WO2016073866A1 - Lymphocytes polyfonctionnels à titre de biomarqueurs d'une activité antitumorale - Google Patents

Lymphocytes polyfonctionnels à titre de biomarqueurs d'une activité antitumorale Download PDF

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WO2016073866A1
WO2016073866A1 PCT/US2015/059490 US2015059490W WO2016073866A1 WO 2016073866 A1 WO2016073866 A1 WO 2016073866A1 US 2015059490 W US2015059490 W US 2015059490W WO 2016073866 A1 WO2016073866 A1 WO 2016073866A1
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tumor
patient
cns
treatment
metastatic
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Christopher Jackson
Charles Drake
Drew Pardoll
Michael Lim
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The Johns Hopkins University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/495—Transforming growth factor [TGF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • Brain metastases afflict 20% to 40% of patients with advanced cancer and represent a major source of morbidity and mortality (1).
  • Patients receiving chemotherapy and radiation for high-grade gliomas exhibit impaired T cell homeostasis (3), and lymphopenia has been identified as a negative prognostic indicator in these patients (4). While it is unclear if these observations represent sequelae of pathology and/or treatment effect, location in the immunologically distinct CNS may play an important role in clinical outcome.
  • RNA interference RNA interference
  • the presently disclosed subject matter provides a method for the treatment of a metastatic central nervous system (CNS) tumor comprising
  • a combination treatment comprising: (a) radiotherapy; and (b) a Listeria-based vaccination.
  • the presently disclosed subject matter provides a composition for the treatment of a metastatic central nervous system (CNS) tumor comprising a radiotherapeutic agent and a Listeria-based vaccine.
  • CNS central nervous system
  • the presently disclosed subject matter provides a kit comprising: (a) a radiotherapeutic agent; (b) a Listeria-based vaccine; and (c) a package insert or label with directions to treat a patient with a metastatic central nervous system (CNS) tumor by administering a combination treatment comprising the radiotherapeutic agent and the Listeria-based vaccine.
  • a radiotherapeutic agent comprising: (a) a radiotherapeutic agent; (b) a Listeria-based vaccine; and (c) a package insert or label with directions to treat a patient with a metastatic central nervous system (CNS) tumor by administering a combination treatment comprising the radiotherapeutic agent and the Listeria-based vaccine.
  • CNS central nervous system
  • the presently disclosed subject matter provides a method of monitoring treatment efficacy of a combination treatment regimen comprising radiotherapy and Listeria-based vaccination to treat a metastatic central nervous system (CNS) tumor in a patient, the method comprising: (a) detecting the level of at least one cytokine produced from a polyfunctional lymphocyte in a patient before the patient begins the combination treatment regimen to treat the metastatic CNS tumor to obtain a baseline level of the at least one cytokine produced from the polyfunctional lymphocyte; (b) detecting the level of the at least one cytokine produced from the polyfunctional lymphocyte in the patient at one or more time intervals after the patient begins the combination treatment regimen to treat the metastatic CNS tumor; and (c) informing the patient regarding the treatment efficacy of the combination treatment, wherein the patient is informed that the treatment is effective when the level of the at least one cytokine produced from the polyfunctional lymphocyte is increased relative to the baseline level at the one or more time intervals, and wherein the patient is informed that the treatment is ineffective when
  • the presently disclosed subject matter provides a method of monitoring treatment efficacy of a combination treatment regimen comprising radiotherapy and Listeria-based vaccination to treat a metastatic central nervous system (CNS) tumor in a patient, the method comprising: (a) detecting the level of TGF- ⁇ in a patient before the patient begins the combination treatment regimen to treat the metastatic CNS tumor to obtain a baseline level of the TGF- ⁇ ; (b) detecting the level of the TGF- ⁇ in the patient at one or more time intervals after the patient begins the combination treatment regimen to treat the metastatic CNS tumor; and (c) informing the patient regarding the treatment efficacy of the combination treatment, wherein the patient is informed that the treatment is effective when the level of the TGF- ⁇ is decreased relative to the baseline level at the one or more time intervals, and wherein the patient is informed that the treatment is ineffective when the level of the TGF- ⁇ is increased relative to, or remains at or near, the baseline level, at the one or more time intervals.
  • CNS central nervous system
  • the method of monitoring treatment efficacy further comprises administering the combination treatment to the patient after the treatment efficacy is monitored.
  • treatment efficacy is monitored using a sample from the patient comprising serum, and/or blood.
  • At least one cytokine is selected from the group consisting of Granzyme B (GB), Interferon- ⁇ (IFN- ⁇ ), Tumor Necrosis Factor (TNF)- a, and Interleukin-2 (IL-2). In some embodiments, at least four cytokines are detected.
  • the metastatic CNS tumor is a brain tumor. In some embodiments, the brain tumor is not a glioma. In some embodiments, the metastatic CNS tumor metastasized from a cancer selected from melanoma, lung, breast, kidney, large intestine, small intestine, rectal, urinary tract, genital, osteosarcoma, head and neck, gastrointestinal, esophageal, and lymphoma. In some embodiments, the metastatic CNS tumor is a melanoma.
  • the combination therapy stimulates tumor infiltration by the polyfunctional lymphocyte.
  • the polyfunctional lymphocyte is a CD8+ T cell.
  • the Listeria-bassd vaccine is a live-attenuated vaccine.
  • the Listeria is Listeria monocytogenes.
  • the Listeria-bassd vaccine comprises ovalbumin (OVA) or an immunogenic part thereof.
  • the Listeria-bassd vaccine further comprises an adjuvant.
  • the radiotherapy is focal.
  • the radiotherapy is stereotactic radiosurgery, fractionated stereotactic radiosurgery, and/or intensity-modulated radiation therapy (IMRT).
  • the radiotherapy has a radiation source selected from the group consisting of a particle beam (proton), cobalt-60 (photon), and a linear accelerator (x-ray).
  • the dosage of radiotherapy ranges from about 1 Gy to about 30 Gy. In some embodiments, the dosage of radiotherapy is about 8 Gy to about 16 Gy.
  • the presently disclosed subject matter provides for the use of a composition comprising a radiotherapeutic agent and a Listeria-bassd vaccine for the treatment of a metastatic central nervous system (CNS) tumor.
  • CNS central nervous system
  • the presently disclosed subject matter provides for the use of a composition comprising a radiotherapeutic agent and a Listeria-bassd vaccine for the manufacture of a medicament for the treatment of a metastatic central nervous system (CNS) tumor.
  • a composition comprising a radiotherapeutic agent and a Listeria-bassd vaccine for the manufacture of a medicament for the treatment of a metastatic central nervous system (CNS) tumor.
  • CNS central nervous system
  • the presently disclosed subject matter provides a method for identifying a candidate agent that can be used to treat a metastatic central nervous system (CNS) tumor, the method comprising: (a) inducing metastatic central nervous system (CNS) tumor formation in a mammal; (b) determining levels of at least one cytokine produced from a polyfunctional lymphocyte in the mammal; (c) administering a test agent to the mammal; and (d) determining levels of at least one cytokine produced from a polyfunctional lymphocyte in the mammal after administration of the test agent, wherein increased levels of the at least one cytokine produced from a polyfunctional lymphocyte after administration are indicative that the test agent is a candidate agent for treating a metastatic CNS tumor.
  • the mammal comprises a rodent.
  • the rodent comprises a mouse.
  • FIG. 1 shows brain and flank tumors are of equivalent mass. Brain and flank tumor mass was not different on day 17 when tissues were harvested for analysis. Experiment conducted x 2 with > 5 mice/group; FIG. 2A, FIG. 2B, FIG. 2C and FIG. 2D show that adoptively transferred tumor antigen-specific CD8+ T cells are tolerized by CNS melanoma.
  • FIG. 2A shows representative FACS plots of Pmel (CD45.2+) CD8 T cells isolated from spleens, brain tumor DLN, and TIL.
  • FIG. 2B shows summary graphs showing percentages and numbers of Pmel CD8 T cells.
  • FIG. 2C shows
  • FIG. 2D shows summary graphs showing percentages and numbers of CD8 T cells represented by the adoptively transferred OT-1 population.
  • N 5 mice / group, repeated x 3;
  • FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D show tumor-specific CD8 T cells undergo fewer divisions and produce less IFN- ⁇ in response to CNS melanoma as compared to equivalent flank tumors.
  • FIG. 3A shows representative histograms of CFSE in cancer-specific T cells. The skilled artisan will appreciate that the values used in the histograms have been deleted from the x and y axes for purposes of compliance with formatting requirements for patent applications, and the x and y values that would be presented on the graphs are described briefly here. In particular, the longest hashmarks perpendicular to the y axis represent the values of 10°, 10 1 , 10 2 , 10 3 , and 10 4 .
  • the longest hashmarks perpendicular to the x axis represent the values of: top row, from left to right: 0, 30, 60, 90, 120 (naive, spleen); 0, 200, 400, 600, 800 (vac-OVA, spleen); 0, 30, 60, 90, 120 (flank tumor, spleen); 0, 50, 100, 150, 200, 250 (brain tumor, spleen); second row, from left to right: 0, 200, 400, 600, 800 (naive and vac-OVA, DLN); 0, 300, 600, 900, 1200 (flank tumor, DLN); 0, 500, 1000, 1500, 2000 (brain tumor, DLN); third row from left to right: 0, 5, 10, 15, 20, 25 (flank tumor, TIL); 0, 10, 20, 30 (brain tumor, TIL).
  • FIG. 3B shows summary graphs showing percentages and numbers of specific T cells undergoing > 1 division.
  • FIG. 3C shows representative FACS plots of division vs. IFN- ⁇ .
  • the skilled artisan will appreciate that the values used in the FACS plots have been deleted from the x and y axes for purposes of compliance with formatting requirements for patent applications, and the x and y values that would be presented on the graphs are described briefly here. In particular, the longest hashmarks perpendicular to the x axis and
  • FIG. 4A and FIG. 4B show that brain tumors are more tolerogenic than flank or lung tumors.
  • FIG. 4A shows representative FACS plots from tumor draining lymph nodes of mice with B 16-OVA brain, flank, or lung tumors.
  • FIG. 4B shows summary graphs of the percentage of daughter cells producing IFN- ⁇ recovered from the tumor draining lymph nodes of mice with Bl 6-OVA brain, flank, or lung tumors (5 mice/group);
  • FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, and FIG. 5F show that CNS melanoma impairs a systemic tumor-antigen directed lytic response.
  • FIG. 5A shows representative histograms showing numbers of peptide-pulsed (CSFE-high) and control (CFSE-low) cells recovered from unvaccinated mice bearing Bl 6-OVA brain or flank tumors.
  • FIG. 5B shows summary graphs showing percent target lysis in unvaccinated mice with brain or flank tumors. Each data point represents one animal.
  • FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, and FIG. 5F show that CNS melanoma impairs a systemic tumor-antigen directed lytic response.
  • FIG. 5A shows representative histograms showing numbers of peptide-pulsed (CSFE-high) and control (CFSE-low) cells recovered from unvaccinated mice bearing Bl 6-OVA brain or flank
  • 5C shows representative histograms showing OVA-pulsed and control peaks in mice with B 16-OVA brain or flank tumors after adoptive transfer of 100 OT-1 cells and vaccination with Vac-OVA.
  • FIG. 5D shows summary graphs showing percent target lysis in mice receiving adoptive transfer of 100 OT-1 cells and vaccination with Vac-OVA.
  • FIG. 5E shows representative histograms showing OVA-pulsed and control peaks in mice with B16- OVA brain and flank tumors after vaccination with LM-OVA.
  • FIG.5F shows summary graphs showing percent target lysis in mice with Bl 6-OVA brain and flank tumors after vaccination with LM-OVA. Experiments repeated x 2 with similar results.
  • N 3-10 animals per group;
  • FIG. 6A, FIG. 6B, FIG. 6C, and FIG. 6D show that elevated TGF- ⁇ is associated with systemic tolerance in animals with CNS melanoma.
  • FIG. 6A shows concentration of TGF- ⁇ in serum of mice with B16 brain or flank tumors.
  • FIG. 6B shows representative FACS plots demonstrating the percentage of CD8 T cells represented by the adoptively transferred Pmel population in the spleen and cervical lymph nodes after treatment with LY2157299.
  • the skilled artisan will appreciate that the values used in the FACS plots have been deleted from the x and y axes for purposes of compliance with formatting requirements for patent applications, and the x and y values that would be presented on the graphs are described briefly here.
  • FIG. 6C shows summary graphs showing the percentage and number of adoptively transferred Pmel cells recovered from animals with CNS melanoma after treatment with LY2157299.
  • FIG. 6D shows survival of animals with CNS melanoma treated with the TGF- ⁇ signaling inhibitor LY2157299.
  • A-C 5 animals / group, repeated x 2.
  • D 10 animals / group, repeated x 1 ;
  • FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, and FIG. 7E show that combining focal RT with vaccination improves survival in mice with established CNS melanoma.
  • FIG. 7A shows combination treatment with focal RT + TGF- ⁇ signaling inhibition.
  • FIG. 7B shows combination treatment with focal RT + LM-based vaccine.
  • FIG. 7C shows CD3 immunofluorescence in brain sections from treated mice at 20x.
  • FIG. 7D shows representative H&E micrographs of brain tissue from mice with treated brain tumors at 20x (top row) and 40x (bottom row).
  • FIG. 7E shows representative ex vivo MRI slices from mice with treated brain tumors. Experiments performed x 3 with 10 mice/group, typical results shown;
  • FIG. 8A and FIG. 8B show that TGF- ⁇ blockade does not add to RT + vaccination.
  • FIG. 1 OA and FIG. 10B show tumor morphology and volume upon death.
  • FIG. 1 OA and FIG. 10B show tumor morphology and volume upon death.
  • FIG. 10A shows morphology of tumors calculated using ex vivo MRI as described.
  • FIG. 1 1A, FIG. 1 IB, FIG. 1 1C, FIG. 1 ID, FIG. 1 IE, FIG. 1 IF, FIG. 1 1G, FIG. 1 1H, FIG. 1 II, FIG. 1 1J, FIG. 1 IK, FIG. 11L, FIG. 1 1M, and FIG. 1 IN show that combination therapy with LM-based vaccination and focal RT is associated with polyfunctional CD8 T cells, an increased Teff to Treg ratio, FIG. 11L, FIG. 1 1M, and FIG. 1 IN show that combination therapy with LM-based vaccination and focal RT is associated with polyfunctional CD8 T cells, an increased Teff to Treg ratio, FIG. 11L, FIG. 1 1M, and FIG. 1 IN show that combination therapy with LM-based vaccination and focal RT is associated with polyfunctional CD8 T cells, an increased Teff to Treg ratio, FIG. 11L, FIG. 1 1M, and FIG. 1 IN show that combination therapy with LM-based vaccination and focal RT is associated with polyfunctional CD8 T cells,
  • FIG. 1 1B and FIG. 11C show percentages and numbers, respectively, of CD8 TIL producing single and multiple cytokines.
  • FIG. 1 ID shows quantification of Treg (FoxP3+, CD4+) in treated animals.
  • FIG. 1 IE shows quantification of CD4 TIL producing IL-2.
  • FIG. 1 IF and FIG. 1 1G show Teff to Treg ratios in TIL, with effectors defined as IFNy+, or IFNg TNFa double positive respectively.
  • FIG. 1 II, and FIG. 1 1 J show CFSE dilution of OT-1 T cells cultured with pulsed APC from Spleen, DLN and microglia (CD1 lb+ CD45-mid).
  • the skilled artisan will appreciate that the values used in the histograms have been deleted from the x and y axes for purposes of compliance with formatting requirements for patent applications, and the x and y values that would be presented on the graphs are described briefly here.
  • the longest hashmarks perpendicular to the y axis and perpendicular to the x axis represent the values of 10°, 10 1 , 10 2 , 10 3 , and 10 4 .
  • FIG. UK, FIG. 11L, and FIG. 1 1M show summary graphs of the percentage of OT-1 responders producing IFN- ⁇ when co- cultured with the indicated APC populations.
  • FIG. 1 IN shows TGF- ⁇
  • FIG. 12 A, FIG. 12B, and FIG. 12C show B16-OVA brain tumors stimulate secretion of TGF- ⁇ from microglia.
  • the presently disclosed subject matter provides methods, compositions, and kits for a combination treatment comprising radiotherapy and Listeria-bassd vaccination to treat a metastatic central nervous system (CNS) tumor in a patient.
  • the presently disclosed subject matter also provides methods for monitoring treatment efficacy of a treatment regimen comprising immunotherapy (e.g., Listeria-bassd vaccination) to treat a metastatic tumor (e.g., a metastatic CNS tumor) in a patient, for example, by detecting the levels of cytokines produced from polyfunctional lymphocytes.
  • the presently disclosed subject matter also contemplates monitoring the treatment efficacy of combination treatment regiment comprising the
  • immunotherapy e.g., an immunotherapeutic agent, such as a Lister ia-based vaccination
  • radiotherapy to treat a metastatic tumor (e.g., a metastatic CNS tumor) in a patient, for example, by detecting the levels of cytokines (e.g., any combination of any three or four cytokines) produced from polyfunctional lymphocytes.
  • cytokines e.g., any combination of any three or four cytokines
  • the presently disclosed subject matter provides a method for the treatment of a metastatic central nervous system (CNS) tumor comprising administering to a patient with a metastatic CNS tumor an effective amount of a combination treatment comprising: (a) radiotherapy; and (b) an immunotherapy.
  • the immunotherapy comprises an immunotherapeutic agent.
  • immunotherapeutic agent refers to a molecule that can aid in the treatment of a disease by inducing, enhancing, or suppressing an immune response.
  • immunotherapeutic agents include, but are not limited to, interleukins (e.g., IL-2, IL-7, IL-12, IL-15), cytokines (e.g., interferons, G- CSF, imiquimod), chemokines (e.g., CCL3, CCL26, CXCL7), vaccines (e.g., peptide vaccines, dendritic cell (DC) vaccines, EGFRvIII vaccines, mesothilin vaccine, G- VAX, listeria vaccines), and adoptive T cell therapy including chimeric antigen receptor T cells (CAR T cells).
  • interleukins e.g., IL-2, IL-7, IL-12, IL-15
  • cytokines e.g., interferons, G- CSF, imiquimod
  • chemokines e
  • the immunotherapeutic agent is not an immune checkpoint inhibitor. In some embodiments, the immunotherapeutic agent is not an anti-PDl antibody, such as AMP -224, lambrolizumab, nivolumab, pidilizumab, BMS-936559, MEDI-4736, and MPDL3280A.
  • the presently disclosed subject matter provides a method for the treatment of a metastatic central nervous system (CNS) tumor comprising administering to a patient with a metastatic CNS tumor an effective amount of a combination treatment comprising: (a) radiotherapy; and (b) a Listeria- based vaccination.
  • CNS central nervous system
  • central nervous system is the complex of nerve tissues that controls the activities of a body, such as the brain and spinal cord in vertebrates.
  • a “cancer” in a patient refers to the presence of cells possessing characteristics typical of cancer-causing cells, for example, uncontrolled proliferation, loss of specialized functions, immortality, significant metastatic potential, significant increase in anti-apoptotic activity, rapid growth and proliferation rate, and certain characteristic morphology and cellular markers.
  • cancer cells will be in the form of a tumor; such cells may exist locally within an animal, or circulate in the blood stream as independent cells, for example, leukemic cells.
  • Cancer as used herein includes newly diagnosed or recurrent cancers, including without limitation, blastomas, carcinomas, gliomas, leukemias, lymphomas, melanomas, myeloma, and sarcomas.
  • Cancer as used herein includes, but is not limited to, head cancer, neck cancer, head and neck cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, esophageal cancer, stomach cancer, leukemia/lymphoma, uterine cancer, skin cancer, endocrine cancer, urinary cancer, pancreatic cancer, gastrointestinal cancer, ovarian cancer, cervical cancer, and adenomas.
  • the cancer comprises Stage 0 cancer.
  • the cancer comprises Stage I cancer.
  • the cancer comprises Stage II cancer.
  • the cancer comprises Stage III cancer.
  • the cancer comprises Stage rv cancer.
  • the cancer is refractory and/or metastatic.
  • the presently disclosed methods can be used for treating any kind of cancer.
  • the combination treatment is useful for treating metastatic cancers other than CNS cancers, such as esophageal cancers.
  • a “solid tumor”, as used herein, is an abnormal mass of tissue that generally does not contain cysts or liquid areas.
  • a "metastatic CNS tumor” refers to a tumor that originated somewhere else in the body and metastasized to the CNS.
  • the metastatic CNS tumor metastasized from a cancer selected from melanoma, lung, breast, kidney, large intestine, small intestine, rectal, urinary tract, genital, osteosarcoma, head and neck, gastrointestinal, esophageal, and lymphoma.
  • the metastatic CNS tumor is a melanoma, a tumor consisting of melanin-forming cells.
  • the metastatic CNS tumor is a brain tumor, such that a cancer from somewhere else metastasized to the brain.
  • the brain tumor is not a glioma, a tumor of the glial tissue of the nervous system.
  • the radiotherapy is focal, such that the radiotherapy is locally administered in the treatment of the tumor, such as intratumorally and/or within the tumor bed.
  • Radiation methods suitable for use with the presently disclosed methods include, but are not limited to, stereotactic radiosurgery, fractionated stereotactic radiosurgery, and intensity-modulated radiation therapy (IMRT).
  • stereotactic radiosurgery involves the precise delivery of radiation to a tumorous tissue, for example, a brain tumor, while avoiding the surrounding non-tumorous, normal tissue. Because stereotactic radiosurgery is so precise, it allows a higher dose of radiation to be given with more sparing of normal tissue than can be achieved with conventional radiotherapy techniques.
  • MRI magnetic resonance imaging
  • CT computed tomography
  • 3-D three-dimensional
  • a complex radiation delivery planning system is used to target a high dose of radiation at the tumor while greatly limiting the dose to nearby normal tissue. Special devices are used to keep the subject still so that the radiation will be aimed with great accuracy at the targeted tumor.
  • stereotactic radiation need not be delivered in a single treatment.
  • the treatment plan can be reliably duplicated day-today, thereby allowing multiple fractionated doses of radiation to be delivered.
  • the radiosurgery is referred to as "fractionated stereotactic radiosurgery" or FSR.
  • stereotactic radiosurgery refers to a one-session treatment.
  • fractionation allows higher doses to be delivered to tumorous tissue because of an increased tolerance of the surrounding normal tissue to these smaller fractionated doses. Accordingly, while single-dose stereotactic radiation takes advantage of the pattern of radiation given, fractionated stereotactic radiation takes advantage of not only the pattern of radiation, but also of the differing radiosensitivities of normal and surrounding tumorous tissues. Another advantage of fractionated stereotactic radiation is so-called "iterative" treatment, in which the shape and intensity of the treatment plan can be modified during the course of therapy.
  • Fractionated stereotactic radiosurgery can result in a high therapeutic ratio, i.e., a high rate of killing of tumor cells and a low effect on normal tissue.
  • the tumor and the normal tissue respond differently to high single doses of radiation vs. multiple smaller doses of radiation.
  • Single large doses of radiation can kill more normal tissue than several smaller doses of radiation can. Accordingly, multiple smaller doses of radiation can kill more tumor cells while sparing normal tissue.
  • multiple smaller doses are administered every day over weeks, such as for 1, 2, 3, 4, 5, 6, 7 or more weeks.
  • multiple smaller doses are administered several times a day, several times a week, weekly, bimonthly, or monthly, for example.
  • the frequency of administration of the fractionated radiotherapy varies depending on the size of the tumor, the location of the tumor, the aggressiveness of the tumor, the intensity of the radiation, and the like.
  • stereotactic radiation treatment Another advance in stereotactic radiation treatment is the development of three-dimensional images of the tumor and surrounding tissues.
  • Sophisticated software can take small, e.g., 2-mm, cuts from either CT or MRI scans and converts them into three-dimensional images.
  • Three-dimensional treatment planning delivers a high-precision dose to the tumor, while sparing normal tissue, and can achieve more efficacious results than can be achieved with two-dimensional planning.
  • stereotactic radiosurgery can be characterized by the source of radiation used, including particle beam (proton), cobalt-60 (photon-Gamma Knife.RTM.), and linear accelerator (x- ray).
  • a linear accelerator produces high-energy X-ray radiation and is capable of delivering precise and accurate doses of radiation required for radiosurgery.
  • Radiosurgery using a linear accelerator is typically carried out in multi-session, smaller dose treatments so that healthy surrounding tissue is not damaged from too high a dose of radiation. Radiosurgery using linear accelerator technology also is able to target larger brain cancers with less damage to healthy tissues.
  • a "gamma knife” uses multiple, e.g., 192 or 201, highly-focused x-ray beams to make up the "knife” that cuts through diseased tissue.
  • the gamma knife uses precisely targeted beams of radiation that converge on a single point to painlessly “cut” through brain tumors.
  • a gamma knife makes it possible to reach the deepest recesses of the brain and correct disorders not treatable with conventional surgery.
  • proton beam radiation offers certain theoretical advantages over other modalities of stereotactic radiosurgery (e.g., Gamma Knife.RTM. and linear accelerators), because it makes use of the quantum wave properties of protons to reduce doses of radiation to surrounding tissue beyond the target tissue.
  • stereotactic radiosurgery e.g., Gamma Knife.RTM. and linear accelerators
  • the proton beam radiation offers advantages for treating unusually shaped brain tumors.
  • the homogeneous doses of radiation delivered by a proton beam source also make fractionated therapy possible.
  • Proton beam radiosurgery also has the ability to treat tumors outside of the cranial cavity. These properties make proton beam radiosurgery efficacious for post- resection therapy for many chordomas and certain chondrosarchomas of the spine and skull base.
  • IMRT intensity-modulated radiation therapy
  • 3DCRT three-dimensional conformal radiation therapy
  • MLC multileaf collimator
  • IMRT allows the radiation dose to conform more precisely to the three-dimensional (3-D) shape of the tumor by modulating the intensity of the radiation beam in multiple small volumes.
  • IMRT allows higher radiation doses to be focused to regions within the tumor while minimizing the dose to surrounding normal critical structures.
  • IMRT improves the ability to conform the treatment volume to concave tumor shapes, for example, when the tumor is wrapped around a vulnerable structure, such as the spinal cord.
  • IMRT Treatment with IMRT is planned by using 3-D computed tomography (CT) or magnetic resonance (MRI) images of the patient in conjunction with computerized dose calculations to determine the dose intensity pattern that will best conform to the tumor shape.
  • CT computed tomography
  • MRI magnetic resonance
  • combinations of multiple intensity-modulated fields coming from different beam directions produce a custom tailored radiation dose that maximizes tumor dose while also minimizing the dose to adjacent normal tissues.
  • IMRT typically is used to treat cancers of the prostate, head and neck, and central nervous system.
  • the dosage of radiation applied can vary. In some embodiments, the dosage can range from 1 Gy to about 30 Gy, and can encompass intermediate ranges including, for example, from 1 to 5, 10, 15, 20, 25, up to 30 Gy in dose. In some embodiments, the dosage of radiotherapy is about 8 Gy to about 16 Gy.
  • a "vaccine” is a substance that promotes the immune system to attack a tumor, such as a metastatic CNS tumor.
  • the vaccine increases the immune response against cancer cells that are already in the body.
  • the vaccine can be combined with other substances or cells called adjuvants that help boost the immune response.
  • adjuvants include cholera toxin, cytokines, chemokines, and bacterial nucleic acid sequences, like CpG.
  • the combination treatment comprises a vaccine, such as a Listeria-based vaccine.
  • a vaccine such as a Listeria-based vaccine.
  • any Listeria species capable of producing infectious disease can be genetically attenuated according to the methods of the presently disclosed subject matter to yield a useful and safe vaccine.
  • the Listeria can be a pathogenic organism
  • the Listeria is "attenuated” or “live-attenuated”.
  • attenuated refers to a process by which a pathogen is modified to lessen or eliminate its pathogenicity, but retains its ability to act as a prophylactic or therapeutic for the disease of interest.
  • live-attenuated refers to a process by which a pathogen is modified to lessen or eliminate its pathogenicity, but it is still kept viable or alive.
  • Bacterial attenuation can be achieved by different mechanisms. One is to introduce mutations into one or more metabolic pathways, the function of which is essential for bacteria to survive and grow in vivo to cause disease.
  • the bacterium is mutated to lessen or prevent the ability to grow and spread intracellularly.
  • the uvrAB genes are deleted from the Listeria genome.
  • the Listeria can comprise a mutation that inactivates ActA.
  • the Listeria can comprise a mutation that inactivates InlB.
  • the Listeria genome comprises a
  • an attenuated, metabolically active Listeria is deleted for its native ActA, inlB, and uvrAB genes (AactAAinlBAuvr).
  • the presently disclosed subject matter employs a Listeria that is killed but metabolically active ("KBMA").
  • the Listeria is Listeria monocytogenes, a small, cocci shaped Gram-positive rod shaped bacterium that is a member of the Family
  • the use of L. monocytogenes in generating attenuated mutants for the vaccines of the presently disclosed subject matter may be substituted by other suitable Listeria species to generate similar attenuated mutants.
  • the Listeria-based vaccine comprises a live-attenuated Listeria monocytogenes vaccine.
  • the live-attenuated Listeria monocytogenes vaccine comprises a deletion of a gene, such as ActA, inlB, or uvrAB.
  • the live-attenuated Listeria monocytogenes vaccine comprises a deletion of a combination of two or more genes, such as two genes selected from ActA, inlB, and uvrAB.
  • the live-attenuated Listeria monocytogenes vaccine comprises a deletion of a combination of three or more genes, such as ActA, inlB, and uvrAB.
  • the Listeria-based vaccine comprising a selected attenuated strain of Listeria expresses a foreign antigen capable of causing the production of a cell- mediated immune response.
  • the Listeria- based vaccine comprises ovalbumin (OVA) or an immunogenic part thereof.
  • the ovalbumin gene is incorporated into the Listeria genome.
  • the combination treatment increases the
  • polyfunctional lymphocytes is stimulated.
  • lymphocyte refers to a type of white blood cell that is part of the immune system, such as a T cell, a B cell, and a natural killer (NK) cell.
  • NK natural killer
  • polyfunctional lymphocyte refers to a lymphocyte that produces more than one cytokine, such as 2, 3, 4, or more cytokines.
  • the polyfunctional lymphocyte is a T cell.
  • the polyfunctional lymphocyte is a CD 8+ T cell.
  • Non- limiting examples of cytokines include TNF-a, Granzyme B (GB); an interleukin, such as IL-1, IL-2, IL-4, IL-3, IL-4, 1 IL-5, IL-6, IL-8, IL-10, and IL-12; and an interferon, such as IFN- a, IFN- ⁇ , and IFN- ⁇ .
  • the number of cytokines being produced by the polyfunctional lymphocytes is used for the presently disclosed methods, such as to determine the efficacy of an immunotherapy treatment regiment (e.g., combination treatment). Accordingly, in some embodiments, any combination of cytokines can be used for the presently disclosed methods.
  • At least one cytokine is selected from the group consisting of Granzyme B (GB), Interferon- ⁇ (IFN- ⁇ ), Tumor Necrosis Factor (TNF)-a, and Interleukin-2 (IL- 2) ⁇
  • GB Granzyme B
  • IFN- ⁇ Interferon- ⁇
  • TNF Tumor Necrosis Factor
  • IL-2 Interleukin-2
  • a metastatic CNS tumor results in an increase in levels of TGF- ⁇ produced from microglia but a decrease in the polyfunctionality of lymphocytes, resulting in a decrease in the levels of other cytokines. It has been found herein that the combination treatment decreases the levels of TGF- ⁇ from microglia, but increases the levels of other cytokines produced from polyfunctional lymphocytes.
  • the combination treatment increases the polyfunctionality of lymphocytes resulting in an increase in the production of cytokines from the lymphocytes, but also decreases the production of the cytokine TGF- ⁇ produced from microglia.
  • TGF- ⁇ refers to any isoform of TGF- ⁇ , such as TGF- ⁇ , TGF- ⁇ 2, and TGF- ⁇ 3.
  • the TGF- ⁇ produced from the microglia is TGF- ⁇ .
  • the TGF- ⁇ 2 isoform is measured using the presently disclosed methods.
  • the presently disclosed subject matter also provides a composition for the treatment of a metastatic CNS tumor comprising a
  • the presently disclosed subject matter provides a composition for the treatment of a metastatic CNS tumor comprising a radiotherapeutic agent and a Listeria-bassd vaccine.
  • a radiotherapeutic agent refers to those agents
  • the radiation may be high-LET (linear energy transfer) or low-LET.
  • LET is the energy transferred per unit length of the distance.
  • High LET is said to be densely ionizing radiation and
  • Low LET is said to be sparsely ionizing radiation.
  • Representative examples of high-LET are neutrons and alpha particles.
  • Representative examples of low-LET are x-ray and gamma rays.
  • Low LET radiation including both x-rays and ⁇ - rays is most commonly used for radiotherapy of cancer patients.
  • the radiation may be used for external radiation therapy that is usually given on an outpatient basis or for internal radiation therapy that uses radiation that is placed very close to or inside the tumor.
  • the radiation source is usually sealed in a small holder called an implant.
  • Implants may be in the form of thin wires, plastic tubes called catheters, ribbons, capsules, or seeds. The implant is put directly into the body. Internal radiation therapy may require a hospital stay.
  • the ionizing radiation source is provided as a unit dose of radiation and is preferably an x-ray tube since it provides many advantages, such as convenient adjustable dosing where the source may be easily turned on and off, minimal disposal problems, and the like.
  • a unit dose of radiation is generally measured in gray (Gy).
  • the ionizing radiation source may also comprise a radioisotope, such as a solid radioisotopic source (e.g., wire, strip, pellet, seed, bead, or the like), or a liquid radioisotopic filled balloon. In the latter case, the balloon has been specially configured to prevent leakage of the radioisotopic material from the balloon into the body lumen or blood stream.
  • the ionizing radiation source may comprise a receptacle in the catheter body for receiving radioisotopic materials like pellets or liquids.
  • the radioisotopic material may be selected to emit ⁇ , ⁇ and ⁇ .
  • a and ⁇ radiations are preferred since they may be quickly absorbed by the surrounding tissue and will not penetrate substantially beyond the wall of the body lumen being treated. Accordingly, incidental irradiation of the heart and other organs adjacent to the treatment region can be substantially eliminated.
  • the total number of units provided will be an amount determined to be therapeutically effective by one skilled in treatment using ionizing radiation. This amount will vary with the subject and the type of malignancy or neoplasm being treated. The amount may vary but a patient may receive a dosage of about 30-75 Gy over several weeks.
  • Radiotherapeutic agents include factors that cause DNA damage, such as .gamma. -rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells.
  • Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
  • Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the target cell.
  • the radiotherapeutic agent is selected from the group consisting of 47 Sc, 67 Cu, 90 Y, 109 Pd, 123 1, 125 I, 131 I, 186 Re, 188 Re, 199 Au, 211 At, 212 Pb, 212 B, 32 P and 33 P, 71 Ge, 77 As, 103 Pb, 105 Rh, m Ag, 119 Sb, 121 Sn, 131 Cs, 143 Pr, 161 Tb, 177 Lu, 191 Os, 193 MPt, 197 H, 43 K, 52 Fe, 57 Co, 67 Cu, 67 Ga, 68 Ga, 77 Br, 81 Rb/.
  • the presently disclosed subject matter provides the use of a composition for the treatment of a metastatic central nervous system (CNS) tumor comprising a radiotherapeutic agent and an immunotherapeutic agent, such as a Listeria -based vaccine.
  • the presently disclosed subject matter provides the use of a composition for the treatment of a metastatic central nervous system (CNS) tumor comprising a radiotherapeutic agent and an immunotherapeutic agent, such as a Listeria-bassd vaccine, for the manufacture of a medicament for the treatment of a metastatic central nervous system (CNS) tumor.
  • the composition does not include an anti-PD 1 antibody.
  • treating can include reversing, alleviating, inhibiting the progression of, preventing or reducing the likelihood of the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder or condition (e.g., cancer).
  • the combination treatment reduces the likelihood of tumor progression and/or mediates tumor regression.
  • the combination treatment can reduce the likelihood of tumor progression and/or mediate tumor regression by at least 5%, 10%, 15%, 20%, 25%, 30%, 33%, 35%, 40%, 45%, 50%, 55%, 60%, 66%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more as compared to the likelihood of tumor progression and/or tumor regression in the patient when the combination therapy is not given, such as if the patient is treated with monotherapy treatment, such as radiotherapy alone or an immunotherapeutic agent, such as a Listeria vaccine alone.
  • monotherapy treatment such as radiotherapy alone or an immunotherapeutic agent, such as a Listeria vaccine alone.
  • the combination treatment completely inhibits tumor progression in the patient. In some embodiments, the combination treatment mediates complete tumor regression. In some embodiments, the combination treatment reduces the likelihood of tumor progression and/or mediates tumor regression by at least approximately 50% as compared to the likelihood of tumor progression and/or mediation of tumor regression in the patient when the combination treatment is not given, such as if the patient is treated with monotherapy treatment, such as radiotherapy alone, or an
  • immunotherapeutic agent such as a Listeria vaccine alone.
  • the combination treatment extends survival of the patient.
  • the combination treatment can extend survival (e.g., progression free survival) of the patient by 5%, 10%, 15%, 20%, 25%, 30%, 33%, 35%, 40%, 45%, 50%, 55%, 60%, 66%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 1-fold, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0 fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 5.0-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more as compared to survival of the patient when the combination therapy is not given, such as if the patient is treated with monotherapy treatment, such as radiotherapy alone or an immunotherapeutic agent, such as a Listeria vaccine alone.
  • monotherapy treatment
  • the combination treatment extends survival of the patient by at least approximately 40% as compared to survival of the patient when the combination treatment is not given, such as if the patient is treated with monotherapy treatment, such as with radiotherapy alone or an immunotherapeutic agent, such as a Listeria vaccine alone. In some embodiments, the combination treatment extends progression free survival of the patient until the patient succumbs to another disease, disorder, or condition, or dies naturally as a result of old age.
  • the presently disclosed subject matter provides a method for extending survival of a patient with a metastatic CNS tumor, the method comprising administering to the patient an effective amount of a combination treatment comprising: (a) radiotherapy; and (b) an immunotherapy (e.g., an immunotherapeutic agent.
  • a combination treatment comprising: (a) radiotherapy; and (b) an immunotherapy (e.g., an immunotherapeutic agent.
  • the presently disclosed subject matter provides a method for extending survival of a patient with a metastatic CNS tumor, the method comprising administering to the patient an effective amount of a combination treatment comprising: (a) radiotherapy; and (b) a Listeria-bassd vaccination.
  • the survival is progression-free survival. In some embodiments, survival is extended by at least 40%.
  • the term “reduce” or “inhibit,” and grammatical derivations thereof, refers to the ability of an agent to block, partially block, interfere, decrease, reduce or deactivate a biological molecule, pathway or mechanism of action.
  • the term “inhibit” encompasses a complete and/or partial loss of activity, e.g., a loss in activity by at least 10%, in some embodiments, a loss in activity by at least 20%, 30%, 50%, 75%, 95%, 98%, and up to and including 100%.
  • a “subject” can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes.
  • Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like.
  • mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; cap
  • An animal may be a transgenic animal.
  • the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects.
  • a "subject" can include a patient afflicted with or suspected of being afflicted with a condition or disease.
  • compositions e.g., comprising a radiotherapeutic agent and an immunotherapeutic agent, such as a Listeria-bassd vaccine
  • a subject for therapy can be administered by any suitable route of administration, including orally, nasally, transmucosally, ocularly, rectally, intravaginally, parenterally, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intra-articular, intra-sternal, intra-synovial, intra-hepatic, intralesional, intracranial, intraperitoneal, intranasal, or intraocular injections, intracisternally, topically, as by powders, ointments or drops (including eyedrops), including buccally and sublingually, transdermally, through an inhalation spray, or other modes of delivery known in the art.
  • any suitable route of administration including orally, nasally, transmucosally, ocularly, rectally, intravagina
  • the presently disclosed compositions are administered locally, such as intratumorally, so that the compositions are directly administered into a solid tumor (or injected or implanted into a microenvironment in which the solid tumor resides).
  • intratumoral administration comprises injection into a solid tumor of the patient or injection or implantation into a microenvironment in which the solid tumor resides or resided.
  • the means of administration into a solid tumor include a needle, needle-less injection device, or any other means by which the radiotherapeutic agent and an immunotherapeutic agent, such as a Listeria-based vaccine, can be administered locally. It should be appreciated that all or a portion of the solid tumor may be surgically removed prior to administration of the presently disclosed combination treatment. In some embodiments, the methods further comprise surgically removing all or a portion of the solid tumor prior to administration of the combination treatment.
  • peripheral administration and “administered peripherally” as used herein mean the administration of compositions comprising a radiotherapeutic agent and an immunotherapeutic agent, such as a Listeria-based vaccine, such that they enter the patient's system and, thus, are subject to metabolism and other like processes, for example, subcutaneous administration.
  • an immunotherapeutic agent such as a Listeria-based vaccine
  • parenteral administration and “administered parenterally” as used herein mean modes of administration other than enteral and topical
  • administration usually by injection, and includes, without limitation, intravenous, intramuscular, intarterial, intrathecal, intracapsular, intraorbital, intraocular, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • compositions can be manufactured in a manner known in the art, e.g. by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • the presently disclosed pharmaceutical compositions can be administered by rechargeable or biodegradable devices.
  • a variety of slow-release polymeric devices have been developed and tested in vivo for the controlled delivery of drugs, including proteinacious biopharmaceuticals.
  • Suitable examples of sustained release preparations include semipermeable polymer matrices in the form of shaped articles, e.g., films or microcapsules.
  • Sustained release matrices include polyesters, hydrogels, polylactides (U.S. Patent No.
  • Sustained release compositions also include liposomally entrapped compositions comprising a radiotherapeutic agent combined with an immunotherapeutic agent, such as a Listeria-bassd vaccine, which can be prepared by methods known in the art (Epstein et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:3688; Hwang et al. (1980) Proc. Natl. Acad. Sci. U.S.A. 77:4030; U.S. Patent Nos. 4,485,045 and 4,544,545; and EP 102,324A).
  • a radiotherapeutic agent such as a Listeria-bassd vaccine
  • the liposomes are of the small (about 200-800 angstroms) unilamelar type in which the lipid content is greater than about 30 mol % cholesterol, the selected proportion being adjusted for the optimal therapy.
  • Such materials can comprise an implant, for example, for sustained release of the presently disclosed compositions, which, in some embodiments, can be implanted at a particular, pre-determined target site, such as at a solid tumor, or at a site at which a solid tumor has been surgically removed.
  • the presently disclosed pharmaceutical compositions may comprise PEGylated therapeutics (e.g., PEGylated antibodies).
  • PEGylation is a well established and validated approach for the modification of a range of antibodies, proteins, and peptides and involves the attachment of polyethylene glycol (PEG) at specific sites of the antibodies, proteins, and peptides (Chapman (2002) Adv. Drug Deliv. Rev. 54:531-545).
  • PEGylation results include: (a) markedly improved circulating half-lives in vivo due to either evasion of renal clearance as a result of the polymer increasing the apparent size of the molecule to above the glomerular filtration limit, and/or through evasion of cellular clearance mechanisms; (b) improved pharmacokinetics; (c) improved solubility— PEG has been found to be soluble in many different solvents, ranging from water to many organic solvents such as toluene, methylene chloride, ethanol and acetone; (d) PEGylated antibody fragments can be concentrated to 200 mg/ml, and the ability to do so opens up formulation and dosing options such as subcutaneous administration of a high protein dose; this is in contrast to many other therapeutic antibodies which are typically administered intravenously; (e) enhanced proteolytic resistance of the conjugated protein (Cunningham-Rundles et.al.
  • compositions for parenteral administration include aqueous solutions of compositions comprising a radiotherapeutic agent and an
  • the presently disclosed pharmaceutical compositions can be formulated in aqueous solutions, for example, in some embodiments, in physiologically compatible buffers, such as Hank's solution, Ringer's solution, or physiologically buffered saline.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of compositions include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • the suspension also can contain suitable stabilizers or agents that increase the solubility of the compositions comprising a radiotherapeutic agent and an immunotherapeutic agent, such as a Listeria-based vaccine, to allow for the preparation of highly concentrated solutions.
  • suitable stabilizers or agents that increase the solubility of the compositions comprising a radiotherapeutic agent and an immunotherapeutic agent, such as a Listeria-based vaccine, to allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • the agents of the disclosure also can be formulated by methods known to those of skill in the art, and may include, for example, but not limited to, examples of solubilizing, diluting, or dispersing substances such as, saline, preservatives, such as benzyl alcohol, absorption promoters, and fluorocarbons.
  • fragrances, opacifiers, antioxidants, gelling agents, stabilizers, surfactants, emollients, coloring agents, preservatives, buffering agents, and the like can be present.
  • the pH of the presently disclosed topical composition can be adjusted to a physiologically acceptable range of from about 6.0 to about 9.0 by adding buffering agents thereto such that the composition is physiologically compatible with a subject's skin.
  • compositions are formulated into pharmaceutically acceptable dosage forms such as described herein or by other conventional methods known to those of skill in the art.
  • the "effective amount” or “therapeutically effective amount” of an active agent or drug delivery device refers to the amount necessary to elicit the desired biological response.
  • the effective amount of an agent or device may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the encapsulating matrix, the target tissue, and the like.
  • the term “combination” is used in its broadest sense and means that a subject is administered at least two agents, more particularly a radiotherapeutic agent and an immunotherapeutic agent, such as a Listeria-bassd vaccine. More particularly, the term “in combination” refers to the concomitant administration of two (or more) active agents for the treatment of a, e.g., single disease state.
  • the active agents may be combined and administered in a single dosage form, may be administered as separate dosage forms at the same time, or may be administered as separate dosage forms that are administered alternately or sequentially on the same or separate days. In some embodiments of the presently disclosed subject matter, the active agents are combined and administered in a single dosage form.
  • the active agents are administered in separate dosage forms (e.g., wherein it is desirable to vary the amount of one but not the other).
  • the single dosage form may include additional active agents for the treatment of the disease state.
  • a radiotherapeutic agent is administered before an
  • an immunotherapeutic agent such as a Listeria-based vaccine.
  • an immunotherapeutic agent such as a Listeria-based vaccine
  • the combination treatment is performed and then one of the active agents, such as the immunotherapeutic agent, e.g. a Listeria-based vaccine, is given separately, for e.g., as a booster vaccine.
  • compositions can be administered alone or in combination with adjuvants that enhance stability of the agents, facilitate
  • compositions containing them in certain embodiments, provide increased dissolution or dispersion, increase activity, provide adjuvant therapy, and the like, including other active ingredients.
  • combination therapies utilize lower dosages of the conventional therapeutics, thus avoiding possible toxicity and adverse side effects incurred when those agents are used as monotherapies.
  • a radiotherapeutic agent combined with an immunotherapeutic agent such as a Listeria-based vaccine
  • an immunotherapeutic agent such as a Listeria-based vaccine
  • additional agents either simultaneously, sequentially, or a combination thereof.
  • a subject administered a combination of a radiotherapeutic agent and an immunotherapeutic agent such as a Listeria-bassd vaccine, and, optionally, additional agents can receive a radiotherapeutic agent combined with an immunotherapeutic agent, such as a Listeria-based vaccine and, optionally, additional agents at the same time (i.e., simultaneously) or at different times (i.e., sequentially, in either order, on the same day or on different days), so long as the effect of the combination of all agents is achieved in the subject.
  • agents administered sequentially can be administered within 1, 5, 10, 30, 60, 120, 180, 240 minutes or longer of one another. In other embodiments, agents administered sequentially, can be administered within 1, 2, 3, 4, 5, 10, 15, 20 or more days of one another. Where the agents are administered simultaneously, they can be administered to the subject as separate pharmaceutical compositions, each comprising either a radiotherapeutic agent or an immunotherapeutic agent, such as a Listeria-based vaccine and, optionally, additional agents, or they can be administered to a subject as a single pharmaceutical composition comprising all agents. In some embodiments, one agent, such as the radiotherapeutic agent, is administered and the other agent, such as the immunotherapeutic agent, e.g. a Listeria-based vaccine, is administered three days later. In some embodiments, one agent, such as the radiotherapeutic agent, is administered and the other agent, such as the
  • immunotherapeutic agent e.g. a Listeria-based vaccine
  • a booster immunotherapeutic agent such as a Listeria-based vaccine
  • the effective concentration of each of the agents to elicit a particular biological response may be less than the effective concentration of each agent when administered alone, thereby allowing a reduction in the dose of one or more of the agents relative to the dose that would be needed if the agent was administered as a single agent.
  • the effects of multiple agents may, but need not be, additive or synergistic.
  • the agents may be administered multiple times.
  • the two or more agents can have a synergistic effect.
  • “synergistic,” “synergistically” and derivations thereof, such as in a “synergistic effect” or a “synergistic combination” or a “synergistic composition” refer to circumstances under which the biological activity of a combination of an agent and at least one additional therapeutic agent is greater than the sum of the biological activities of the respective agents when administered individually.
  • Synergy can be expressed in terms of a "Synergy Index (SI)," which generally can be determined by the method described by F. C. Kull et al. Applied Microbiology 9, 538 (1961), from the ratio determined by:
  • SI Synergy Index
  • Q A is the concentration of a component A, acting alone, which produced an end point in relation to component A;
  • Q a is the concentration of component A, in a mixture, which produced an end point
  • Q B is the concentration of a component B, acting alone, which produced an end point in relation to component B;
  • Qb is the concentration of component B, in a mixture, which produced an end point.
  • a "synergistic combination” has an activity higher that what can be expected based on the observed activities of the individual components when used alone.
  • a “synergistically effective amount" of a component refers to the amount of the component necessary to elicit a synergistic effect in, for example, another therapeutic agent present in the composition.
  • the presently disclosed subject matter provides a pharmaceutical composition including a radiotherapeutic agent combined with an immunotherapeutic agent, such as a Listeria-based vaccine, optionally, additional agents, alone or in combination with one or more additional therapeutic agents in admixture with a pharmaceutically acceptable excipient.
  • a radiotherapeutic agent such as a Listeria-based vaccine
  • additional agents alone or in combination with one or more additional therapeutic agents in admixture with a pharmaceutically acceptable excipient.
  • the presently disclosed subject matter provides a pharmaceutical composition
  • a pharmaceutical composition comprising a radiotherapeutic agent combined with an immunotherapeutic agent, such as a Listeria-based vaccine and, optionally, additional agents and a pharmaceutically acceptable carrier.
  • the compounds of the disclosure can be formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remington: The Science and Practice of Pharmacy (20 th ed.) Lippincott, Williams and Wilkins (2000).
  • compositions of the present disclosure in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection.
  • the compounds can be formulated readily using
  • Such carriers enable the compounds of the disclosure to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject (e.g., patient) to be treated.
  • the agents of the disclosure also may be formulated by methods known to those of skill in the art, and may include, for example, but not limited to, examples of solubilizing, diluting, or dispersing substances, such as saline; preservatives, such as benzyl alcohol; absorption promoters; and fluorocarbons.
  • compositions suitable for use in the present disclosure include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. Generally, the compounds according to the disclosure are effective over a wide dosage range. For example, in the treatment of adult humans, dosages from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, and from 5 to 40 mg per day are examples of dosages that may be used. A non-limiting dosage is 10 to 30 mg per day. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician.
  • these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.
  • a method for monitoring treatment efficacy of a reatment regimen comprising an immunotherapy (e.g., immunotherapeutic agent) to treat a metastatic tumor in a patient comprising: (a) detecting the level of at least one cytokine produced from a polyfunctional lymphocyte in a patient before the patient begins the treatment regimen to treat the metastatic tumor to obtain a baseline level of the at least one cytokine produced from the polyfunctional lymphocyte; (b) detecting the level of the at least one cytokine produced from the polyfunctional lymphocyte in the patient at one or more time intervals after the patient begins the treatment regimen to treat the metastatic tumor; and (c) informing the patient regarding the treatment efficacy of the treatment, wherein the patient is informed that the treatment is effective when the level of the at least one cytokine produced from the polyfunctional lymphocyte is increased relative to the baseline level at the one or more time intervals, and wherein the patient is informed that the treatment is ineffective when the level of the at least one cytokin
  • an immunotherapy e.g
  • the immunotherapy does not comprise an immune checkpoint inhibitor, such as anti-PD l antibody.
  • the method is used to monitor treatment efficacy of a combination treatment regimen comprising the immunotherapy and radiotherapy.
  • a method for monitoring treatment efficacy of a combination treatment regimen comprising radiotherapy and an
  • the method comprising: (a) detecting the level of at least one cytokine produced from a polyfunctional lymphocyte in a patient before the patient begins the combination treatment regimen to treat the metastatic CNS tumor to obtain a baseline level of the at least one cytokine produced from the polyfunctional lymphocyte; (b) detecting the level of the at least one cytokine produced from the polyfunctional lymphocyte in the patient at one or more time intervals after the patient begins the combination treatment regimen to treat the metastatic CNS tumor; and (c) informing the patient regarding the treatment efficacy of the combination treatment, wherein the patient is informed that the treatment is effective when the level of the at least one cytokine produced from the polyfunctional lymphocyte is increased relative to the baseline level at the one or more time intervals, and wherein the patient is informed that the treatment is ineffective when the level of the at least one cytokine produced from the polyfunctional lymphocyte is decreased relative to, or remains at or near, the baseline level
  • a method for monitoring treatment efficacy of a combination treatment regimen comprising radiotherapy and Listeria-bassd vaccination to treat a metastatic central nervous system (CNS) tumor in a patient comprising: (a) detecting the level of at least one cytokine produced from a polyfunctional lymphocyte in a patient before the patient begins the combination treatment regimen to treat the metastatic CNS tumor to obtain a baseline level of the at least one cytokine produced from the polyfunctional lymphocyte; (b) detecting the level of the at least one cytokine produced from the polyfunctional lymphocyte in the patient at one or more time intervals after the patient begins the combination treatment regimen to treat the metastatic CNS tumor; and (c) informing the patient regarding the treatment efficacy of the combination treatment, wherein the patient is informed that the treatment is effective when the level of the at least one cytokine produced from the polyfunctional lymphocyte is increased relative to the baseline level at the one or more time intervals, and wherein the patient is informed that the treatment is ineffective when the level
  • CNS central nervous system
  • the levels of more than one cytokine are detected, such as 2, 3, 4, 5, 6, 7, 8 or more. In some embodiments, the levels of at least one cytokine are detected. In some embodiments, the levels of at least two cytokines are detected. In some embodiments, the levels of at least three cytokines are detected. In some embodiments, the levels of at least four cytokines are detected. In some
  • the levels of at least five cytokines are detected.
  • at least one cytokine is selected from the group consisting of Granzyme B (GB), IFN- ⁇ , Tumor Necrosis Factor (TNF)-a, and IL-2.
  • the presently disclosed subject matter provides a method of monitoring treatment efficacy of a treatment regimen comprising immunotherapy (e.g., an immunotherapeutic agent administered as a monotherapy, a combination treatment regiment comprising an immunotherapeutic agent and radiotherapy) to treat a tumor (e.g., metastatic central nervous system (CNS) tumor) in a patient, the method comprising: (a) detecting the level of TGF- ⁇ in a patient before the patient begins the combination treatment regimen to treat the tumor (e.g., metastatic CNS tumor) to obtain a baseline level of the TGF- ⁇ ; (b) detecting the level of the TGF- ⁇ in the patient at one or more time intervals after the patient begins the treatment regimen to treat the tumor (e.g., metastatic CNS tumor); and (c) informing the patient regarding the treatment efficacy of the treatment, wherein the patient is informed that the treatment is effective when the level of the TGF- ⁇ is decreased relative to the baseline level at the one or more time intervals, and wherein the patient
  • immunotherapy
  • the presently disclosed subject matter provides a method of monitoring treatment efficacy of a combination treatment regimen comprising radiotherapy and Listeria-bassd vaccination to treat a metastatic central nervous system (CNS) tumor in a patient, the method comprising: (a) detecting the level of TGF- ⁇ in a patient before the patient begins the combination treatment regimen to treat the metastatic CNS tumor to obtain a baseline level of the TGF- ⁇ ; (b) detecting the level of the TGF- ⁇ in the patient at one or more time intervals after the patient begins the combination treatment regimen to treat the metastatic CNS tumor; and (c) informing the patient regarding the treatment efficacy of the combination treatment, wherein the patient is informed that the treatment is effective when the level of the TGF- ⁇ is decreased relative to the baseline level at the one or more time intervals, and wherein the patient is informed that the treatment is ineffective when the level of the TGF- ⁇ is increased relative to, or remains at or near, the baseline level, at the one or more time intervals.
  • CNS central nervous system
  • the time interval for monitoring treatment efficacy will depend on certain conditions, such as how aggressive the tumor is, the size of the tumor, the time required for the treatment to be effective, and the like.
  • the time interval will be weekly.
  • the time interval will be biweekly.
  • the time interval will be every three weeks.
  • the time interval will be every month.
  • the time interval will be more than one month apart.
  • the time interval will vary depending on the effectiveness of the treatment and other factors, for e.g., monitoring may occur every week and then change to every month.
  • the treatment is considered “effective" when the metastatic CNS tumor is regressing or not progressing or the tumor appears to be regressing or not progressing. In some embodiments, the treatment is considered “effective” when the patient has progression- free survival that is longer than if the patient was not undergoing the combination treatment.
  • sample encompasses a variety of sample types obtained from a patient and useful in the procedure of the presently disclosed subject matter.
  • the sample comprises whole blood, hemocytes, serum, or plasma.
  • samples may include, but are not limited to, solid tissue samples, liquid tissue samples, biological fluids, aspirates, cells and cell fragments.
  • specific examples of samples include, but are not limited to, solid tissue samples obtained by surgical removal, pathology specimens, archived samples, or biopsy specimens, tissue cultures or cells derived therefrom and the progeny thereof, and sections or smears prepared from any of these sources.
  • Samples also include any material derived from the body of the patient, including, but not limited to, blood, cerebrospinal fluid, serum, plasma, urine, nipple aspirate, fine needle aspirate, tissue lavage such as ductal lavage, saliva, sputum, ascites fluid, liver, kidney, breast, bone, bone marrow, testes, brain, ovary, skin, lung, prostate, thyroid, pancreas, cervix, stomach, intestine, colorectal, brain, bladder, colon, nares, uterine, semen, lymph, vaginal pool, synovial fluid, spinal fluid, head and neck, nasopharynx tumors, amniotic fluid, breast milk, pulmonary sputum or surfactant, urine, fecal matter and other liquid samples of biologic origin.
  • samples may be, but are not limited to, fresh, frozen, fixed, formalin fixed, paraffin embedded, or formalin fixed and paraffin embedded.
  • the sample may be, but are
  • monitoring treatment efficacy further comprises administering the combination treatment to the patient after the treatment efficacy is monitored.
  • the presently disclosed subject matter also provides a method for identifying a candidate agent that can be used to treat a metastatic tumor, the method comprising: (a) inducing a metastatic tumor formation in a mammal; (b) determining levels of at least one cytokine produced from a polyfunctional lymphocyte in the mammal; (c) administering a test agent to the mammal; and (d) determining levels of at least one cytokine produced from a polyfunctional lymphocyte in the mammal after administration of the test agent, wherein increased levels of the at least one cytokine produced from a polyfunctional lymphocyte after administration are indicative that the test agent is a candidate agent for treating the metastatic tumor.
  • the method can be used to identify candidate agents for treating any metastatic tumor, including metastatic CNS tumors.
  • the presently disclosed subject matter also provides a method for identifying a candidate agent that can be used to treat a metastatic central nervous system (CNS) tumor, the method comprising: (a) inducing metastatic central nervous system (CNS) tumor formation in a mammal; (b) determining levels of at least one cytokine produced from a polyfunctional lymphocyte in the mammal; (c) administering a test agent to the mammal; and (d) determining levels of at least one cytokine produced from a polyfunctional lymphocyte in the mammal after administration of the test agent, wherein increased levels of the at least one cytokine produced from a polyfunctional lymphocyte after administration are indicative that the test agent is a candidate agent for treating a metastatic CNS tumor.
  • the mammal comprises a rodent.
  • the rodent comprises a mouse.
  • the test agent is selected from the group consisting of small molecules, such as small organic or inorganic molecules; saccharides;
  • oligosaccharides oligosaccharides; polysaccharides; a biological macromolecule selected from the group consisting of peptides, proteins, peptide analogs and derivatives;
  • test agent comprises an immunotherapeutic agent.
  • test agent is not an anti-PDl antibody.
  • kits for practicing the methods of the presently disclosed subject matter.
  • a presently disclosed kit contains some or all of the components, reagents, supplies, and the like to practice a method according to the presently disclosed subject matter.
  • the term "kit” refers to any intended any article of manufacture (e.g., a package or a container) comprising at at least one radiotherapeutic agent and an immunotherapeutic agent, such as a Listeria-based vaccine, and a set of particular instructions for practicing the methods of the presently disclosed subject matter.
  • the kit can be packaged in a divided or undivided container, such as a carton, bottle, ampule, tube, etc.
  • compositions can be packaged in dried, lyophilized, or liquid form.
  • Additional components provided can include vehicles for reconstitution of dried components.
  • Preferably all such vehicles are sterile and apyrogenic so that they are suitable for injection into a subject without causing adverse reactions.
  • the kit comprises (a) a radiotherapeutic agent; (b) an immunotherapeutic agent; and (c) a package insert or label with directions to treat a patient with a metastatic central nervous system (CNS) tumor by administering a combination treatment comprising the radiotherapeutic agent and the Listeria-bassd vaccine.
  • the kit further includes an adjuvant.
  • the immunotherapeutic agent in the kit does not comprise an anti-PD 1 antibody.
  • the kit comprises (a) a radiotherapeutic agent; (b) a Listeria-bassd vaccine; and (c) a package insert or label with directions to treat a patient with a metastatic central nervous system (CNS) tumor by administering a combination treatment comprising the radiotherapeutic agent and the Listeria-bassd vaccine.
  • the kit further includes an adjuvant.
  • the term "about,” when referring to a value can be meant to encompass variations of, in some embodiments, ⁇ 100% in some embodiments ⁇ 50%, in some embodiments ⁇ 20%, in some embodiments ⁇ 10%, in some embodiments ⁇ 5%, in some embodiments ⁇ 1%, in some embodiments ⁇ 0.5%, and in some embodiments ⁇ 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.
  • MHC Major Histocompatibility Complex
  • TGF Tumor Growth Factor
  • B16 a poorly immunogenic murine melanoma cell line that expresses no MHC II and low levels of MHC I (8) and/or a more immunogenic variant which expresses a class I (H- 2Kb) restricted epitope of ovalbumin.
  • H- 2Kb class I restricted epitope of ovalbumin.
  • brain tumors are more tolerogenic than equivalently advanced tumors located outside the CNS and that mice harboring brain tumors have higher local and circulating levels of TGF- ⁇ , although blocking TGF- ⁇ failed to mediate tumor regression.
  • Vaccination alone had a modest effect on survival, but combination immunotherapy using a recombinant Listeria monocytogenes (LM) based vector and focal radiation therapy (RT) significantly prolonged survival.
  • LM Listeria monocytogenes
  • RT focal radiation therapy
  • LM-OVA Recombinant LM-OVA was constructed in the Lm AactA AinlB AuvrAB background by integrating pPL2- OVA as described (10, 1 1) (Aduro Biotech, Berkeley, CA). LM-OVA was grown in BHI to mid-log, washed, and stored in PBS/8% glycerol at -80°C in single-use aliquots.
  • LM-OVA was thawed, diluted in PBS to 1x107 cfu per mouse (0.1 LD50), and administered by intraperitoneal injection.
  • Vac-OVA or Vac-GPlOO mice received 1x106 pfu (0.1 LD50).
  • the G4 hybridoma was used to produce hamster antimurine PD-1 monoclonal antibodies as described at 10 mg/kg (12).
  • B 16-OVA cells were maintained in culture under continuous selection.
  • cells were resuspended at either 1,000 cells ⁇ L for survival experiments or 5,000 cells ⁇ L for immunology experiments.
  • For flank tumor and lung tumor implantation cells were resuspended at 50 cells ⁇ L and 500 cells ⁇ L, respectively.
  • Flank tumors were established by injecting 200 ⁇ , subcutaneously in the right flank.
  • Lung tumors were established by injecting 200 by tail vein injection.
  • Intracranial tumors were established as previously described (9). No cell line authentication was done.
  • Flow cytometry was carried out on a FACSCalibur or LSR II (BD Biosciences).
  • the following antibodies were used: CD45.2 PE, PB (Biolegend), CD8a PerCP, Pac Orange (Invitrogen), CD4 PerCP (BD), IFN- ⁇ APC, PE-Cy7 (Biolegend), Granzyme B PE (eBio), TNF-a PE
  • BD IL-2 APC
  • FoxP3 AF700 BioLegend
  • CDl lb AF700 CDl lb AF700
  • PE eBio
  • IL-17 PerCP/Cy5.5 BioLegend
  • Tumor-infiltrating lymphocyte immunophenotyping, pathology, and immunohistochemistry 2,000 F10 B16-OVA cells were implanted in the left hemisphere of C57BL/6 mice. RT was delivered on day 7, LM- OVA was administered on day 10, and mice were sacrificed on day 18. Tumors were excised from surrounding brain tissue and homogenized. TIL were isolated using density gradient centrifugation (Percoll). Cells were stimulated for 4 hours with
  • mice underwent transcardial perfusion with 10 mL PBS followed by 4%
  • RT and LM-OVA were administered as described (day 10).
  • mice were sacrificed and serum, brains, spleens and tumor draining lymph nodes were collected. Red blood cells were lysed in spleens and CD1 lc+ cells were isolated by positive selection (Miltenyi). Monocytes were isolated from brains by density gradient centrifugation, stained for CD1 lb AF700 (eBio) and CD45 PE (BioLegend), and sorted using a FACSAria (BD). CD 1 lb+/CD45-mid cells, as well as CD1 lc+ splenocytes and unsorted DLN cells were plated in a 96-well plate (lxlO 4 cells/well).
  • OT-1 CD8 cells were CFSE-labeled (0.5 ⁇ ) and plated with APCs at a ratio of 1 :5 for CD 1 lb+/CD45 microglia, a ratio of 1 :5 for CD 1 lc+ splenocytes, and a 1 : 1 ratio for DLNs.
  • SII FEKL SEQ ID NO: 1 peptide was added to the wells (2 ⁇ ) and plates were maintained in an incubator for 48 hours. GolgiStop (BDBiosciences) was added for the last 6 hours and supernatants were collected and stored at - 80°C. Cells were collected and stained for CD8, CD45.2, and IFN- ⁇ and analyzed by FACS. Supernatants and serum samples were analyzed for concentrations of IFN- ⁇ , IL-2, IL- 12, GM-CSF, IL-10, and TGF- ⁇ by multiplex (Luminex, Austin, TX).
  • CD8 T cells recognizing an endogenous tumor-associated melanoma antigen are deleted, while CD8 T cells recognizing a tumor-restricted neo-antigen persist.
  • Antigen-specific tolerance is an early event in tumor progression (14), and prior studies have shown that naive CD 8 T cells specific for the endogenous self/tumor antigen gplOO (Pmel) are rapidly tolerized upon adoptive transfer into C57BL/6 mice with established B16 flank tumors (15).
  • Pmel self/tumor antigen gplOO
  • FIG. 1 B 16 flank or brain tumors. Five days post- transfer very few tumor-infiltrating Pmel CD8 T cells persisted in brain or flank tumors (FIG. 2A).
  • CSF cerebrospinal fluid
  • CSF cerebrospinal fluid
  • TIL tumor- infiltrating lymphocytes
  • OT-1 CD8 T cells in the brain tumor DLN also showed clear evidence of division, with a significant fraction of cells undergoing greater than 4 divisions (FIG 3A, FIG. 3B). Although division was attenuated in brain tumor DLNs as compared with flank tumor DLNs, these results provide clear evidence of CNS tumor antigen recognition, though we cannot determine whether initial recognition occurred in the DLN or upon entry into the tumor itself.
  • IFN interferon
  • Antigen-specific cytotoxicity is systemically impaired in animals with CNS melanoma
  • CD8 T cell priming in the context of a brain tumor or flank tumor on in vivo effector function by performing a series of cytotoxic T lymphocyte (CTL) assays in mice bearing either B16-OVA brain or flank tumors.
  • CTL cytotoxic T lymphocyte
  • TGF- ⁇ is elevated in the serum of mice with CNS melanoma
  • TGF- ⁇ family cytokines are pluripotent molecules involved in regulating tissue homeostasis (21) and TGF- ⁇ has been implicated as a critical driver of melanoma progression (22).
  • CNS melanoma is associated with systemically elevated levels of TGF- ⁇
  • TGF- ⁇ signaling inhibitor LY2157299
  • RT is a standard treatment modality for CNS lesions, and in previous studies using an orthotopic glioma model we found that combining RT with other immunotherapies had a synergistic effect on OS (9).
  • SARRP small animal radiation research platform
  • mice receiving LM alone or LM in combination with radiation therapy confirmed the presence of an intratumoral T-cell infiltrate in mice receiving LM alone or LM in combination with radiation therapy (FIG. 7C).
  • Corresponding H&E staining demonstrated a lack of inflammation in untreated tumors and minimal inflammation in tumors treated with RT alone (FIG. 7D).
  • LM vaccination by contrast, was associated with increased perivascular inflammation, and combination therapy was associated with a marked peritumoral lymphocytic infiltrate.
  • MRI magnetic resonance imaging
  • Tumor borders were delineated in each slice and volumes were calculated using ImageJ software by a blinded observer (FIG. 7E).
  • Combination therapy is associated with poiyfunctionai CD8 T cells, an increased Teff to Treg ratio, and increased APC function.
  • mice with established CNS disease were treated with RT, LM or the combination of RT and LM.
  • Tumors were harvested 17 days after implantation and endogenous TIL were analyzed for cytokine production by flow cytometry.
  • RT or LM alone generated a modest increase in Granzyme B, IFN- ⁇ and TNF-a production, whilst with combination therapy, the majority of cells produced Granzyme B (GB), IFN- ⁇ and Tumor Necrosis Factor (TNF)-a (FIG. 1 IB).
  • the intratumoral Teff/Treg ratio correlates with clinical outcome. Indeed, we found that LM-based vaccination significantly increased this ratio, but RT did not add further to this, suggesting that increases in Teff / Treg were not the sole mechanism explaining the efficacy of the combination regimen. To further elucidate the mechanism(s) underlying the combination treatment effect, we tested whether the combination regimen affected the ability of various APC populations to present relevant tumor antigens ex vivo.
  • focal RT alone decreased antigen presentation by microglia and splenic APCs, with a trend towards decreased presentation noted in the tumor DLN as well (FIG.11A, FIG. 1 IB, FIG. 1 1C, FIG. 1 ID, FIG. 1 IE, FIG. 1 IF, FIG. 11G, FIG. 11H, FIG. 1 II, FIG. 1 1J, FIG. 1 IK, FIG. 11L, FIG. 1 1M, FIG. 1 IN), whereas LM-based vaccination largely corrected this APC defect in both distant (splenic) and local sites.
  • Brain metastases are a negative prognostic indicator in patients with metastatic melanoma (26) despite the fact that most patients succumb to systemic disease progression rather than neurologic compromise (10).
  • metastasis to the brain is a relatively late event and thus a harbinger for widely disseminated disease.
  • This hypothesis is challenged, however, by the finding that patients presenting with isolated melanoma brain metastases have shorter life expectancies than patients presenting with visceral metastases or synchronous brain and visceral lesions (27).
  • a hypothesis consistent with these clinical data is that brain metastases accelerate systemic disease progression, potentially through an immune- mediated mechanism. Immunosuppression has been extensively studied in patients with high- grade gliomas, but little is known about the systemic immunologic effects of metastatic brain tumors.
  • OT-1 T cells were more readily deleted in mice with brain tumors compared with equivalent B 16 flank tumors, OT-1 T cells underwent fewer divisions, and daughter cells produced less IFN- ⁇ in response to brain tumors compared with flank tumors.
  • OT-1 TIL were divided in both brain and flank tumors, consistent with the hypothesis that naive T cells have limited access to the CNS in the absence of inflammation (6) and that tumor antigens originating in the CNS are presented in secondary lymphoid organs.
  • Dysfunctional myeloid cells have been identified as key mediators of immunosuppression in cancer patients (31) and M2-differentiated microglia may be drivers of glioma progression (32).
  • TGF- ⁇ is a pleotropic cytokine that induces immune suppression and drives tumor progression in several solid tumors, including melanoma and glioma (8,33).
  • TGF- ⁇ has also been shown to enhance IL-4 mediated, M2 microglial activation (34).
  • Our data indicate that microglia isolated from mice with B16-OVA brain tumors express significantly higher levels of TGF- ⁇ than microglia from naive mice.
  • TGF- ⁇ signaling blockade rescued deletional tolerance in the Pmel adoptive transfer model. Based on these data, we suspect that CNS melanoma may drive microglia into an alternatively activated phenotype characterized by TGF- ⁇ expression. However, TGF-beta blockade in this model was unable to mediate a significant anti-tumor effect, either alone, combined with RT or with LM-based vaccination.
  • MDSC have been described in a CNS glioma model (36); and additional work will be required to address the role of MDSC populations in this model, both in the systemic tolerance mediated by implanted CNS tumors, as well as in the response of those tumors to RT, vaccination, or combination regimens.
  • Live-attenuated LM vaccines have demonstrated efficacy in several preclinical cancer models (37,38) and safety in Phase I and II clinical trials (39,40) .
  • the ability of LM to generate adaptive T cell- mediated immunity is based on its intracellular lifecycle and propensity to infect CD8+ dendritic cells (DCs), where bacterial antigens are processed through both MHC class I and class II pathways (40).
  • DCs dendritic cells
  • Liau and colleagues have previously reported that a different strain of LM delays progression of intracranial B16 tumors (41).
  • LM-based vaccination restored CTL activity in a majority of B16 brain tumor- bearing mice; however, a trend remained toward impaired CTL function compared with flank tumor- bearing animals.
  • RT has been associated with a mix of pro-inflammatory and inhibitory immunologic effects (15,42), but may have particular utility in potentiating the activity of immunotherapy (43). Demaria and colleagues showed that RT in combination with FLt3-ligand impairs growth of irradiated tumors as well as tumors outside the radiation field (44,45) and that local RT combined with cytotoxic T lymphocyte antigen- 4 (CTLA-4) blockade inhibits metastasis in a breast cancer model (46).
  • CTLA-4 cytotoxic T lymphocyte antigen- 4
  • Combination therapy had mixed effects: focal RT abrogated the systemic reduction in Tregs stimulated by LM-OVA vaccination while maintaining a favorable intratumoral Teff to Treg ratio.
  • Oligodendrocytes Enforce Immune Tolerance of the Uninfected Brain by
  • combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B 16 melanoma tumors. Proceedings of the National Academy of Sciences. 2010; 107:4275-80.
  • Allogeneic GM-CSF- secreting tumor cell immunotherapies generate potent anti-tumor responses comparable to autologous tumor cell immunotherapies.

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Abstract

Cette invention concerne des méthodes, des compositions et des kits destinés à traiter une tumeur métastasique du système nerveux central (SNC) comprenant un polytraitement associant la radiothérapie à une vaccination à base de Listeria. Des méthodes destinées à surveiller l'efficacité du polytraitement sont en outre décrites.
PCT/US2015/059490 2014-11-07 2015-11-06 Lymphocytes polyfonctionnels à titre de biomarqueurs d'une activité antitumorale WO2016073866A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110223187A1 (en) * 2010-02-15 2011-09-15 Vafa Shahabi Live listeria-based vaccines for central nervous system therapy
WO2012125551A1 (fr) * 2011-03-11 2012-09-20 Advaxis Adjuvants à base de listeria
US20130315950A1 (en) * 2010-05-23 2013-11-28 Aduro Biotech Methods and compositions using listeria for adjuvant treatment of cancer
US20140234370A1 (en) * 2009-11-11 2014-08-21 Advaxis, Inc. Compositions and methods for prevention of escape mutation in the treatment of her2/neu over-expressing tumors
US20150196628A1 (en) * 2009-11-11 2015-07-16 The Trustees Of The University Of Pennsylvania Combination immuno therapy and radiotherapy for the treatment of her-2-positive cancers

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140234370A1 (en) * 2009-11-11 2014-08-21 Advaxis, Inc. Compositions and methods for prevention of escape mutation in the treatment of her2/neu over-expressing tumors
US20150196628A1 (en) * 2009-11-11 2015-07-16 The Trustees Of The University Of Pennsylvania Combination immuno therapy and radiotherapy for the treatment of her-2-positive cancers
US20110223187A1 (en) * 2010-02-15 2011-09-15 Vafa Shahabi Live listeria-based vaccines for central nervous system therapy
US20130315950A1 (en) * 2010-05-23 2013-11-28 Aduro Biotech Methods and compositions using listeria for adjuvant treatment of cancer
WO2012125551A1 (fr) * 2011-03-11 2012-09-20 Advaxis Adjuvants à base de listeria

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JACKSON, C. M. ET AL.: "Systemic Tolerance Mediated by Melanoma Brain Tumors is Reversible by Radiotherapy and Vaccination", CLINICAL CANCER RESEARCH, vol. 22, no. 5, 21 October 2015 (2015-10-21), pages 1 - 23, XP055279422 *

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