 85, wherein the immune stimulatory cell is selected from T cells, cytotoxic T lymphocytes, T helper cells, natural killer (NK) cells, natural killer T (NKT) cells, B cells, and dendritic cells.
88. The method of any one of claims 84-87, wherein the modulation of the immune response increases a ratio of immune stimulatory cells to immune inhibitory cells.
89. The method of any one of claims 84-87, wherein the modulation of the immune response increases a ratio of cytotoxic T cells (Tc) to regulatory T cell (Tregs).
90. The method of any one of claims 84-87, wherein the modulation of the immune response comprises a reduction in checkpoint inhibition.
91. The method of any one of claims 71-90, wherein the infection is a microbial infection and/or chronic infection.
92. The method of claim 91 , wherein the infection is selected from Chagas disease, HIV/AIDS, tuberculosis, osteomyelitis, hepatitis B, hepatitis C, Epstein-Barr virus or parvovirus, T cell leukemia virus, bacterial overgrowth syndrome, fungal and parasitic infections.
93. The method of any one of claims 71-92, wherein the method is in combination with an anti-infective agent.
94. The method of claim 93, wherein the anti-infective agent is an anti-viral agent including, but not limited to, abacavir, acyclovir, adefovir, amprenavir, atazanavir, cidofovir, darunavir, delavirdine, didanosine, docosanol, efavirenz, elvitegravir, emtricitabine, enfuvirtide, etravirine, famciclovir, and foscarnet.
95. The method of claim 93, wherein the anti-infective agent is an anti-bacterial agent selected from cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics (cipro, Levaquin, floxin, tequin, avelox, and norflox); tetracycline antibiotics (tetracycline, minocycline, oxytetracycline, and doxycycline); penicillin antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, and methicillin); monobactam antibiotics (aztreonam); and carbapenem antibiotics (ertapenem, doripenem, imipenem/cilastatin, and meropenem). In some embodiments, the anti-infectives include anti-malarial agents (e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline, artemether/lumefantrine, atovaquone/proguanil and sulfadoxine/pyrimethamine), metronidazole, tinidazole, ivermectin, pyrantel pamoate, and albendazole.
96. A method of making an immunomodulatory cancer treatment, comprising:
(a) identifying an immunomodulatory anti-cancer agent by:
(i) determining whether the agent binds to or interacts with 6-phosphogluconate dehydrogenase (6PGD); and
(ii) classifying the agent as immunomodulatory based on an ability to bind to or interact with 6PGD; and
(b) formulating the agent for cancer treatment.
97. The method of claim 96, wherein the compound binds to or interacts with 6PGD.
98. The method of claims 96 or 97, wherein the agent stimulates an increase in levels or activity of a granzyme.
99. The method of claim 98, wherein the granzyme is selected from granzyme A and granzyme B.
100. The method of any one of claims 96-99, wherein the agent stimulates an increase in levels or activity of an interferon as compared to levels without administration of the compound.
101. The method of claim 100, wherein the interferon is selected from IFNa, IFN , and IFNy.
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