WO2016073866A1

WO2016073866A1 - Polyfunctional lymphocytes as a biomarker of antitumor activity

Info

Publication number: WO2016073866A1
Application number: PCT/US2015/059490
Authority: WO
Inventors: Christopher Jackson; Charles Drake; Drew Pardoll; Michael Lim
Original assignee: Johns Hopkins University
Current assignee: Johns Hopkins University
Priority date: 2014-11-07
Filing date: 2015-11-06
Publication date: 2016-05-12
Anticipated expiration: 2017-05-07

Abstract

The presently disclosed subject matter provides methods, compositions, and kits for the treatment of a metastatic central nervous system (CNS) tumor comprising a combination treatment comprising radiotherapy and Listeria-based vaccination. Methods for monitoring efficacy of the combination treatment are also provided.

Description

POLYFUNCTIONAL LYMPHOCYTES AS A BIOMARKER OF ANTITUMOR

ACTIVITY

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No.

62/076,721, filed November 7, 2014, which is incorporated herein by reference in its entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under CA 127153 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED

ELECTRONICALLY

This application contains a sequence listing. It has been submitted electronically via EFS-Web as an ASCII text file entitled "11 1232-00467_ST25.txt". The sequence listing is 585 bytes in size, and was created on November 3, 2015. It is hereby incorporated by reference in its entirety.

BACKGROUND

Brain metastases afflict 20% to 40% of patients with advanced cancer and represent a major source of morbidity and mortality (1). The basic mechanisms constraining immune responses against CNS tumor antigens, however, remain poorly defined (2). 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.

SUMMARY

The practice of the present invention will typically employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant nucleic acid (e.g., DNA) technology, immunology, and RNA interference (RNAi) which are within the skill of the art. Non- limiting descriptions of certain of these techniques are found in the following publications: Ausubel, F., et al, (eds.), Current Protocols in Molecular Biology, Current Protocols in Immunology, Current Protocols in Protein Science, and Current Protocols in Cell Biology, all John Wiley & Sons, N.Y., edition as of December 2008; Sambrook, Russell, and Sambrook, Molecular Cloning. A Laboratory Manual, 3^rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. and Lane, D., Antibodies— A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988; Freshney, R. I., "Culture of Animal Cells, A Manual of Basic Technique", 5th ed., John Wiley & Sons, Hoboken, N.J., 2005. Non-limiting information regarding therapeutic agents and human diseases is found in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 11th Ed., McGraw Hill, 2005, Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw-Hill/Appleton & Lange 10^th ed. (2006) or 11th edition (July 2009). Non-limiting information regarding genes and genetic disorders is found in McKusick, V. A.: Mendelian Inheritance in Man. A Catalog of Human Genes and Genetic Disorders. Baltimore: Johns Hopkins University Press, 1998 (12th edition) or the more recent online database: Online Mendelian Inheritance in Man, OMIM™. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), as of May 1, 2010, World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim/ and in Online Mendelian Inheritance in Animals (OMIA), a database of genes, inherited disorders and traits in animal species (other than human and mouse), at

http ://omia. angis . org. au/contact. shtml.

All patents, patent applications, and other publications (e.g., scientific articles, books, websites, and databases) mentioned herein are incorporated by reference in their entirety. In case of a conflict between the specification and any of the incorporated references, the specification (including any amendments thereof, which may be based on an incorporated reference), shall control. Standard art-accepted meanings of terms are used herein unless indicated otherwise. Standard abbreviations for various terms are used herein.

In an aspect, 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.

In an aspect, 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.

In an aspect, 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.

In an aspect, 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 level of the at least one cytokine produced from the polyfunctional lymphocyte is decreased relative to, or remains at or near, the baseline level, at the one or more time intervals.

In an aspect, 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-β 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-β; (b) detecting the level of the TGF-β 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-β 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-β is increased relative to, or remains at or near, the baseline level, at the one or more time intervals.

In some embodiments, the method of monitoring treatment efficacy further comprises administering the combination treatment to the patient after the treatment efficacy is monitored. In some embodiments, treatment efficacy is monitored using a sample from the patient comprising serum, and/or blood.

In some embodiments, at least one cytokine is selected from the group consisting of Granzyme B (GB), Interferon-γ (IFN-γ), Tumor Necrosis Factor (TNF)- a, and Interleukin-2 (IL-2). In some embodiments, at least four cytokines are detected.

In some embodiments, 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.

In some embodiments, the combination therapy stimulates tumor infiltration by the polyfunctional lymphocyte. In some embodiments, the polyfunctional lymphocyte is a CD8+ T cell.

In some embodiments, the Listeria-bassd vaccine is a live-attenuated vaccine.

In some embodiments, the Listeria is Listeria monocytogenes. In some embodiments, the Listeria-bassd vaccine comprises ovalbumin (OVA) or an immunogenic part thereof. In some embodiments, the Listeria-bassd vaccine further comprises an adjuvant.

In some embodiments, the radiotherapy is focal. In some embodiments, the radiotherapy is stereotactic radiosurgery, fractionated stereotactic radiosurgery, and/or intensity-modulated radiation therapy (IMRT). In some embodiments, 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). In some embodiments, 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.

In an aspect, 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.

In an aspect, 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.

In an aspect, 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. In some embodiments, the mammal comprises a rodent. In some embodiments, the rodent comprises a mouse.

Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

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. 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 perpendicular to the y axis represent the values of no value, 0, 10², 10³, 10⁴, and 10⁵. FIG. 2B shows summary graphs showing percentages and numbers of Pmel CD8 T cells. FIG. 2C shows

representative FACS plots of the percentage of OT-1 (CD45.2+) CD8+ T cells isolated from spleens, brain tumor DLN, and TIL, animals bearing B16-OVA tumors. 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-γ 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°, 10¹, 10², 10³, and 10⁴. 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-γ. 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

perpendicular to the y axis represent the values of 10°, 10¹, 10², 10³, and 10⁴. FIG. 3D shows summary graphs showing percentages and numbers of divided cells producing IFN-γ. N = 5 mice/group, repeated x 3 with similar results;

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-γ 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. 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-βΙ is associated with systemic tolerance in animals with CNS melanoma. FIG. 6A shows concentration of TGF-βΙ 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. In particular, the longest hashmarks perpendicular to the x axis and perpendicular to the y axis represent the values of no value, 0, 10², 10³, 10⁴, and 10⁵. 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-β 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-βΙ 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-β blockade does not add to RT + vaccination. TGF-β blockade with 1D11 (FIG. 8A) or LY2157299 (FIG. 8B) does not extend survival when combined with RT + rLM-OVA in mice with B16-OVA brain tumors. (N= 10 animals / group);

FIG. 9 shows that PD-1 blockade does not add to RT + Vaccination. Adding PD-1 blockade to RT + rLM-OVA does not significantly improve survival in mice with B16-OVA brain tumors. (N = 10 animals / group);

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. 10B shows calculated brain tumor volumes at the time of death as determined by ex vivo MRI (N = 3 mice/group);

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, and improved APC function. FIG. 11A shows FACS plots demonstrating the effects of combination therapy on cytokine production from adoptively transferred, tumor-infiltrating CD8 T cells. 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 perpendicular to the y axis represent the values of no value, 0, 10², 10³, 10⁴, and 10⁵. 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. 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 perpendicular to the y axis represent the values of no value, 0, 10², 10³, 10⁴, and 10⁵. 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. 11H, 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. In particular, the longest hashmarks perpendicular to the y axis and perpendicular to the x axis represent the values of 10°, 10¹, 10², 10³, and 10⁴. FIG. UK, FIG. 11L, and FIG. 1 1M show summary graphs of the percentage of OT-1 responders producing IFN-γ when co- cultured with the indicated APC populations. FIG. 1 IN shows TGF-βΙ

concentrations in supernatants from FIG. J, FIG. M. FIG. A, FIG. 1 IB, FIG. 11C, FIG. 1 ID, FIG. 1 IE, FIG. 1 IF, and FIG. 1 1G, N repeated x 2, N = 3-5 animals, group, FIG. 1 1H, FIG. 1 II, FIG. 11 J, FIG. 1 IK, FIG. 11L, and FIG. 1 1M repeated x 1, N = 10 animals / group; and

FIG. 12 A, FIG. 12B, and FIG. 12C show B16-OVA brain tumors stimulate secretion of TGF-βΙ from microglia. Cytokine concentrations in the supernatants from OT-1 cells co-cultured with: FIG. 12A) CDl lc+ APCs isolated from the spleen at a ratio of 1 :5; FIG. 12B) APC from brain tumor draining lymph nodes at a ratio of 1 : 1; and FIG. 12C) CDl lb+/CD45-mid microglia isolated from the brain at a ratio of 1 :5.

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the inventions are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated Figures. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

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) and 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.

I. METHODS FOR TREATING A METASTATIC TUMOR (E.G.,

METASTATIC CENTRAL NERVOUS SYSTEM (CNS) TUMOR)

In some embodiments, 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. In some embodiments, the immunotherapy comprises an immunotherapeutic agent.

As used herein, the term "immunotherapeutic agent" refers to a molecule that can aid in the treatment of a disease by inducing, enhancing, or suppressing an immune response. Examples of 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). In some embodiments, 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.

In some embodiments, 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.

As used herein, the "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 "tumor," as used herein, refers to all neoplastic cell growth and proliferation and all precancerous and cancerous cells and tissues. 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. In some circumstances, 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. In some embodiments, the cancer comprises Stage 0 cancer. In some embodiments, the cancer comprises Stage I cancer. In some embodiments, the cancer comprises Stage II cancer. In some embodiments, the cancer comprises Stage III cancer. In some embodiments, the cancer comprises Stage rv cancer. In some embodiments, the cancer is refractory and/or metastatic. In some embodiments, the presently disclosed methods can be used for treating any kind of cancer. In some embodiments, 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. 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, a tumor consisting of melanin-forming cells. In some embodiments, the metastatic CNS tumor is a brain tumor, such that a cancer from somewhere else metastasized to the brain. In some embodiments, the brain tumor is not a glioma, a tumor of the glial tissue of the nervous system.

In some embodiments, 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). It will be understood by those of ordinary skill in the art that 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. To achieve this precision, specific procedures for identifying the position of the tumorous tissue are used. For example, information from magnetic resonance imaging (MRI) and/or computed tomography (CT) scans can be transferred directly to a treatment-planning computer system to create a three-dimensional (3-D) model of the tumor and surrounding normal tissue. The 3-D image allows the position of the abnormality to be treated to be identified and targeted. 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.

Because of noninvasive fixation devices, 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. When used to treat a tumor over time, the radiosurgery is referred to as "fractionated stereotactic radiosurgery" or FSR. In contrast, stereotactic radiosurgery refers to a one-session treatment.

The main advantage of fractionation is that it 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. In some

embodiments, multiple smaller doses are administered every day over weeks, such as for 1, 2, 3, 4, 5, 6, 7 or more weeks. In some embodiments, multiple smaller doses are administered several times a day, several times a week, weekly, bimonthly, or monthly, for example. In some embodiments, 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.

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.

It will be understood by those of ordinary skill in the art that 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.

As used with the presently disclosed methods and compositions provided herein, 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.

As used with the presently disclosed methods and compositions, use of 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. In practice, 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.

In some embodiments, intensity-modulated radiation therapy (IMRT) can be used. IMRT is an advanced mode of high-precision three-dimensional conformal radiation therapy (3DCRT), which uses computer-controlled linear accelerators to deliver precise radiation doses to a malignant tumor or specific areas within the tumor. In 3DCRT, the profile of each radiation beam is shaped to fit the profile of the target from a beam's eye view (BEV) using a multileaf collimator (MLC), thereby producing a number of beams. More particularly, 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.

Accordingly, 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.

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. Typically, 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. Because the ratio of normal tissue dose to tumor dose is reduced to a minimum with the IMRT approach, higher and more effective radiation doses can safely be delivered to tumors with fewer side effects compared with conventional radiotherapy techniques. IMRT typically is used to treat cancers of the prostate, head and neck, and central nervous system.

In some embodiments, 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.

As used herein, a "vaccine" is a substance that promotes the immune system to attack a tumor, such as a metastatic CNS tumor. In some embodiments, the vaccine increases the immune response against cancer cells that are already in the body. In some embodiments, the vaccine can be combined with other substances or cells called adjuvants that help boost the immune response. Non-limiting examples of adjuvants include cholera toxin, cytokines, chemokines, and bacterial nucleic acid sequences, like CpG.

In some embodiments, the combination treatment comprises a vaccine, such as a Listeria-based vaccine. In some embodiments, 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.

In some embodiments, as Listeria can be a pathogenic organism, the Listeria is "attenuated" or "live-attenuated". As used herein, the term "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. As used herein, the term "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. In the case of Listeria, in certain embodiments the bacterium is mutated to lessen or prevent the ability to grow and spread intracellularly. In some embodiments, the uvrAB genes are deleted from the Listeria genome. In some embodiments, the Listeria can comprise a mutation that inactivates ActA. In some embodiments, the Listeria can comprise a mutation that inactivates InlB. In some embodiments, the Listeria genome comprises a

combination of more than one mutation. In some embodiments, an attenuated, metabolically active Listeria is deleted for its native ActA, inlB, and uvrAB genes (AactAAinlBAuvr). In some embodiments, the presently disclosed subject matter employs a Listeria that is killed but metabolically active ("KBMA").

In some embodiments, the Listeria is Listeria monocytogenes, a small, cocci shaped Gram-positive rod shaped bacterium that is a member of the Family

Listeriaceae. In some embodiments, 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. In some embodiments, the Listeria-based vaccine comprises a live-attenuated Listeria monocytogenes vaccine. In some embodiments, the live-attenuated Listeria monocytogenes vaccine comprises a deletion of a gene, such as ActA, inlB, or uvrAB. In some embodiments, 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. In some embodiments, the live-attenuated Listeria monocytogenes vaccine comprises a deletion of a combination of three or more genes, such as ActA, inlB, and uvrAB.

In some embodiments, 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. In some embodiments, the Listeria- based vaccine comprises ovalbumin (OVA) or an immunogenic part thereof. In some embodiments, the ovalbumin gene is incorporated into the Listeria genome.

In some embodiments, the combination treatment increases the

polyfunctionality of lymphocytes, such that the tumor infiltration by the

polyfunctional lymphocytes is stimulated. As used herein, the term "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. As used herein, the term "polyfunctional lymphocyte" refers to a lymphocyte that produces more than one cytokine, such as 2, 3, 4, or more cytokines. In some embodiments, the polyfunctional lymphocyte is a T cell. In some embodiments, 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-β, and IFN-γ. In some embodiments, 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. In some embodiments, at least one cytokine is selected from the group consisting of Granzyme B (GB), Interferon-γ (IFN-γ), Tumor Necrosis Factor (TNF)-a, and Interleukin-2 (IL- 2)· In some embodiments, a metastatic CNS tumor results in an increase in levels of TGF-β 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-β from microglia, but increases the levels of other cytokines produced from polyfunctional lymphocytes. Accordingly, in some embodiments, 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-β produced from microglia. As used herein, "TGF- β" refers to any isoform of TGF- β, such as TGF- βΐ, TGF- β2, and TGF- β3. In some embodiments, the TGF- β produced from the microglia is TGF- βΐ . In some embodiments, the TGF- β2 isoform is measured using the presently disclosed methods.

In some embodiments, the presently disclosed subject matter also provides a composition for the treatment of a metastatic CNS tumor comprising a

radiotherapeutic agent and an immunotherapeutic agent. In some embodiments, 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. As used herein, a "radiotherapeutic agent" refers to those agents

conventionally adopted in the therapeutic field of cancer treatment and includes photons having enough energy for chemical bond ionization such as, for instance, alpha (a), beta (β), and gamma (γ) rays from radioactive nuclei as well as x-rays. 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 γ- 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. In case of internal radiation therapy, 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. Still further, 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 α, β and γ. Usually, a and β 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.

Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. 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. In some embodiments, the radiotherapeutic agent is selected from the group consisting of ⁴⁷Sc, ⁶⁷Cu, ⁹⁰Y, ¹⁰⁹Pd, ¹²³1, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁹Au, ²¹¹At, ²¹²Pb, ²¹²B, ³²P and ³³P, ⁷¹Ge, ⁷⁷As, ¹⁰³Pb, ¹⁰⁵Rh, ^mAg, ¹¹⁹Sb, ¹²¹Sn, ¹³¹Cs, ¹⁴³Pr, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹¹Os, ¹⁹³MPt, ¹⁹⁷H, ⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br, ⁸¹Rb/.⁸¹MKr, ⁸⁷MSr, ⁹⁹MTc, ^mIn, ¹¹³MIn, ¹²⁷Cs, ¹²⁹Cs, ¹³²I, ¹⁹⁷Hg, ²⁰³Pb and ²⁰⁶Bi, as described in U.S. Pat. No. 8,946, 168, the entirety of which is incorporated herein by reference.

In some embodiments, 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. In some embodiments, 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. In some embodiments, the composition does not include an anti-PD 1 antibody.

As used herein, the term "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).

In some embodiments, the combination treatment reduces the likelihood of tumor progression and/or mediates tumor regression. For example, 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. In some embodiments, 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.

In some embodiments, the combination treatment extends survival of the patient. For example, 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. In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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. In some embodiments, the survival is progression-free survival. In some embodiments, survival is extended by at least 40%.

As used herein, 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. Thus, one of ordinary skill in the art would appreciate that 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%.

The terms "subject" and "patient" are used interchangeably herein. The subject treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term "subject." Accordingly, 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. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a "subject" can include a patient afflicted with or suspected of being afflicted with a condition or disease.

Generally, the presently disclosed compositions (e.g., comprising a radiotherapeutic agent and an immunotherapeutic agent, such as a Listeria-bassd vaccine) can be administered to a subject for therapy 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. However, in some particular embodiments, 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). In some embodiments, 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.

The phrases "systemic administration", "administered systemically",

"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.

The phrases "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.

The presently disclosed pharmaceutical 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.

In some embodiments, the presently disclosed pharmaceutical compositions can be administered by rechargeable or biodegradable devices. For example, 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. 3,773,919; EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al, Biopolymers 22:547, 1983), poly (2-hydroxyethyl-methacrylate) (Langer et al. (1981) J. Biomed. Mater. Res. 15: 167; Langer (1982), Chem. Tech. 12:98), ethylene vinyl acetate (Langer et al. (1981) J. Biomed. Mater. Res. 15: 167), or poly-D-(-)-3- hydroxybutyric acid (EP 133,988A). 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). Ordinarily, 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.

In another embodiment, 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). Some effects of PEGylation 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. (1992) J. Immunol. Meth. 152: 177-190); (f) improved bioavailability via reduced losses at subcutaneous injection sites; (g) reduced toxicity has been observed; for agents where toxicity is related to peak plasma level, a flatter pharmacokinetic profile achieved by sub-cutaneous administration of PEGylated protein is advantageous; proteins that elicit an immune response which has toxicity consequences may also benefit as a result of PEGylation; and (h) improved thermal and mechanical stability of the PEGylated molecule.

Pharmaceutical compositions for parenteral administration include aqueous solutions of compositions comprising a radiotherapeutic agent and an

immunotherapeutic agent, such as a Lister ia-based vaccine. For injection, 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.

Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.

Additionally, suspensions of compositions include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.

Optionally, 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.

For nasal or transmucosal administration generally, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For inhalation delivery, 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.

Additional ingredients can be added to compositions for topical

administration, as long as such ingredients are pharmaceutically acceptable and not deleterious to the epithelial cells or their function. Further, such additional ingredients should not adversely affect the epithelial penetration efficiency of the composition, and should not cause deterioration in the stability of the composition. For example, 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.

Regardless of the route of administration selected, the presently disclosed 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.

In general, 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. As will be appreciated by those of ordinary skill in this art, 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. As used herein, 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. In some embodiments, 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. In some embodiments, a radiotherapeutic agent is administered before an

immunotherapeutic agent, such as a Listeria-based vaccine. In some embodiments, an immunotherapeutic agent, such as a Listeria-based vaccine, is administered before at least one radiotherapeutic agent. In some embodiments, 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.

Further, the presently disclosed compositions can be administered alone or in combination with adjuvants that enhance stability of the agents, facilitate

administration of pharmaceutical compositions containing them in certain embodiments, provide increased dissolution or dispersion, increase activity, provide adjuvant therapy, and the like, including other active ingredients. Advantageously, such 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.

The timing of administration of a radiotherapeutic agent combined with an immunotherapeutic agent, such as a Listeria-based vaccine can be varied so long as the beneficial effects of the combination of these agents are achieved. Accordingly, the phrase "in combination with" refers to the administration of a radiotherapeutic agent combined with an immunotherapeutic agent, such as a Listeria-based vaccine, and, optionally, additional agents either simultaneously, sequentially, or a combination thereof. Therefore, 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.

When administered sequentially, the agents 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, is administered 4, 5, 6, 7, 8, 9, 10, 15, 20 days or more later. In some embodiments, a booster immunotherapeutic agent, such as a Listeria-based vaccine, is administered 1, 2, 3, 4, 5, 6 or more weeks after the initial immunotherapeutic agent, such as a Listeria-bassd vaccine is administered.

When administered in combination, 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. In some embodiments, when administered in combination, the two or more agents can have a synergistic effect. As used herein, the terms "synergy,"

"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:

QaQ_A + QbQ_B = Synergy Index (SI) wherein:

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; and

Qb is the concentration of component B, in a mixture, which produced an end point.

Generally, when the sum of Q₃/Q_A and Q_I/Q_B is greater than one, antagonism is indicated. When the sum is equal to one, additivity is indicated. When the sum is less than one, synergism is demonstrated. The lower the SI, the greater the synergy shown by that particular mixture. Thus, a "synergistic combination" has an activity higher that what can be expected based on the observed activities of the individual components when used alone. Further, 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.

In another aspect, 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.

More particularly, the presently disclosed subject matter provides 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.

In therapeutic and/or diagnostic applications, 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).

Use of pharmaceutically acceptable inert carriers to formulate the compounds herein disclosed for the practice of the disclosure into dosages suitable for systemic administration is within the scope of the disclosure. With proper choice of carrier and suitable manufacturing practice, the 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

pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. 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.

For nasal or inhalation delivery, 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.

Pharmaceutical 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.

In addition to the active ingredients, 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. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.

II. METHODS FOR MONITORING EFFICACY OF TREATING A TUMOR (E.G., METASTATIC CENTRAL NERVOUS SYSTEM (CNS) TUMOR)

In some embodiments, a method is provided for monitoring treatment efficacy of a reatment regimen comprising an immunotherapy (e.g., immunotherapeutic agent) to treat a metastatic 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 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 cytokine produced from the polyfunctional lymphocyte is decreased relative to, or remains at or near, the baseline level, at the one or more time intervals. It should be appreciated that the method can be used to monitor treatment efficacy of any form of immunotherapy. In some embodiments, the immunotherapeutic agent administered as the form of

immunotherapy does not comprise an immune checkpoint inhibitor, such as anti-PD l antibody. In some embodimetns, the method is used to monitor treatment efficacy of a combination treatment regimen comprising the immunotherapy and radiotherapy.

In some embodiments, a method is provided for monitoring treatment efficacy of a combination treatment regimen comprising radiotherapy and an

immunotherapeutic agent 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 level of the at least one cytokine produced from the polyfunctional lymphocyte is decreased relative to, or remains at or near, the baseline level, at the one or more time intervals. It should be appreciated that the method can be used to monitor treatment efficacy of any form of immunotherapy. In some embodimetns, the method is used to monitor treatment efficacy of an immunotherapy treatment regiment in the absence of performing radiotherapy.

In some embodiments, a method is provided 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, 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, at the one or more time intervals. In some embodiments, 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

embodiments, the levels of at least five cytokines are detected. In some embodiments, at least one cytokine is selected from the group consisting of Granzyme B (GB), IFN- γ, Tumor Necrosis Factor (TNF)-a, and IL-2.

In some embodiments, 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-β 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-β; (b) detecting the level of the TGF-β 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-β 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-β is increased relative to, or remains at or near, the baseline level, at the one or more time intervals.

In some embodiments, 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-β 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-β; (b) detecting the level of the TGF-β 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-β 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-β is increased relative to, or remains at or near, the baseline level, at the one or more time intervals.

In some embodiments, 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. In some embodiments, the time interval will be weekly. In some embodiments, the time interval will be biweekly. In some embodiments, the time interval will be every three weeks. In some embodiments, the time interval will be every month. In some embodiments, the time interval will be more than one month apart. In some embodiments, 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.

In some embodiments, 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.

In some embodiments, treatment efficacy is monitored using a sample from the patient. As used herein, the term "sample" encompasses a variety of sample types obtained from a patient and useful in the procedure of the presently disclosed subject matter. In some embodiments of the presently disclosed subject matter, the sample comprises whole blood, hemocytes, serum, or plasma. However, 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. In some embodiments, samples may be, but are not limited to, fresh, frozen, fixed, formalin fixed, paraffin embedded, or formalin fixed and paraffin embedded. In some embodiments, the sample comprises tumor tissue. In some embodiments, the sample comprises serum and/or blood.

In some embodiments, monitoring treatment efficacy further comprises administering the combination treatment to the patient after the treatment efficacy is monitored.

III. METHODS FOR IDENTIFYING A CANDIDATE AGENT FOR

TREATING A METASTATIC CENTRAL NERVOUS SYSTEM (CNS) TUMOR

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. In some embodiments, the mammal comprises a rodent. In some embodiments, the rodent comprises a mouse. In some embodiments, the test agent is selected from the group consisting of small molecules, such as small organic or inorganic molecules; saccharides;

oligosaccharides; polysaccharides; a biological macromolecule selected from the group consisting of peptides, proteins, peptide analogs and derivatives;

peptidomimetics; nucleic acids, such as RNA interference molecules, selected from the group consisting of siRNAs, shRNAs, antisense RNAs, ribozymes, dendrimers and aptamers; antibodies, including antibody fragments and intrabodies; an extract made from biological materials selected from the group consisting of bacteria, plants, fungi, animal cells, and animal tissues; naturally occurring or synthetic compositions; and any combination thereof. In some embodiments, the test agent comprises an immunotherapeutic agent. In some embodiments, the test agent is not an anti-PDl antibody.

IV. KITS FOR FOR TREATING A METASTATIC TUMOR (E.G., CENTRAL NERVOUS SYSTEM (CNS) TUMOR)

The presently disclosed subject matter also relates to kits for practicing the methods of the presently disclosed subject matter. In general, 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. In some embodiments, 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. The presently disclosed 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.

In some embodiments, 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. In some embodiments, the kit further includes an adjuvant. In some embodiments, the immunotherapeutic agent in the kit does not comprise an anti-PD 1 antibody.

In some embodiments, 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. In some embodiments, the kit further includes an adjuvant.

Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this presently described subject matter belongs.

Following long-standing patent law convention, the terms "a," "an," and "the" refer to "one or more" when used in this application, including the claims. Thus, for example, reference to "a subject" includes a plurality of subjects, unless the context clearly is to the contrary (e.g., a plurality of subjects), and so forth.

Throughout this specification and the claims, the terms "comprise,"

"comprises," and "comprising" are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the term "include" and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.

For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, parameters, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term "about" even though the term "about" may not expressly appear with the value, amount or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact, but may be approximate and/or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art depending on the desired properties sought to be obtained by the presently disclosed subject matter. For example, 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.

Further, the term "about" when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

EXAMPLES

The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are only intended for the purposes of illustration, and are not to be construed as limiting in any manner to make compounds of the disclosure by other methods.

EXAMPLE 1

Systemic Tolerance Mediated by Melanoma Brain Tumors is

Reversible by Radiotherapy and Vaccination

Introduction

Although the presence of the blood-brain barrier, lack of conventional lymphatics, paucity of antigen presenting cells, and low basal expression of Major Histocompatibility Complex (MHC) molecules qualify the CNS as immunologically unique, peripheral leukocytes access the brain and orchestrate robust immune responses under inflammatory conditions (5). Unlike peripheral lymphocytes, however, these cells interact with a variety of tissue-resident cells, including astrocytes, neurons, and microglia, which modulate lymphocyte function in a highly context-dependent manner (6). Location within the CNS may be important for immunosuppression in animal models as extracranially implanted gliomas are characterized by lower levels of Tumor Growth Factor (TGF)- transcription, increased infiltration by CD4 and CD8 T cells, decreased T regulatory cell (Treg) accumulation, and slower growth as compared with intracranial gliomas (7).

To investigate the effects of tumor location on immune function we used 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. We found that 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-β, although blocking TGF-β failed to mediate tumor regression. Having established intracranial B16 as a challenging model, we tested the induction/expansion of anti-tumor T cells by vaccination. 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.

Although previous studies demonstrated glioma regression after treatment with RT and PD- 1 blocking antibodies (9), we found that LM and RT were superior to anti-PD- 1 and RT against established intracranial melanoma. Mechanistically, vaccination combined with focal RT significantly decreased secretion of TGF-βΙ from microglia and increased intratumoral polyfunctional CD8 T cell density. Based on these data, we propose a mechanism by which microglia in the brain tumor microenvironment mediate systemic immune tolerance and describe how

appropriately primed T cells can reverse this effect. These findings have implications for systemic disease control as well as designing and implementing effective immunotherapies for patients with metastatic brain tumors.

Materials and Methods

Mice, cell lines, antibodies, and vaccines. Female C57BL/6 (Jackson

Laboratory) or LY5.2 (NCI) mice (6-8 weeks) were housed in pathogen-free conditions under approved animal protocols (Institutional Animal Care and Use Committee of Johns Hopkins University). OT-l/CD45.2/Rag -/-and Pmel/CD45.2 mice were used as donors for adoptive transfer experiments. 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. For vaccination, LM-OVA was thawed, diluted in PBS to 1x107 cfu per mouse (0.1 LD50), and administered by intraperitoneal injection. For 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).

Tumor models. B 16-OVA cells were maintained in culture under continuous selection. For intracranial implantation, 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 μΐ, 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. Flow cytometry was carried out on a FACSCalibur or LSR II (BD Biosciences). For adoptive transfer experiments, the following antibodies were used: CD45.2 PE, PB (Biolegend), CD8a PerCP, Pac Orange (Invitrogen), CD4 PerCP (BD), IFN-γ APC, PE-Cy7 (Biolegend), Granzyme B PE (eBio), TNF-a PE

(BD), IL-2 APC (BioLegend), FoxP3 AF700 (BioLegend), CDl lb AF700, PE (eBio), and IL-17 PerCP/Cy5.5 (BioLegend). Data were analyzed using FlowJo software (Tree Star).

Adoptive transfer experiments. Spleens and lymph nodes from OT-1, Rag-/- or Pmel mice were collected and homogenized. For wild-type Pmel mice, CD8 T cells were isolated by positive selection (Miltenyi Biotech) and labeled with CFSE (Invitrogen). Cells were resuspended in PBS at 1.25x107 cells/mL then transferred by retro-orbital injection (2.5x106 cells) 12 days after implantation of 1x104 F 10 B 16- OVA cells in the brain or flank of mice expressing the congenic marker CD45.1 (LY5.2). Five days after adoptive transfer, brains, draining lymph nodes, and spleens were collected and homogenized. Brain and flank tumors were excised and TIL were isolated using Percoll (Sigma) density gradient centrifugation per manufacturer instructions. Cells were isolated and stimulated with 2 μΜ H-2Kb restricted class I epitope SIINFEKL (OVA257-264) (SEQ ID NO: l) or the human epitope KVPRNQDWL (gpl0025-33) (SEQ ID NO:2) in the presence of GolgiStop

(BDBiosciences), then analyzed by FACS.

In vivo CTL assays. Assays were performed as previously described (13). Splenocytes from wild-type C57BL/6 mice were isolated and divided into two groups. One group was labeled CFSEio (0.5 μΜ), while a second group was labeled CFSEhi (5 μΜ) and loaded with SIINFEKL (SEQ ID NO: 1) peptide at a concentration of 2 μΜ. Cells were combined and transferred by retro-orbital injection. For tumor- bearing mice, lxl0⁴ F10 B16- OVA cells were implanted in the brain or flank 17 days prior to target transfer. Vac-OVA or LM-OVA were administered as described above. Spleens were harvested from recipient mice 18 hours after target transfer and splenocytes analyzed by FACS.

Radiation therapy. 16 Gy was delivered using the small animal radiation research platform (SARRP) (Xstrahl, Suwanee, GA) as previously described (9).

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

PMA/Ionomycin, washed, stained for CD8, CD4, IFN-γ, TNF-a, IL-2, Granzyme B, FoxP3, and IL-17, and analyzed by flow cytometry. For immunohistochemistry, mice underwent transcardial perfusion with 10 mL PBS followed by 4%

paraformaldehyde/PBS. Brains were removed and cryoprotected in 30% sucrose/PBS for 48 hours at 4°C, snap frozen, and stored at -80°C prior to sectioning. H&E staining was performed by the histology core facility. For immunostaining, slides were washed twice for 5 minutes in PBS and blocked in 5% NGS/PBS for 1 hour. Tissues were incubated with anti-CD3e antibody (Dako, Carpinteria, CA) (1 : 10 diluted in 3% NGS/PBS) overnight at 4°C and washed in PBS before incubating with goat anti-rabbit secondary (1 : 1000) for one hour at room temperature.

APC co-culture experiments. lxlO⁴ F10 B16-OVA cells were implanted and

RT and LM-OVA were administered as described (day 10). On day 17, 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 (lxlO⁴ cells/well). OT-1 CD8 cells were CFSE-labeled (0.5 μΜ) 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 μΜ) 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-γ and analyzed by FACS. Supernatants and serum samples were analyzed for concentrations of IFN-γ, IL-2, IL- 12, GM-CSF, IL-10, and TGF-βΙ by multiplex (Luminex, Austin, TX).

Survival experiments and tumor volume analysis. 2,000 F10 B16-OVA cells were implanted in the left hemisphere of C57BL/6 mice. RT was delivered (16 Gy) 7 days after tumor implantation and LM-OVA was administered on day 10. Mice received a LM-OVA boost on day 31. Mice were sacrificed according to protocol upon development of motor deficits or sustained hunched posture. In studies involving post- mortem tumor volume assessment, brains were removed at the time of death and stored in 4% paraformaldehyde/PBS. Tissues were transferred to PFPE (Fomblin®, Sigma-Aldrich) and MR images were acquired by the Small Animal Imaging Resource Program at Johns Hopkins. Tumor borders were delineated slice- by-slice using ImageJ software and tumor volumes were calculated.

Statistics. Data were analyzed by 2-tailed Student's T-test or ANOVA using GraphPad Prism software. Results

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). To test whether tumor location affects antigen-specific tolerance, we adoptively transferred Pmel CD8 T cells into mice bearing equivalently sized (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). However, there was a significantly lower percentage (p < 0.001) and number (p < 0.001) of adoptively transferred cells in the cervical lymph nodes (brain tumor DLN) compared with flank tumor DLN (FIG. 2B). This effect was systemic, as there were also fewer Pmel CD8 T cells in the spleens of brain tumor- bearing mice as compared with cancer- free (p < 0.001) or flank tumor-bearing mice (p = 0.03).

To extend these results to a tumor-restricted antigen, we implanted B16-OVA brain and flank tumors. Here, ovalbumin models a mutated neo-antigen, to which preexisting tolerance is not expected. This model has the advantage that tolerance is unlikely to be primarily deletional, as suggested by previous studies (16). We found a significant decrease in OT-1 percentage (p = 0.007) and number (p = 0.002) in brain tumor DLNs compared with flank tumor DLNs (FIG. 2C, FIG. 2D). Unlike the Pmel model, however, differences in the spleens did not reach statistical significance (FIG. 2D).

Immunological tolerance to CNS melanoma antigens is not the result of ignorance

Antigens exit the CNS via cerebrospinal fluid (CSF) drainage along olfactory nerves passing through the cribriform plate and along perivascular spaces, including channels associated with dural venous sinuses (17), en route to the cervical lymph nodes (2). Given that antigens in the CNS parenchyma are poorly recognized by naive lymphocytes (18), we hypothesized that differences in tumor antigen recognition might underlie the observed differences in brain and flank tumor-bearing animals. To test this hypothesis, we adoptively transferred CFSE-labeled OT-1 CD8 T cells to mice bearing B16-OVA brain or flank tumors. These studies could not be performed with Pmel cells, since so few cells escape deletional tolerance (FIG. 2B). As shown in FIG. 3A, the majority of tumor- infiltrating lymphocytes (TIL) were divided in both brain and flank tumors, consistent with antigen recognition. 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. To determine if brain tumor antigen recognition impaired acquisition of CD8 T cell effector function, we analyzed interferon (IFN)-y production by antigen- specific CD 8 T cells (FIG. 3C) and found a significantly lower percentage (p < 0.001) and number (p = 0.01) of divided, iFN-y+ CD 8 T cells in the DLNs of brain tumor-bearing mice compared with flank tumor-bearing mice (FIG. 3D). Extending these data to a third tumor site, we found that established lung tumors also stimulated IFN-γ secretion more readily than brain tumors (p < 0.001) and similar to flank tumors (p = 0.56) (FIG. 4A, FIG. 4B).

Antigen-specific cytotoxicity is systemically impaired in animals with CNS melanoma

We next tested the effects of 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. In the absence of tumor antigen-specific vaccination or adoptive T cell transfer, recognition of tumor antigen was insufficient to confer effector function (p = 0.14) (FIG. 5A, FIG. 5B). To test whether adoptively transferred, vaccine-primed antigen- specific T cells respond differentially in the context of a brain or flank tumor, we adoptively transferred a physiologically relevant number (approximately 100) OT-1 CD8 T cells (19) to mice bearing established B16-OVA tumors, then vaccinated with a recombinant OVA-expressing vaccinia-based vaccine (Vac-OVA) (15). In cancer- free mice targets persisted in untreated animals, while essentially all targets were lysed after adoptive transfer of antigen-specific CD8 T cells plus vaccination (FIG. 5C, FIG. 5D). Lysis was attenuated in tumor-bearing mice, and CNS melanoma attenuated CD8 T cell function to a greater degree than flank tumors (FIG. 5D). We next examined whether this relative resistance to vaccination applied to therapy with a novel listeria-based vaccine (20), and found that even in the absence of adoptive transfer, this vaccine strain mediated lysis with a non-significant trend towards decreased killing in mice bearing CNS melanoma as compared to mice bearing flank tumors (p = 0.052) (FIG. 5E, FIG. 5F).

TGF-β is elevated in the serum of mice with CNS melanoma

TGF-β family cytokines are pluripotent molecules involved in regulating tissue homeostasis (21) and TGF-βΙ has been implicated as a critical driver of melanoma progression (22). To determine whether CNS melanoma is associated with systemically elevated levels of TGF-β, we measured serum levels of TGF-βΙ . As shown in FIG. 6A, mice with B 16-OVA brain tumors had significantly higher levels of serum TGF-βΙ compared to mice with B 16-OVA flank tumors (p = 0.039). Based on these data, we hypothesized that blocking TGF-β signaling might reverse brain tumor-mediated tolerance. To test this hypothesis, we returned to the Pmel adoptive transfer model and treated brain tumor-bearing mice with a small molecule TGF-β signaling inhibitor (LY2157299) (23). We found that inhibiting TGF-β signaling significantly increased the percentage and number of Pmel CD8 T cells in the spleens of brain tumor- bearing mice (p < 0.001, p = 0.001, respectively) and significantly increased the percentage of Pmel CD8 T cells in the DLN (p = 0.031) (FIG. 6B, FIG. 6C). Somewhat surprisingly, TGF-β blockade did not increase the number of TIL, nor did treatment affect overall survival (FIG. 6D). Nearly complete blockade of TGF-β signaling was confirmed via western blotting (data not shown). Treatment with the pan TGF-β neutralizing antibody 1D11 also did not improve overall survival in these animals (data not shown). Taken together, these results show that CNS melanoma is associated with elevated TGF-β levels, but that blockade alone is not sufficient to alter pre-clinical outcome.

Combination treatment regimens in animals with CNS melanoma

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). We thus tested whether the combination of TGF-β signaling inhibition + focal RT delivered using the small animal radiation research platform (SARRP) could mediate treatment effects in this CNS melanoma model. Unfortunately, this was not the case; although RT enhanced survival significantly (HR = 0.02, p = 0.004), TGF-β inhibition did not add to this effect (FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E). Since LM-based vaccination alone appeared to partially reverse brain tumor-mediated tolerance (FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F), we next tested whether LM-based vaccination could prolong survival. Indeed, the LM-based vaccine mediated a relatively modest increase in OS, but the combination of vaccination + RT significantly increased survival, with a number of animals showing long- term non-progression (FIG. 7B). The addition of TGF-beta blockade to this combination regimen (using either 1D1 1 or LY2157299) did not extend survival compared with LM-OVA + RT (FIG. 8A, FIG. 8B), nor did the addition of PD-1 blockade (FIG. 9). To explore the mechanism of action of this efficacious combination regimen, we performed immunostaining with anti-CD3e. The results 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. To test whether combination immunotherapy altered patterns of brain tumor progression, brains from representative animals (3 per group) were imaged ex vivo with magnetic resonance imaging (MRI). Tumor borders were delineated in each slice and volumes were calculated using ImageJ software by a blinded observer (FIG. 7E). To test for differences in morphology, we identified the dorsal-ventral midplane of each tumor and calculated the ratio of tumor volumes superior and inferior to this plane. Although no differences were observed with RT, LM vaccination appeared to constrain tumor growth, with untreated tumors and tumors treated with RT alone exhibiting more variable morphology at the time of death (FIG. 10A). Additionally, we found that there was no difference in tumor volume between groups at the time of death (FIG. 1 OB).

Combination therapy is associated with poiyfunctionai CD8 T cells, an increased Teff to Treg ratio, and increased APC function.

To elucidate the immunological mechanisms by which focal RT and systemic LM mediate treatment effects in CNS melanoma, 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. As show in in FIG. 1 1A, RT or LM alone generated a modest increase in Granzyme B, IFN-γ and TNF-a production, whilst with combination therapy, the majority of cells produced Granzyme B (GB), IFN-γ and Tumor Necrosis Factor (TNF)-a (FIG. 1 IB). In addition to stimulating cytokine production, combination therapy increased the density of tumor- infiltrating poiyfunctionai CD 8 T cells (p = 0.001 by two-way ANOVA) (FIG. 11C).

Accumulation of Tregs is an important mechanism of immunosuppression in cancer (24), so we next evaluated the effects of this regimen on peripheral and intratumoral Tregs. In the periphery, we observed a modest decrease in the percentage of CD4 T cells expressing FoxP3 with LM vaccination (FIG. 11D). Similarly, LM vaccination decreased the percentage of Tregs within the tumor. Conversely, RT approximately doubled the percentage of CD4 TIL expressing FoxP3, consistent with prior data (25). Combination therapy resulted in an intratumoral Treg profile similar to LM alone, while favorably increasing the percentage of CD4 T cells producing IL- 2 compared with either RT or LM monotherapy (FIG. 1 IE). In several human caner types, 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. We also quantified concentrations of the pro- inflammatory cytokines IFN-γ, IL-2, GM-CSF, and IL-12 as well as the inhibitory cytokines IL-10 and TGF-β in the supernatants of OT-1 CD8 T cells co-cultured with either splenic dendritic cells, tumor DLNs, or microglia (FIG. 12A, FIG. 12B, FIG. 12C). We found that microglia isolated from mice with brain tumors secreted significantly more TGF-β compared with microglia from naive animals and that combination therapy significantly decreased TGF-β secretion from microglia

(FIG.1 1A, FIG. 1 1B, FIG. 11C, FIG. 1 1D, FIG. HE, FIG. 11F, FIG. 11G, FIG. 1 1H, FIG. I ll, FIG. 11 J, FIG. UK, FIG. 11L, FIG. 1 1M, FIG. UN). Interestingly, 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.

Discussion

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). One potential explanation is that 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.

Using a B 16 model we demonstrated that brain tumors inhibit cellular immunity to a greater degree than flank or lung tumors in mice. Pmel CD8 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-γ in response to brain tumors compared with flank tumors. Of note, 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. Furthermore, although re-stimulation of OVA-directed cells in the CNS is sufficient to restore effector function in a CD8-mediated EAE model (28), our data indicate that dividing TIL produce less IFN-γ in brain tumors than in flank tumors. Although caution is warranted in interpreting these pooled TIL data; this observation indicates that tolerance acquired upon priming in secondary lymphoid organs may persist in the brain tumor microenvironment.

Impaired cellular immunity has long been suspected in GBM patients and may be compounded by interventions such as chemotherapy and steroids (4,29). Our data demonstrate that tumor location within the CNS may be an independent mediator of systemic immunosuppression. We used a series of in vivo CTL experiments to explore the functional significance of this finding. Tumor-reactive lymphocytes

characteristically express the exhaustion markers PD-1, LAG-3 and TIM-3 (30), so it was not surprising that tumor antigen processing was insufficient to stimulate a cytotoxic response. This finding is important, however, as it demonstrated that antigen recognition on B 16 tumors was insufficient to mediate significant cytotoxicity. We next applied immunologic pressure by adoptively transferring a physiologically relevant number of OT- 1 cells and vaccinating with Vac-OVA. Here, we found that mice with B16-OVA brain tumors exhibited impaired target lysis, indicating that the presence of a CNS tumor may blunt systemic responses to some immunotherapies.

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-β is a pleotropic cytokine that induces immune suppression and drives tumor progression in several solid tumors, including melanoma and glioma (8,33). TGF-β 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-β than microglia from naive mice. We found that this elevation in TGF-β was also systemic, as B 16-OVA brain tumor bearing mice had significantly elevated serum levels of TGF-βΙ compared with tumor- free or flank tumor-bearing mice. Further, we found that TGF-β 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-β 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. Those data are perhaps somewhat contradictory to recent studies demonstrating that blocking TGF-β prior to hypofractionated radiation enhances preclinical responses (35), and additional work will be required to determine whether these differences reflect the use of different TGF-β blocking agents, the location of the tumor, or the cancer model under study. While our studies focused on microglia and antigen-presenting cells in the tumor draining lymph nodes and spleen, it is clear that tolerance to tumor antigens is mediated by a number of additional myeloid cell types, particularly myeloid derived suppressor cells (MDSC) (31). 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). Liau and colleagues have previously reported that a different strain of LM delays progression of intracranial B16 tumors (41). In our studies, 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.

Interestingly, treatment with LM vaccines increased lysis in mice with brain or flank tumors as compared to tumor-free mice. This effect was not observed with a vaccinia- based vaccine and suggests that LM may be have been superior to vaccinia in boosting a low level of T cell priming that occurs upon recognition of antigen on tumor cells. These data are consistent with the notion that brain tumors may be more systemically tolerogenic than flank tumors, but also show that a potent vaccine may reverse CTL tolerance. Both vaccine platforms, however, failed to completely abrogate brain tumor- mediated tolerance, indicating that combination therapy may be required to achieve maximum efficacy.

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). Newcomb and colleagues showed that radiation therapy combined with GVAX generates long-term survival and protective immunity in an orthotopic glioma model (47) and our group demonstrated that combining focal RT with PD- 1 blockade prolongs survival of mice with orthotopic gliomas and protects against flank tumor re- challenge (9).

Consistent with these data, in some studies we found that, while anti-PD-1 alone or in combination with LM-OVA vaccination had no effect on survival, the combination of RT + anti-PD-1 produced a small percentage of long-term survivors (data not shown). The translational relevance of these findings should be interpreted with caution given that PD-1 or CTLA-4 blockade alone have a modest effect on B 16 progression (48), while multiple clinical trials have proven efficacy of checkpoint blockade in human melanoma. Further, immune checkpoint blockade may represent a form of vaccination in humans; CTLA- 4 blockade has been shown to boost T cell responses to shared tumor antigens (49), and responses to PD-1 blockade in humans may represent recognition of mutated tumor antigens (50). Additional experiments in other melanoma models are warranted to more clearly delineate the role of checkpoint blockade in the setting of tumor antigen-specific vaccination.

We found that combining focal RT with LM-OVA vaccination significantly prolonged survival over either monotherapy. Combination therapy stimulated tumor infiltration by polyfunctional CD8 T cells and increased Teff to Treg ratios, an immune profile that has been associated with tumor regression (24,51). Furthermore, combination treatment with focal RT and LM-OVA altered the cytokine profile of microglia, reducing TGF-βΙ secretion to levels indistinguishable from naive microglia. Consistent with previous reports, RT increased intratumoral Treg density (52). Conversely, LM-OVA decreased the percentage of Tregs both within the tumor and peripherally. 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. These data suggest a therapeutic mechanism by which LM vaccination decreased the number of Tregs locally and systemically and bolstered APC function, while focal RT promoted polyfunctionality of CTLs and increased intratumoral Teff density.

Based on these data, it is reasonable to hypothesize that development of a melanoma brain metastasis curbs immunologic pressure against tumor antigens and accelerates systemic disease progression. This model affords a plausible explanation for why brain metastases carry a grave prognosis, even in the setting of stable CNS disease. In summary, these data suggest that CNS tumors may impair systemic antitumor immunity and consequently accelerate cancer progression locally as well as outside the CNS while antitumor immunity may be restored by combining vaccination with radiation therapy.

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All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

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Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.

Claims

THAT WHICH IS CLAIMED:

1. 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.

2. A composition for the treatment of a metastatic central nervous system (CNS) tumor comprising a radiotherapeutic agent and a Listeria-based vaccine.

3. 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.

4. 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 level of the at least one cytokine produced from the polyfunctional lymphocyte is decreased relative to, or remains at or near, the baseline level, at the one or more time intervals.

5. 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-β 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-β;

(b) detecting the level of the TGF-β 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-β 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-β is increased relative to, or remains at or near, the baseline level, at the one or more time intervals.

6. The method, composition, or kit of any one of claims 1-5, wherein the method of monitoring treatment efficacy further comprises administering the combination treatment to the patient after the treatment efficacy is monitored.

7. The method, composition, or kit of any one of claims 1-6, wherein treatment efficacy is monitored using a sample from the patient comprising serum, and/or blood.

8. The method, composition, or kit of any one of claims 1-7, wherein at least one cytokine is selected from the group consisting of Granzyme B (GB), Interferon-γ (IFN-γ), Tumor Necrosis Factor (TNF)-a, and Interleukin-2 (IL-2).

9. The method, composition, or kit of any one of claims 1-8, wherein at least four cytokines are detected.

10. The method, composition, or kit of any one of claims 1-9, wherein the metastatic CNS tumor is a brain tumor.

11. The method, composition, or kit of any one of claims 1-10, wherein the brain tumor is not a glioma.

12. The method, composition, or kit of any one of claims 1-1 1, wherein 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.

13. The method, composition, or kit of any one of claims 1-12, wherein the metastatic CNS tumor is a melanoma.

14. The method, composition, or kit of any one of claims 1-13, wherein the combination therapy stimulates tumor infiltration by the polyfunctional lymphocyte.

15. The method, composition, or kit of any one of claims 1-14, wherein the polyfunctional lymphocyte is a CD8+ T cell.

16. The method, composition, or kit of any one of claims 1-15, wherein the Listeria-bassd vaccine is a live-attenuated vaccine.

17. The method, composition, or kit of any one of claims 1-16, wherein the Listeria is Listeria monocytogenes.

18. The method, composition, or kit of any one of claims 1-17, wherein the Listeria-bassd vaccine comprises ovalbumin (OVA) or an immunogenic part thereof.

19. The method, composition, or kit of any one of claims 1-18, wherein the Listeria-based vaccine further comprises an adjuvant.

20. The method, composition, or kit of any one of claims 1-19, wherein the radiotherapy is focal.

21. The method, composition, or kit of any one of claims 1 -20, wherein the radiotherapy is stereotactic radiosurgery, fractionated stereotactic radiosurgery, and/or intensity-modulated radiation therapy (IMRT).

22. The method, composition, or kit of any one of claims 1-21, wherein 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).

23. The method, composition, or kit of any one of claims 1 -22, wherein the dosage of radiotherapy ranges from about 1 Gy to about 30 Gy.

24. The method, composition, or kit of any one of claims 1-23, wherein the dosage of radiotherapy is about 8 Gy to about 16 Gy.

25. Use of the composition of claim 2 for the treatment of a metastatic central nervous system (CNS) tumor.

26. Use of the composition of claim 2 for the manufacture of a

medicament for the treatment of a metastatic central nervous system (CNS) tumor.

27. 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 method of claim 27, wherein the mammal comprises a rodent. The method of claim 28, wherein the rodent comprises a mouse.