In SITU tumor vaccine to promote Anti-tumor immunity

Abstract

Provided herein are methods and compositions for treating cancer by administering an in-situ cancer vaccine, such as comprising tumor directed radiation therapy and a Bromodomain and Extra-Terminal motif (BET) inhibitor.

Description

IN SITU TUMOR VACCINE TO PROMOTE ANTI TUMOR IMMUNITYRELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 641,218, filed May 1, 2024, which is hereby incorporated by reference in its entirety.BACKGROUND

[0002] The present disclosure is generally directed to compositions and methods for an in- situ cancer vaccine combining administration of radiation therapy and Bromodomain and Extra-Terminal motif (BET) inhibitors to a subject in need thereof.

[0003] Although immunotherapies have revolutionized the treatment of patients with many types of cancers, most patients do not respond to existing immunotherapies. Furthermore, existing immunotherapies require prolonged treatment to afford any benefit to patients. Tumor vaccines that afford long lasting tumor control with a brief duration of administration are currently lacking. There is a strong need to develop novel chemotherapy- free, vaccine-like immunotherapy regimens that achieve superior tumor control with minimal toxicity.SUMMARY OF THE INVENTION

[0004] In accordance with an aspect of the disclosure, provided herein is a method of treating a cancer or a tumor in a subject in need thereof, the method comprising administering to the subject an in-situ cancer vaccine as described herein, e.g., (a) administering one or more doses of a tumor directed radiation therapy; and (b) administering to the subject one or more doses of a Bromodomain and Extra-Terminal motif (BET) inhibitor.

[0005] In an aspect, provided herein is a method of inducing a cancer specific or tumor specific immune memory response in a subject in need thereof, the method comprising administering to the subject an in-situ cancer vaccine as described herein, e.g., (a) administering to the subject one or more doses of a tumor directed radiation therapy; and (b) administering to the subject one or more doses of a BET inhibitor.

[0006] In an aspect, provided herein is a method of activating host innate immunity against a cancer or tumor in a subject in need thereof, the method comprising administering to the subject an in-situ cancer vaccine as described herein, e.g., (a) administering to the subject one or more doses of a tumor directed radiation therapy; and (b) administering to the subject one or more doses of a Bromodomain and BET inhibitor. The activating of host innate immunity may comprise activating the cGAS / STING signaling pathway in the subject.

[0007] In an aspect, provided herein is a method of increasing neoantigen release in a subject in need thereof, the method comprising administering to the subject an in-situ cancer vaccine as described herein, e.g., (a) administering to the subject one or more doses of a tumor directed radiation therapy; and (b) administering to the subject one or more doses of a BET inhibitor, wherein the neoantigen is specific for a cancer or tumor in the subject.

[0008] In an aspect, provided herein is a method of reducing or preventing immunosuppressive effects of a cancer or tumor in a subject in need thereof, the method comprising administering to the subject an in-situ cancer vaccine as described herein, e.g.,(a) administering to the subject one or more doses of a tumor directed radiation therapy; and(b) administering to the subject one or more doses of a BET inhibitor. The reducing or preventing immunosuppressive effects may comprise reducing or preventing of PD-L1 at the cancer or tumor. The reducing or preventing of PD-L1 may comprise reducing PD-L1 expression or activity. Any of the methods as described herein may comprise administering 2 to 4 or more doses of tumor directed radiation therapy, optionally 2 to 3 doses of tumor directed radiation therapy. Each dose of radiation therapy may be an absorbed dose of about 5 to 10 Gy, optionally the absorbed dose can be 8 Gy. For any of the methods as described herein, the tumor directed radiation therapy may comprise external-beam radiation therapy, interstitial implantation of radioisotopes (1-125, palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and / or total abdominal and pelvic radiation therapy.

[0009] In an aspect, a BET inhibitor as described herein may be selected from OTX015, ZEN-3694, JQ1, TEN-010 (JQ2), Molibresib (I-BET762), I-BET 151 (GSK1210151A), I- BET 762 (GSK525762), CPI-203, or CPI-0610, or any combination thereof. The radiation therapy and BET inhibitor may be administered concomitantly or concurrently, sequentially,0or a combination thereof. The BET inhibitor may be administered daily for the duration of the treatment (e.g., daily for 3 to 5 days, or daily for 4 days).

[0010] In an aspect, any of the methods as described herein may comprise administering the tumor radiation therapy and the BET inhibitor in less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, or less than 5 days. In some embodiments, the administering the tumor radiation therapy and the BET inhibitor occurs in 4 days. The radiation therapy may be administered twice, every other day, over four days and the BET inhibitor is administered daily for the four days.

[0011] In an aspect, the cancer or tumor is of a cancer type as described herein. The tumor may be solid tumor. The solid tumor is selected from testicular tumor, ovarian tumor, cervical tumor, a kidney tumor, bladder tumor, head-and-neck tumor, skin tumor, bone tumor, brain tumor, thyroid tumor, pancreatic tumor, liver tumor, stomach tumor, lung tumor, endometrial tumor, esophageal tumor, breast tumor, cervical tumor, central nervous system tumor, germ cell tumor, prostate tumor, Hodgkin's lymphoma, non-Hodgkin's lymphoma, neuroblastoma, sarcoma, multiple myeloma, melanoma, mesothelioma, osteogenic sarcoma, or any combination thereof.

[0012] The cancer or tumor may be a breast cancer or tumor, optionally wherein the breast cancer or tumor is a triple negative breast cancer or an ER+ breast cancer. The cancer or tumor may be a sarcoma, optionally wherein the sarcoma is a soft tissue sarcoma or bone sarcoma.

[0013] In an aspect, a method as described herein may further comprise administering one or more immunotherapies and / or chemotherapies, optionally selected from pembrolizumab, carboplatin, paclitaxel, doxorubicin / epirubicin, cyclophosphamide, or any combination thereof.

[0014] In an aspect, the subject may have improved survival and / or the tumor or cancer has reduced growth and / or metastasis after administering an in-situ cancer vaccine as described herein (e.g., radiation therapy and BET inhibitor) compared to a subject receiving a standard of care. The standard of care may comprise administering one or more immunotherapies and / or chemotherapies, optionally selected from pembrolizumab,carboplatin, paclitaxel, doxorubicin / epirubicin, cyclophosphamide, or any combination thereof.

[0015] The method of claim 28, wherein the standard of care further comprises administering the one or more immunotherapies and / or chemotherapies over the course of 60 weeks.

[0016] In an aspect, provided herein is a kit comprising one or more components of an in- situ cancer vaccine as described herein, and instructions for use according to the any one of the methods as described herein. The kit may comprise a BET inhibitor and / or a tumor directed radiation therapy; and instructions for use according to the any one of the methods as described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office by request and payment of the necessary fee.

[0018] Embodiments of the present inventive concept are illustrated by way of example in which like reference numerals indicate similar elements, and in which:

[0019] FIG. 1 depicts a schematic describing the multi-step process of cancer cell immunity according to the disclosures herein. An RT-BET inhibitor treatment can affect multiple steps of the cycle to produce a stronger immune response. The top left and bottom right bubbles indicate possible anti -tumor immune effects (e.g., activation of cGAS / STING and antigen release) of the treatment, and the top right bubble indicates possible pro-tumor or immunosuppressive effects (e.g., suppressing PD-L1 increase).

[0020] FIG. 2 depicts data showing that BET inhibition enhances radiation-induced DNA damage.

[0021] FIG. 3A-3B depict data showing that BET inhibition reverses silencing of radiation induced PD-L1 expression.

[0022] FIGS. 4A-4B show protocols for BET inhibition plus radiation therapy as a novel cancer vaccine. FIG. 4A depicts a schematic of a combination of radiation and BET inhibition (e.g., RT+OTX). FIG. 4B depicts a schematic of a standard of care protocol.

[0023] FIGS. 5A-5B show results of RT+ BET inhibitor administration exerting superior tumor control than SOC or RT only. FIG. 5A depicts a schematic illustrating different treatment protocols (, SOC “standard of care”, OTX x 4 (BET inhibitor alone), 8 Gy radiation therapy x 2 (radiation therapy alone), and 8 Gy radiation therapy x2 plus OTX (combination of RT+ BET inhibitor)). FIG. 5B shows a plot of tumor volume up to 50 days after treatment in mice following different treatment protocols.

[0024] FIGS. 6A-6B show results of RT+ BET inhibitor administration promoting antitumor activity. FIG. 6A depicts a schematic of 4T1 TNBC mice treated with RT+BET inhibitor and then rechallenged (n=2), or naive mice (n=4). FIG. 6B shows a plot of tumor volume after tumor injection following the 4T1 rechallenge in these mice.

[0025] FIGS. 7A-7B show that BET inhibition synergizes with radiation therapy to cause potent tumor growth inhibition in a syngeneic ER+ BC model. FIG. 7A depicts results from biochemical assays validating MXT cells and FIG. 7B shows a plot of tumor volume over time in mice injected with MXT cells following treatment with BET inhibitor (OTX), radiation therapy (RT), or a combination of the two.

[0026] FIGS. 8A-8C show the response to RT+BET inhibition is CD8 T cell-dependent. FIG. 8A depicts a representative schematic of an experimental protocol to deplete IgG or CD8 T cells in animals after the RT+BET inhibitor combination protocol and FIGS. 8B-8C illustrate flow cytometry plots indicating successful depletion of IgG and CD8 cells in mice.

[0027] FIG. 9 demonstrates that the response to RT+BET inhibition is CD8 T celldependent. FIG. 9 shows a plot of tumor volume in IgG depleted or CD8 T cell depleted animals over time after treatment with RT+BET inhibitor.

[0028] FIGS. 10A-10B depict plots of lung and liver metastases observed in IgG or CD8 depleted mice following a RT+BET inhibitor combination treatment protocol.

[0029] FIGS. 11A-11B depict results from an in vivo drug compound screen for inhibitors of various targets as described in TABLE 1. FIG. 11A shows the treatment regimen.FIG. 11B shows the results of the drug screen determined by relative tumor volume, identifying BET inhibitor (i.e., OTX015) as the most effective.

[0030] FIGS. 12A-12B depict a schematic of experimental designs for testing treatment regimen against cancer cell lines (e.g., TNBC cell lines). FIG. 12A depicts a four-day treatment regimen, used for 4T1, EMT6, and other cell lines of the disclosure using a vehicle, OTX alone, RT alone, or RT+OTX. FIG. 12B depicts an eight-week standard-of- care treatment.

[0031] FIGS. 13A-13B depict the results of experiments comparing the four-day treatment regimen for 4T1 or EMT6 tumor bearing mice. FIG. 13A shows results for vehicle, OTX alone, RT alone, RT+OTX, and standard-of-care (SOC) regimen for 4T1 tumor bearing mice. FIG. 13B shows results for vehicle, OTX, RT alone, or RT+OTX for EMT6 cell - inject mice. Measurements of tumor volume were performed once tumor volume reached 100 mm3 after the four day treatment.

[0032] FIGS. 14A-14C depict exploratory experiments for the syngeneic TNBC mouse model, E0771. FIG. 14A depicts the treatment regimen, and FIG. 14B shows tumor volumes following treatment with RT alone or RT+OTX. FIG. 14C shows tumor masses measured one month post-treatment start.

[0033] FIGS. 15A-15B depict experiments treating an ER+ BC (estrogen receptor-positive breast cancer) syngeneic mouse model (i.e., MXT1 tumor cell bearing). FIG. 15A shows a Western blot comparing expression of estrogen receptor on a human cell line of ER+ BC, MCF7, to the ER+ BC mouse cell line, MXT1 (FIG. 15B) shows the treatment regimen for a vehicle, OTX, RT only, or RT+OTX over six days for the MXT1 bearing mice.

[0034] FIGS. 16A-16B depict results for efficacy of RT+OTX therapy in MXT1 bearing mice. FIG. 16A shows tumor volume growth for vehicle, OTX only, RT only, and RT+OTX therapy. FIG. 16B shows tumor mass measured 29 days after treatment of MXTl-bearing mice with radiation therapy or RT+OTX (FIG. 16A) and the).

[0035] FIGS. 17A-17B depict results for MCA205 mouse fibrosarcoma tumor cell bearing mice. FIG. 17A shows tumor volume growth following RT-OTX treatment. FIG. 17B shows a waterfall survival plot.

[0036] FIGS. 18A-18B depict results from a treatment study comparing RT+OTX to anti- PD1 immunotherapy combined with RT or a BET inhibitor in 4T1 tumor bearing mice. FIG. 18A shows tumor volume growth following PD1+RT and RT-OTX treatment.FIG. 18B shows tumor volume growth following PD1+OTX and RT-OTX treatment.

[0037] FIGS. 19A-19B depict rechallenge experiments for tumor specific immunity using 4T1 and EMT6 tumor cells. FIG. 19A depicts the study design for 4T1 rechallenge (n=2). FIG. 19B shows that neither mouse regrew the tumor (4T1 rechallenge). These cured mice were again rechallenged with EMT6 tumor cells, which grew comparably to mice with no prior tumor or treatment (see EMT6 rechallenge and 4T1 naive)

[0038] FIGS. 20A-20B depict rechallenge experiments for tumor specific immunity using MCA205 mouse fibrosarcoma (FIG. 20A) and E0771 TNBC (FIG. 20B) tumor models, showing that the tumor-specific immune memory and rejection occurs irrespective of cell line or cancer type.

[0039] FIG. 21A-21B depict results for T cell dependency of RT+BET inhibitor therapy. FIG. 21A shows a treatment regimen, followed by flow cytometry at day 14. FIG. 21 shows quantified flow cytometry measurement of immune cell populations (e.g., CD3+; CD4+; and CD8+) within the tumor microenvironment, revealing a T cell enrichment in the tumor microenvironment following RT+OTX treatment.

[0040] FIGS. 22A-22E depict results of a T cell depletion experiment to determine the degree of involvement of CD8+ T cells in the mechanism of the RT+OTX treatment.FIGS. 22A-B show results of a flow cytometry experiment using IgG and CD8 depletion antibodies to confirm that CD8+ T cell depletion was functioning. FIG. 22C shows the tumor volume increase in the CD8+ T cell depleted mice. FIG. 22D shows the metastatic effects of the CD8+ T cell depletion. FIG. 22E shows lungs excised from mice 24 days after treatment.

[0041] FIGS. 23A-23B depict the results of an experiment to determine effects of RT+OTX therapy on DNA damage response. FIG. 23A shows IF staining of yH2AX, a marker of double-stranded DNA breaks. This yH2AX expression is quantified in FIG. 23B, suggesting that RT+OTX increases DNA damage and offers potential for immunogenic mutations.

[0042] FIG. 24 shows that RT+OTX enhances the formation of micronuclei, another sign of DNA damage.

[0043] FIG. 25 shows that RT+OTX treatment alters the recruitment of DNA repair proteins to damaged DNA. A clear reduction in DNA repair proteins bound to the chromatin with RT+OTX was observed.

[0044] FIG. 26 shows that RT+OTX therapy suppresses MYC expression, which is inversely correlated with activation of the cGAS-STING pathway, suggesting another antitumor immune mechanism for RT+OTX therapy.

[0045] FIGS. 27 -27C depict the results of a toxicity study, in which RT+OTX was determined to be minimally toxic. FIG. 27A shows that no difference between RT+OTX and vehicle treatments was observed. FIG. 28B shows a complete blood count analysis, in which no significant difference was observed. FIG. 29C shows a metabolite analysis, where only ALT / ALTV was significantly different (lower) in the RT+OTX treatment.

[0046] The drawing figures do not limit the present inventive concept to the specific embodiments disclosed and described herein. The drawings are not necessarily to scale, emphasis instead being placed on clearly illustrating principles of certain embodiments of the present inventive concept.DETAILED DESCRIPTION

[0047] The following detailed description references the accompanying drawings that illustrate various embodiments of the present inventive concept. The drawings and description are intended to describe aspects and embodiments of the present inventive concept in sufficient detail to enable those skilled in the art to practice the present inventive concept. Other components can be utilized and changes can be made without departing from the scope of the present inventive concept. The following description is, therefore, not to be taken in a limiting sense. The scope of the present inventive concept is defined only by the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0048] The present disclosure is based on the surprising finding of an in-situ cancer vaccine that affords long term anti-tumor immunity. The disclosed in-situ cancer vaccineprovides the following advantages over current standard of care (SOC) treatments. First, it has a brief duration of administration (as opposed to current chemo-10 regimens in breast cancer which are administered over 15 cycles over several months). Second, it is easily administered as it does not require intravenous infusions or intratumoral injections. Third, it has minimal toxicity (especially compared to strong chemotherapy regimens). Finally, it has superior tumor control relative to existing standard of care regimens.

[0049] Accordingly, provided herein is an in-situ cancer vaccine that combines a short course of tumor-directed radiation therapy (2 or 3 total radiation treatments) with a bromodomain and extraterminal domain (BET inhibitor). Although radiation alone or BET inhibitor alone has modest efficacy in the long-term control of tumors, the combination shows evidence of a vaccine like effect and better efficacy than any existing clinically available treatments. In other words, the inventors have discovered that combining radiation therapy with a targeted therapeutic can enhance immune-stimulatory effects and reduce immunosuppressive effects to promote overall anti-tumor immunity.I. Terminology

[0050] The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. For example, the use of a singular term, such as, “a” is not intended as limiting of the number of items. Also, the use of relational terms such as, but not limited to, “top,” “bottom,” “left,” “right,” “upper,” “lower,” “down,” “up,” and “side,” are used in the description for clarity in specific reference to the figures and are not intended to limit the scope of the present inventive concept or the appended claims.

[0051] Further, as the present inventive concept is susceptible to embodiments of many different forms, it is intended that the present disclosure be considered as an example of the principles of the present inventive concept and not intended to limit the present inventive concept to the specific embodiments shown and described. Any one of the features of the present inventive concept may be used separately or in combination with any other feature. References to the terms “embodiment,” “embodiments,” and / or the like in the description mean that the feature and / or features being referred to are included in, at least, one aspect of the description. Separate references to the terms “embodiment,” “embodiments,” and / or thelike in the description do not necessarily refer to the same embodiment and are also not mutually exclusive unless so stated and / or except as will be readily apparent to those skilled in the art from the description. For example, a feature, structure, process, step, action, or the like described in one embodiment may also be included in other embodiments, but is not necessarily included. Thus, the present inventive concept may include a variety of combinations and / or integrations of the embodiments described herein. Additionally, all aspects of the present disclosure, as described herein, are not essential for its practice.Likewise, other systems, methods, features, and advantages of the present inventive concept will be, or become, apparent to one with skill in the art upon examination of the figures and the description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present inventive concept, and be encompassed by the claims.

[0052] Any term of degree such as, but not limited to, “substantially” as used in the description and the appended claims, should be understood to include an exact, or a similar, but not exact configuration. For example, “a substantially planar surface” means having an exact planar surface or a similar, but not exact planar surface. Similarly, the terms “about” or “approximately,” as used in the description and the appended claims, should be understood to include the recited values or a value that is three times greater or one third of the recited values. For example, about 3 mm includes all values from 1 mm to 9 mm, and approximately 50 degrees includes all value from 16.6 degrees to 150 degrees. For example, they can refer to less than or equal to ± 5%, such as less than or equal to ± 2%, such as less than or equal to ± 1%, such as less than or equal to ± 0.5%, such as less than or equal to ± 0.2%, such as less than or equal to ± 0.1%, such as less than or equal to ± 0.05%.

[0053] The term “standard of care” refers to an established treatment approved and normally used to treat a cancer or tumor in a subject. The “standard of care’ may vary depending on the underlying cancer or tumor and would be known by one of skill in the art in medicine. Standard of care can include any combination of chemotherapy, radiation, immunotherapy, hormone therapy, or other anti -cancer therapies known in the art. In certain embodiments, “standard of care” can comprise a combination of chemotherapy and radiation (i.e., targeted tumor radiation). For instance, in one aspect, a “standard of care’ regimen can comprise administering pembrolizumab and carboplatin every three weeks for12 weeks while administering paclitaxel weekly, optionally followed by administering pembrolizumab, doxorubicin / epirubicin and cyclophosphamide every three weeks for an additional 12 weeks, further optionally followed by surgical resection of a tumor, and then further optionally followed by administering pembrolizumab every three weeks for an additional 24 weeks. The standard of care may be provided for in a published clinical trial for the treatment of a cancer as described herein. In an aspect, a subject treated with a “standard of care” regimen such as the one described above, is known as a ‘SOC subject.”

[0054] The terms "comprising," "including" and "having" are used interchangeably in this disclosure. The terms "comprising," "including" and "having" mean to include, but not necessarily be limited to the things so described.

[0055] Lastly, the terms “or” and “and / or,” as used herein, are to be interpreted as inclusive or meaning any one or any combination. Therefore, “A, B or C” or “A, B and / or C” mean any of the following: “A,” “B” or “C”; “A and B”; “A and C”; “B and C”; “A, B and C.” An exception to this definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive.IL In-situ Cancer Vaccines

[0056] As disclosed herein, a cancer vaccine, or oncovaccine, is a vaccine that treats an existing cancer in a subject or prevents development or regrowth of cancer in a subject, with or without a prior cancer treatment which may include any one or any combination of a cancer chemotherapy, a cancer immunotherapy, surgery, and radiation. These vaccines operate by priming the immune system to target the cancer. The in-situ cancer vaccines provided herein have the surprising and unexpected effect of reducing tumor volume following short treatment course of days, as described herein. Without being bound by theory, it is believed that the surprising effects disclosed herein are a result of an overall anti-tumor immunity response in a subject, which is induced by combining radiation therapy with a targeted therapeutic administered as disclosed herein. In one aspect, an in-situ cancer vaccine as disclosed herein is achieved by administering a short course of turn or- directed radiation therapy (e.g., 2 or 3 total radiation treatments) with a bromodomain and extraterminal domain (BET) inhibitor. In one aspect, the combination therapy is delivered over days, or weeks, yet elicits a pronounced decrease in tumor volume.

[0057] Accordingly, in an aspect, the in-situ cancer vaccine may comprise administering to a subject in need thereof one or more doses of a tumor directed radiation therapy and one or more doses of a BET inhibitor.

[0058] The tumor directed radiation therapy can, in an aspect, comprise one or more exposures to a radiation therapy. The radiation used in radiation therapy can be ionizing radiation. Radiation therapy can also be gamma rays, X-rays, or proton beams. Examples of radiation therapy may comprise or be selected from external -beam radiation therapy, interstitial implantation of radioisotopes (1-125, palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and / or total abdominal and pelvic radiation therapy. In some aspects, the radiation therapy can be administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source. In other aspects, the radiation treatment can also be administered as internal therapy or brachytherapy wherein a radioactive source is placed inside the body close to cancer cells or a tumor mass. Also encompassed is the use of photodynamic therapy comprising the administration of photosensitizers, such as hematoporphyrin and its derivatives, vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, demethoxy - hypocrellin A; and 2BA-2-DMHA. Each radiation therapy can, in an aspect, involve administering a total absorbed dose from about 10 to about 40 Gy, about 10 to about 30 Gy, or about 10 to about 20 Gy (e.g., about 16 Gy). The total absorbed dose is preferably administered over two or more sessions (e.g., two sessions at 8 Gy each). These two or more sessions can occur over a few days (i.e., over 4 days, over 2 days). The administration may occur over two sessions, three sessions, four sessions, five sessions, six sessions, eight sessions, nine sessions, or ten sessions. In an aspect, the tumor directed radiation therapy can comprise administering two sessions (or doses) of radiation at 8 Gy over a four-day treatment regimen (i.e., on day 2 and day 4 of the regimen. In an aspect, the tumor directed radiation therapy can comprise administering three sessions (doses) of radiation at 8 Gy over a 4-10 day period. In an aspect, the tumor directed radiation therapy can comprise administering four sessions (doses) of radiation at 8 Gy over a 4-10 day period. Each session (e.g., dose) of radiation may be about 1 Gy, about 2 Gy, about 3 Gy, about 4 Gy, about 5 Gy, about 6 Gy, about 7 Gy, about 8 Gy, about 9 Gy, or about 10 Gy. The totaldose in Gy may be less than comparable SOC therapy. For example, the in-situ cancer vaccine as described herein is more effective, and requires less overall radiation exposure.

[0059] BET inhibitors are a class of drugs that reversibly bind the bromodomains of Bromodomain and Extra-Terminal motif (BET) proteins BRD2, BRD3, BRD4, and / or BRDT, and prevent protein-protein interaction between BET proteins and acetylated histones and transcription factors. The BET inhibitor can be any BET inhibitor (i.e., a pan- BET inhibitor) known in the art. BET inhibitors can target both BRD1 and BRD2 or target just BRD1 or BRD2. BET inhibitors that garget both BRD1 and BRD2 are known as pan- BET inhibitors and include, but are not limited to, OTX015, ZEN-3694, JQ1 , TEN-010 (JQ2), Molibresib (I-BET762), I-BET 151 (GSK1210151A), I-BET 762 (GSK525762), CPI- 203, and CPI-0610. BRD1 specific inhibitors include olinone. BRD2 specific inhibitors include RVX-208 and ABBV-744. Other BET inhibitors include dual-kinase bromodomain inhibitors (E.g., LY294002 which targets P13K and BRD2, BRD4 and BRD4) and bivalent BET inhibitors like AZD5153, MT-1 and MS645. In an aspect, the BET inhibitor can comprise any of the aforementioned BET inhibitors or any combination thereof. In some aspects, the BET inhibitor can comprise or any combination thereof. For example, in an aspect, the BET inhibitor can comprise OTX015, JQ1, TEN-010 (JQ2), Molibresib (I- BET762) or any combination thereof. In some embodiments, the BET inhibitor is selected from: OTX015, TP-3634, TP-472, selisistat, JIB-04, dorsomorphin, talazoparib, alisertib, decitabine, riluzole, curcumin, tazemetostat, fedratinib, GSK332659, panobinostat, Fidas-5, UNC0642, GSK199, SSTM2457, or a combination thereof. These inhibitors are all closely related structurally (as shown in the Table below) and all act as pan-BET inhibitors targeting both BD1 and BD2 domains. In an aspect, the BET inhibitor can comprise OTX015 (referred to herein as “OTX”).

[0060] In an aspect, the BET inhibitor can be administered more than once over a therapeutic regimen that corresponds to the “in-situ cancer vaccine” herein (for example, in one or more doses). For instance, the BET inhibitor can be administered 2, 3, 4, 5, 6, 7, 8, 9, or 10 times over a therapeutic regimen (e.g., in 2, 3 or 4 doses). In an aspect, the BET inhibitor is administered four times (4x) over the course of the disclosed therapeutic regimen. In an aspect, the BET inhibitor is administered on day 1, day 2, day 3, and day 4 of a four-day treatment regimen. In an aspect, the BET inhibitor is administered six times (6x) over the course of the disclosed therapeutic regimen. In an aspect, the BET inhibitor is administered on day 1, day 2, day 3, day 4, day 5, and day 6 of a six-day treatment regimen. In an aspect, the BET inhibitor is administered eight times (8x) over the course of the disclosed therapeutic regimen. In an aspect, the BET inhibitor is administered on day 1, day 2, day 3, day 4, day 5, day 6, day 7, and day 8 of an 8-day treatment regimen. Additional details for administration and dosing protocols are provided below.

[0061] In view of the foregoing, an in-situ cancer vaccine is provided, may have a treatment regimen comprising administering a BET inhibitor (e.g.,, OTX015 (OTX)) on day 1, day 2, day 3 and day 4 and administering a tumor directed radiation therapy (e.g., 8 Gy absorbed radiation) dose on day 2 and day 4. The treatment regimen may comprise administering a BET inhibitor (e.g., OTX015 (OTX)) on day 1, day 2, day 3, day 4, day 5, and day 6 and administering a tumor directed radiation therapy (e.g., 8 Gy absorbed radiation) dose on day 2, day 4, and day 6. The treatment regimen may comprise administering a BET inhibitor (e.g., OTX015 (OTX)) on day 1, day 2, day 3, day 4, day 5, day 6, day 7, and day 8 and administering a tumor directed radiation therapy (e.g., 8 Gy absorbed radiation) dose on day 2, day 4, day 6, and day 8. Other modifications to this protocol (e.g., the order of administering the in-situ vaccine components, or intervals between each) can be envisioned by a person of skill in the art.

[0062] An in-situ cancer vaccine as described herein may further comprise one or more immunotherapies and / or chemotherapies. The one or more immunotherapy or chemotherapy may be selected from pembrolizumab, carboplatin, paclitaxel, doxorubicin / epirubicin, cyclophosphamide, or any regulatory approved cancer therapeutic, or combination thereof. The regulatory approved cancer therapeutic may be at a dosage or treatment regimen as approved by the Food and Drug Administration, European Medicines Agency, or another medical regulatory body.

[0063] In an aspect, the BET inhibitor is administered as a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and / or excipient, which are described further below.Pharmaceutically acceptable carriers and excipients

[0064] Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier” are interchangeably used herein to refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

[0065] In certain embodiments, compositions disclosed herein may further compromise one or more pharmaceutically acceptable diluent(s), excipient(s), and / or carrier(s). As usedherein, a pharmaceutically acceptable diluent, excipient, or carrier, refers to a material suitable for administration to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. Pharmaceutically acceptable diluents, carriers, and excipients can include, but are not limited to, physiological saline, Ringer’s solution, phosphate solution or buffer, buffered saline, and other carriers known in the art.

[0066] In some embodiments, pharmaceutical compositions herein may also include stabilizers, antioxidants, colorants, other medicinal or pharmaceutical agents, carriers, adjuvants, preserving agents, stabilizing agents, wetting agents, emulsifying agents, solution promoters, salts, solubilizers, antifoaming agents, antioxidants, dispersing agents, surfactants, or any combination thereof. Herein, the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.

[0067] In certain embodiments, pharmaceutical compositions described herein may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries to facilitate processing of genetically modified endothelial progenitor cells into preparations which can be used pharmaceutically. In some embodiments, any of the well-known techniques, carriers, and excipients may be used as suitable and / or as understood in the art.

[0068] In certain embodiments, pharmaceutical compositions described herein may be an aqueous suspension comprising one or more polymers as suspending agents. In some embodiments, polymers that may comprise pharmaceutical compositions described herein include: water-soluble polymers such as cellulosic polymers, e.g., hydroxypropyl methylcellulose; water-insoluble polymers such as cross-linked carboxyl-containing polymers; mucoadhesive polymers, selected from, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methylmethacrylate), polyacrylamide, polycarbophil,acrylic acid / butyl acrylate copolymer, sodium alginate, and dextran; or any combination thereof In some embodiments, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% total amount of polymers as suspending agent(s) by total weight of the composition. In some embodiments, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of polymers as suspending agent(s) by total weight of the composition.

[0069] In certain embodiments, pharmaceutical compositions disclosed herein may comprise a viscous formulation. In some embodiments, viscosity of composition herein may be increased by the addition of one or more gelling or thickening agents. In some embodiments, compositions disclosed herein may comprise one or more gelling or thickening agents in an amount to provide a sufficiently viscous formulation to remain on treated tissue. In some embodiments, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% total amount of gelling or thickening agent(s) by total weight of the composition. In some embodiments, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of gelling or thickening agent(s) by total weight of the composition. In some embodiments, suitable thickening agents for use herein can be hydroxypropyl methylcellulose, hydroxyethyl cellulose, polyvinylpyrrolidone, carboxymethyl cellulose, polyvinyl alcohol, sodium chondroitin sulfate, or sodium hyaluronate. In other aspects, viscosity enhancing agents can be acacia (gum arabic), agar, aluminum magnesium silicate, sodium alginate, sodium stearate, bladderwrack, bentonite, carbomer, carrageenan, Carbopol, xanthan, cellulose, microcrystalline cellulose (MCC), ceratonia, chitin, carb oxy methylated chitosan, chondrus, dextrose, furcellaran, gelatin, ghatti gum, guar gum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, maize starch, wheat starch, rice starch, potato starch, gelatin, sterculia gum, xanthum gum, gum tragacanth, ethyl cellulose, ethylhydroxyethyl cellulose, ethylmethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxy ethylmethyl cellulose, hydroxypropyl cellulose, poly(hydroxyethyl methacrylate), oxypolygelatin, pectin, polygeline, povidone, propylenecarbonate, methyl vinyl ether / maleic anhydride copolymer (PVM / MA), poly(methoxyethyl methacrylate), poly(methoxyethoxyethyl methacrylate), hydroxypropyl cellulose, hydroxypropylmethyl-cellulose (HPMC), sodium carboxymethyl-cellulose (CMC), silicon dioxide, polyvinylpyrrolidone (PVP: povidone), Splenda® (dextrose, maltodextrin and sucralose), or any combination thereof.

[0070] In certain embodiments, pharmaceutical compositions disclosed herein may comprise additional agents or additives selected from a group including surface-active agents, detergents, solvents, acidifying agents, alkalizing agents, buffering agents, tonicity modifying agents, ionic additives effective to increase the ionic strength of the solution, antimicrobial agents, antibiotic agents, antifungal agents, antioxidants, preservatives, electrolytes, antifoaming agents, oils, stabilizers, enhancing agents, and the like. In some embodiments, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% total amount of one or more agents by total weight of the composition. In some embodiments, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more agents by total weight of the composition. In some embodiments, one or more of these agents may be added to improve the performance, efficacy, safety, shelf-life and / or other property of the muscarinic antagonist composition of the present disclosure. In some embodiments, additives may be biocompatible, without being harsh, abrasive, and / or allergenic.

[0071] In certain embodiments, pharmaceutical compositions disclosed herein may comprise one or more acidifying agents. As used herein, “acidifying agents” refers to compounds used to provide an acidic medium. Such compounds include, by way of example and without limitation, acetic acid, amino acid, citric acid, fumaric acid and other alpha hydroxy acids, such as hydrochloric acid, ascorbic acid, and nitric acid and others known to those of ordinary skill in the art. In some embodiments, any pharmaceutically acceptable organic or inorganic acid may be used. In some embodiments, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more acidifying agents by total weight of the composition. In some embodiments,pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more acidifying agents by total weight of the composition.

[0072] In certain embodiments, pharmaceutical compositions disclosed herein may comprise one or more alkalizing agents. As used herein, “alkalizing agents” are compounds used to provide alkaline medium. Such compounds include, by way of example and without limitation, ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium bicarbonate, sodium hydroxide, triethanolamine, and trolamine and others known to those of ordinary skill in the art. In some embodiments, any pharmaceutically acceptable organic or inorganic base can be used. In some embodiments, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more alkalizing agents by total weight of the composition. In some embodiments, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more alkalizing agents by total weight of the composition.

[0073] In certain embodiments, pharmaceutical compositions disclosed herein may comprise one or more antioxidants. As used herein, “antioxidants” are agents that inhibit oxidation and thus can be used to prevent the deterioration of preparations by the oxidative process. Such compounds include, by way of example and without limitation, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophophorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite and other materials known to one of ordinary skill in the art. In some embodiments, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more antioxidants by total weight of the composition. In some embodiments, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more antioxidants by total weight of the composition.

[0074] In certain embodiments, pharmaceutical compositions disclosed herein may comprise a buffer system. As used herein, a “buffer system” is a composition comprised of one or more buffering agents wherein “buffering agents” are compounds used to resist change in pH upon dilution or addition of acid or alkali. Buffering agents include, by way of example and without limitation, potassium metaphosphate, potassium phosphate, monobasic sodium acetate and sodium citrate anhydrous and dihydrate and other materials known to one of ordinary skill in the art. In some embodiments, any pharmaceutically acceptable organic or inorganic buffer can be used. In some embodiments, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more buffering agents by total weight of the composition. In some embodiments, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more buffering agents by total weight of the composition.

[0075] In some embodiments, the amount of one or more buffering agents may depend on the desired pH level of a composition. In some embodiments, pharmaceutical compositions disclosed herein may have a pH of about 6 to about 9. In some embodiments, pharmaceutical compositions disclosed herein may have a pH greater than about 8, greater than about 7.5, greater than about 7, greater than about 6.5, or greater than about 6.

[0076] In certain embodiments, pharmaceutical compositions disclosed herein may comprise one or more preservatives. As used herein, “preservatives” refers to agents or combination of agents that inhibits, reduces or eliminates bacterial growth in a pharmaceutical dosage form. Non-limiting examples of preservatives include Nipagin, Nipasol, isopropyl alcohol and a combination thereof. In some embodiments, any pharmaceutically acceptable preservative can be used. In some embodiments, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more preservatives by total weight of the composition. In some embodiments, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more preservatives by total weight of the composition.

[0077] In certain embodiments, pharmaceutical compositions disclosed herein may comprise one or more surface-acting reagents or detergents. In some embodiments, surfaceacting reagents or detergents may be synthetic, natural, or semi -synthetic. In some embodiments, compositions disclosed herein may comprise anionic detergents, cationic detergents, zwitterionic detergents, ampholytic detergents, amphoteric detergents, nonionic detergents having a steroid skeleton, or any combination thereof. In some embodiments, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more surface-acting reagents or detergents by total weight of the composition. In some embodiments, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more surface-acting reagents or detergents by total weight of the composition.

[0078] In certain embodiments, pharmaceutical compositions disclosed herein may comprise one or more stabilizers. As used herein, a “stabilizer” refers to a compound used to stabilize an active agent against physical, chemical, or biochemical process that would otherwise reduce the therapeutic activity of the agent. Suitable stabilizers include, by way of example and without limitation, succinic anhydride, albumin, sialic acid, creatinine, glycine and other amino acids, niacinamide, sodium acetyltryptophonate, zinc oxide, sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethylene glycols, sodium caprylate and sodium saccharin and others known to those of ordinary skill in the art. In some embodiments, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more stabilizers by total weight of the composition. In some embodiments, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more stabilizers by total weight of the composition.

[0079] In some embodiments, pharmaceutical compositions disclosed herein may comprise one or more tonicity agents. As used herein, a “tonicity agents” refers to a compound that can be used to adjust the tonicity of the liquid formulation. Suitable tonicity agents include, but are not limited to, glycerin, lactose, mannitol, dextrose, sodium chloride, sodium sulfate,sorbitol, trehalose and others known to those or ordinary skill in the art. Osmolarity in a composition may be expressed in milliosmoles per liter (mOsm / L). Osmolarity may be measured using methods commonly known in the art. In some embodiments, a vapor pressure depression method is used to calculate the osmolarity of the compositions disclosed herein. In some embodiments, the amount of one or more tonicity agents comprising a pharmaceutical composition disclosed herein may result in a composition osmolarity of about 150 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 350 mOsm / L, about 280 mOsm / L to about 370 mOsm / L or about 250 mOsm / L to about 320 mOsm / L. In some embodiments, a composition herein may have an osmolality ranging from about 100 mOsm / kg to about 1000 mOsm / kg, from about 200 mOsm / kg to about 800 mOsm / kg, from about 250 mOsm / kg to about 500 mOsm / kg, or from about 250 mOsm / kg to about 320 mOsm / kg, or from about 250 mOsm / kg to about 350 mOsm / kg or from about 280 mOsm / kg to about 320 mOsm / kg. In some embodiments, a pharmaceutical composition described herein may have an osmolarity of about 100 mOsm / L to about 1000 mOsm / L, about 200 mOsm / L to about 800 mOsm / L, about 250 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 350 mOsm / L, about 250 mOsm / L to about 320 mOsm / L, or about 280 mOsm / L to about 320 mOsm / L. In some embodiments, pharmaceutical compositions disclosed herein may comprise at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% total amount of one or more tonicity modifiers by total weight of the composition. In some embodiments, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more tonicity modifiers by total weight of the composition.Dosage formulations

[0080] Suitable routes of administration of the BET inhibitors described herein, may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intravenous, intraperitoneal, intranasal injections. Preferably, the BET inhibitor is administered orally. Preferably, the BET inhibitor is not administered via intratumoral injection or via intravenous infusion.

[0081] One may administer the pharmaceutical composition in a local or systemic manner, for example, via local injection of the pharmaceutical composition directly into a tissue region of a patient. In some embodiments, a pharmaceutical composition disclosed herein can be administered parenterally, e.g., by intravenous injection, intracerebroventricular injection, intra- cisterna magna injection, intra-parenchymal injection, or a combination thereof. In some embodiments, a pharmaceutical composition disclosed herein can administered to the human patient via at least two administration routes. In some examples, the combination of administration routes by be intracerebroventricular injection and intravenous injection; intrathecal injection and intravenous injection; intra-cisterna magna injection and intravenous injection; and intra-parenchymal injection and intravenous injection.

[0082] Pharmaceutical compositions of the present disclosure may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0083] Pharmaceutical compositions for use in accordance with the present disclosure thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0084] For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.

[0085] The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0086] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

[0087] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water-based solution, before use.

[0088] Pharmaceutical compositions suitable for use in context of the present disclosure include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. In some embodiments, a therapeutically effective amount means an amount of active ingredients (i.e., modulators and / or inhibitors of BET disclosed herein) effective to prevent, slow, alleviate or ameliorate symptoms of a disorder (e.g., lymphoproliferative disorders, lymphoid malignancy) or prolong the survival of the subject being treated.

[0089] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0090] For any preparation used in the methods of the present disclosure, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays and / or screening platforms disclosed herein. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0091] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage may vary depending upon the dosage form employed and the route of administration utilized. Theexact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1).

[0092] Dosage amount and interval may be adjusted individually to brain or blood levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0093] Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

[0094] The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. Effective doses may be extrapolated from dose- responsive curves derived from in vitro or in vivo test systems.

[0095] Having described several embodiments, it will be recognized by those skilled in the art that various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the present inventive concept. Additionally, a number of well-known processes and elements have not been described in order to avoid unnecessarily obscuring the present inventive concept. Accordingly, this description should not be taken as limiting the scope of the present inventive concept.III. Methods of Use

[0096] The present disclosure provides for methods of treating, attenuating, or preventing cancer and / or metastasis in a subject in need thereof. The present disclosure also provides for methods of impairing, reducing, or preventing tumor growth in a subject in need thereof. The impairing, reducing, or preventing of tumor growth may be in comparison to tumor growth in an untreated subject or SOC subject with identical disease condition and predicted outcome. Insome embodiments, the subject has improved survival. In certain embodiments, a method for treating, attenuating, or preventing a cancer and / or metastasis in a subject or a method for impairing or preventing tumor growth comprises administering to the subject (e.g., a human subject), an in-situ cancer vaccine as provided herein. For instance, in certain embodiments, a method for treating, attenuating, or preventing tumor growth or a method for treating, attenuating, or preventing a cancer and / or metastasis in a subject comprises administering a radiation therapy in combination with one or more BET inhibitors (e.g., a pharmaceutical composition comprising a BET inhibitor). Administering the in-situ cancer vaccine may comprise (a) administering to the subject one or more doses of a tumor directed radiation therapy; and (b) administering to the subject one or more doses of a BET inhibitor.

[0097] The present disclosure provides for methods of inducing a cancer specific or tumor specific immune memory response in a subject in need thereof. In certain embodiments, a method for inducing a cancer specific or tumor specific immune memory response in a subject comprises administering to the subject (e.g., a human subject) an in-situ cancer vaccine as provided herein. For instance, in certain embodiments, a method for inducing a cancer specific or tumor specific immune memory response comprises administering a radiation therapy in combination with one or more BET inhibitors (e.g., a pharmaceutical composition comprising a BET inhibitor). Administering the in-situ cancer vaccine may comprise (a) administering to the subject one or more doses of a tumor directed radiation therapy; and (b) administering to the subject one or more doses of a BET inhibitor.

[0098] In an aspect, the present disclosure provides for methods of activating host innate immunity against a cancer or tumor in a subject in need thereof. In certain embodiments, a method for activating host innate immunity against a cancer or tumor in a subject comprises administering to the subject (e.g., a human subject) an in-situ cancer vaccine as provided herein. The activating of host innate immunity may comprise activating the cGAS / STING signaling pathway in the subj ect. For instance, in certain embodiments, a method for activating host innate immunity against a cancer or tumor comprises administering a radiation therapy in combination with one or more BET inhibitors (e.g., a pharmaceutical composition comprising a BET inhibitor). Administering the in-situ cancer vaccine may comprise (a) administering to the subject one or more doses of a tumor directed radiation therapy; and (b) administering to the subject one or more doses of a BET inhibitor.

[0099] In an aspect, the present disclosure provides for methods of increasing neoantigen release in a subject in a subject in a subject in need thereof. The neoantigen may be specific for the cancer or tumor in the subject. In certain embodiments, a method for increasing neoantigen release in a subject in a subject comprises administering to the subject (e.g., a human subject) an in-situ cancer vaccine as provided herein. For instance, in certain embodiments, a method for increasing neoantigen release comprises administering a radiation therapy in combination with one or more BET inhibitors (e.g., a pharmaceutical composition comprising a BET inhibitor). Administering the in-situ cancer vaccine may comprise (a) administering to the subject one or more doses of a tumor directed radiation therapy; and (b) administering to the subject one or more doses of a BET inhibitor.

[0100] The present disclosure provides for methods of reducing or preventing immunosuppressive effects of a cancer or tumor in a subj ect in need thereof. In certain embodiments, a method for reducing or preventing immunosuppressive effects of a cancer or tumor in a subject comprises administering to the subject (e.g., a human subject) an in-situ cancer vaccine as provided herein. The reducing or preventing immunosuppressive effects may comprise reducing or preventing PD-L1 at the cancer or tumor. In some embodiments, the method of reducing or preventing immunosuppressive effects comprises reducing or preventing PD-L1 expression or activity. For instance, in certain embodiments, a method for reducing or preventing immunosuppressive effects of a cancer or tumor comprises administering a radiation therapy in combination with one or more BET inhibitors (e.g., a pharmaceutical composition comprising a BET inhibitor). Administering the in-situ cancer vaccine may comprise (a) administering to the subject one or more doses of a tumor directed radiation therapy; and (b) administering to the subject one or more doses of a BET inhibitor.

[0101] An in-situ cancer vaccine for use in any of the methods as described herein may comprise (e.g., a combination of) multiple components (e.g., radiation therapy and a BET inhibitor (e.g., OTX15)), as described herein (e.g., in Section II). The components of the in-situ cancer vaccine (e.g., the radiation therapy and BET inhibitor) may be administered to the subject concomitantly or concurrently, sequentially, or a combination thereof. The components of the in-situ cancer vaccine (e.g., the radiation therapy and BET inhibitor) may be administered together, or separately, depending on the treatment regimen suitable for the subject.

[0102] The treatment regimen for the in-situ cancer vaccine may a regimen as described herein (e.g., as demonstrated in the Examples). In some embodiments, the treatment regimen may comprise a first administration, second administration, third administration, and fourth administration; optionally a fifth administration; further optionally a sixth administration; further optionally a seventh administration; further optionally an eighth administration; further optionally a ninth administration; further optionally a tenth administration of one or more components of the in-situ cancer vaccine (e.g., the radiation therapy and BET inhibitor). Each administration may comprise one component of the in-situ cancer vaccine alone (e.g., the radiation therapy or BET inhibitor, but not both), or multiple components of the in-situ cancer vaccine (e.g., both the radiation therapy and BET inhibitor). The treatment regimen may alternate administration the components in a 1 to 1 pattern, 2 to 1 pattern (e.g., two sequential administrations of radiation therapy, followed administration of RT+BET inhibitor), 3 to 1 pattern, or 4 to 1 pattern.

[0103] In some embodiments, one or more of the components of the in-situ cancer vaccine are administered daily, every other day, or with an interval between days (e.g., two days, three days, four days), etc. For instance, one component (e.g., a BET inhibitor) can be administered daily (e.g., for a certain number of days, e.g., any of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 or more days), and another component (e.g., radiation therapy) can be administered at another interval (e.g., every other day, or once everything 3 days). There may be a maximum number of days of administration in the treatment regimen, for instance the in-situ cancer vaccine can be administered for 10 days or less, less than 9 days, less than 8 days, less than 7 days, less than 6 days, or less than 5 days. In some embodiments, the in-situ cancer vaccine (or a component thereof) is administered (e.g., daily or at other interval) for 1 to 10 days, 2 to 8 days, 3 to 7 days, 3 to 5 days, or 4 days. In specific embodiments, the radiation therapy is administered once time, every other day, over two days and the BET inhibitor is administered daily for the two days. In specific embodiments, the radiation therapy is administered twice, every other day, over four days and the BET inhibitor is administered daily for the four days. In specific embodiments, the radiation therapy is administered three times, every other day, over six days and the BET inhibitor is administered daily for the six days. In specific embodiments, the radiation therapy is administered four times, every other day, over eight days and the BET inhibitor isadministered daily for the eight days. In specific embodiments, the radiation therapy is administered five times, every other day, over ten days and the BET inhibitor is administered daily for the 10 days.

[0104] In some embodiments, the treatment regimen may comprise a first administration (e.g., at day 0 or 1) of the BET inhibitor alone; a second administration (e.g., the following day) of the radiation therapy and the BET inhibitor; a third administration (e.g., the following day) of the BET inhibitor alone; and a fourth administration (e.g., the following day) of the radiation therapy and the BET inhibitor, optionally wherein the treatment regimen further comprises a fifth administration (e.g., the following day) of the BET inhibitor alone; and / or a sixth administration (e.g., the following day) of the radiation therapy and the BET inhibitor; further optionally wherein the treatment regimen further comprises a seventh administration (e.g., the following day) of the BET inhibitor alone; and / or an eighth administration (e.g., the following day) of the radiation therapy and the BET inhibitor.

[0105] The treatment regimen may comprise a first administration (e.g., at day 0 or 1) of the radiation therapy and the BET inhibitor; a second administration (e.g., the following day) of the BET inhibitor alone; a third administration (e.g., the following day) of the radiation therapy and the BET inhibitor; and a fourth administration (e.g., the following day) of the BET inhibitor alone, optionally wherein the treatment regimen further comprises a fifth administration (e.g., the following day) of the radiation therapy and the BET inhibitor; and / or a sixth administration (e.g., the following day) of the BET inhibitor alone; further optionally wherein the treatment regimen further comprises a seventh administration (e.g., the following day) of the radiation therapy and the BET inhibitor; and / or an eighth administration (e.g., the following day) of the BET inhibitor alone.

[0106] In an aspect, the subject herein may be a human patient having cancer or tumor (e.g., target solid tumor). The cancer or tumor may be refractory. As used herein, “refractory” refers to the tumor that does not respond to or becomes resistant to a treatment. In some embodiments, the subject herein may have a tumor that is resistant to at least one standard therapy (i.e., chemotherapy). In some embodiments, the subject may be a human patient having a relapsed disease, for example, a relapsed breast cancer (breast tumor) or other cancer. Asused herein, “relapsed” or “relapses” refers to a tumor that returns or progresses following a period of improvement (e.g., a partial or complete response) with treatment.

[0107] A subject having a cancer or tumor (e.g., a target solid tumor) as disclosed herein, (e.g., breast cancer), can be identified by routine medical examination. The identification of the cancer tumor by identified by one or more laboratory tests, organ functional tests, genetic tests, interventional procedure (biopsy, surgery), or any and all relevant imaging modalities. In some embodiments, the subject to be treated by any of the methods described herein is a human cancer patient who has undergone or is subjecting to an anti-cancer therapy, for example, chemotherapy, radiotherapy, immunotherapy, or surgery. In some embodiments, a subject shows disease progression through the treatment. In other embodiments, a subject is resistant to the treatment (either de novo or acquired). In some embodiments, such a subject is demonstrated as having advanced malignancies (e.g., inoperable or metastatic). Alternatively, or in addition, in some embodiments, the subject has no standard therapeutic options available or ineligible for standard treatment options, which refer to therapies commonly used in clinical settings for treating the corresponding solid tumor.

[0108] In certain embodiments, a cancer (e.g., a tumor) to be treated by compositions and methods disclosed herein can be a solid tumor. In some embodiments, a solid tumor can be an abnormal mass of tissue that is devoid of cysts or liquid regions within the tumor. In some embodiments, solid tumors can be benign (not progressed to a cancer), a malignant or metastatic tumor. In some embodiments, a solid tumor herein can be a malignant cancer that has metastasized. In other embodiments, solid tumors contemplated herein can include, but are not limited to, sarcomas, carcinomas, lymphomas, gliomas or any combination thereof. In accordance with some embodiments herein, a tumor to be treated by an in-situ cancer vaccine described herein (e.g., in Section II) can include, but is not limited to, a testicular tumor, ovarian tumor, cervical tumor, a kidney tumor, bladder tumor, head-and-neck tumor, skin tumor, bone tumor, brain tumor, thyroid tumor, pancreatic tumor, liver tumor, stomach tumor, lung tumor, endometrial tumor, esophageal tumor, breast tumor, cervical tumor, central nervous system tumor, germ cell tumor, prostate tumor, Hodgkin's lymphoma, non-Hodgkin's lymphoma, neuroblastoma, sarcoma, multiple myeloma, melanoma, mesothelioma, osteogenic sarcoma or any combination thereof. In some embodiments, a targeted tumor contemplated herein can include a solid tumor such as ovarian tumors, breast tumors, or any combination thereof. Insome embodiments, a targeted tumor contemplated herein can include a solid tumor such as breast cancer, lung adenocarcinoma, colon cancer, melanoma, or any combination thereof. In some embodiments, a targeted tumor is a breast cancer tumor. In some embodiments, the breast cancer is an estrogen receptor positive (ER+) breast cancer. In an aspect, a targeted tumor contemplated herein can include a luminal A breast cancer (i.e., ER+, PR+ and HER2 negative), a luminal B breast cancer (i.e., ER+, PR negative, and HER2 positive), a HER2 positive breast cancer, or a triple negative breast cancer (I.e., ER negative, PR negative and HER2 negative). In some embodiments, a targeted tumor contemplated herein can include a triple negative breast cancer tumor.

[0109] In an aspect, a targeted tumor contemplated herein comprises a sarcoma. The sarcoma may be a bone sarcoma or soft tissue sarcoma. The bone sarcoma may be selected from an osteosarcoma, chondrosarcoma, Ewing sarcoma, fibrosarcoma, angiosarcoma, or myeloid sarcoma. The soft tissue sarcoma may be selected from a liposarcoma, leiomyosarcoma, rhabdomyosarcoma, angiosarcoma, synovial sarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), Kaposi sarcoma, desmoplastic small round cell tumors (DSRCT), epithelioid sarcoma, or myeloid sarcoma.

[0110] The subject of any of the methods as described herein may be described as having the specific cancer type that is associated with any of the tumor type described herein (e.g., the subject has triple negative breast cancer (TNBC) when they have TNBC tumors). The cancer to be treated bv any of the methods described herein may associated with any of the tumor type described herein (e.g., the method is treating TNBC in the subject when the subject has TNBC tumors).[OHl] In certain embodiments, in-situ cancer vaccine disclosed herein can treat and / or prevent cancer in a subject in need. In some embodiments, in-situ cancer vaccine disclosed herein can impair tumor growth compared to tumor growth in an untreated subject or a subject treated with “standard of care” treatment (herein referred to as an “SOC subject”) with identical disease condition and predicted outcome. In some embodiments, tumor growth can be stopped following treatment with compositions disclosed herein. In other embodiments, tumor growth can be impaired at least about 5% or greater to at least about 100%, at least about 10% or greater to at least about 95% or greater, at least about 20% or greater to at least about 80% orgreater, at least about 40% or greater to at least about 60% or greater compared to an untreated subject or SOC subject with identical disease condition and predicted outcome. In other words, tumors in subject treated using a composition of the disclosure have tumors that grow at least 5% less (or more as described above) when compared to an untreated subject or SOC subject with identical disease condition and predicted outcome. In some embodiments, tumor growth can be impaired at least about 5% or greater, at least about 10% or greater, at least about 15% or greater, at least about 20% or greater, at least about 25% or greater, at least about 30% or greater, at least about 35% or greater, at least about 40% or greater, at least about 45% or greater, at least about 50% or greater, at least about 55% or greater, at least about 60% or greater, at least about 65% or greater, at least about 70% or greater, at least about 75% or greater, at least about 80% or greater, at least about 85% or greater, at least about 90% or greater, at least about 95% or greater, at least about 100% compared to an untreated subject or SOC subject with identical disease condition and predicted outcome. In some embodiments, tumor growth can be impaired at least about 5% or greater to at least about 10% or greater, at least about 10% or greater to at least about 15% or greater, at least about 15% or greater to at least about 20% or greater, at least about 20% or greater to at least about 25% or greater, at least about 25% or greater to at least about 30% or greater, at least about 30% or greater to at least about 35% or greater, at least about 35% or greater to at least about 40% or greater, at least about 40% or greater to at least about 45% or greater, at least about 45% or greater to at least about 50% or greater, at least about 50% or greater to at least about 55% or greater, at least about 55% or greater to at least about 60% or greater, at least about 60% or greater to at least about 65% or greater, at least about 65% or greater to at least about 70% or greater, at least about 70% or greater to at least about 75% or greater, at least about 75% or greater to at least about 80% or greater, at least about 80% or greater to at least about 85% or greater, at least about 85% or greater to at least about 90% or greater, at least about 90% or greater to at least about 95% or greater, at least about 95% or greater to at least about 100% compared to an untreated subject or SOC subject with identical disease condition and predicted outcome.

[0112] The standard of care therapy for comparison may comprise administering one or more immunotherapies and / or chemotherapies. The immunotherapy or chemotherapy may be selected from pembrolizumab, carboplatin, paclitaxel, doxorubicin / epirubicin, cyclophosphamide, or any regulatory approved cancer therapeutic, or combination thereof. Theregulatory approved cancer therapeutic may be approved by the Food and Drug Administration, European Medicines Agency, or another medical regulatory body). The standard of care treatment may be administered over the course of 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 24 weeks, 52 weeks, or 60 weeks.

[0113] In some embodiments, treatment of tumors with the in-situ cancer vaccine disclosed herein can result in a shrinking of a tumor in comparison to the starting size of the tumor. In some embodiments, tumor shrinking is at least about 5% or greater to at least about 10% or greater, at least about 10% or greater to at least about 15% or greater, at least about 15% or greater to at least about 20% or greater, at least about 20% or greater to at least about 25% or greater, at least about 25% or greater to at least about 30% or greater, at least about 30% or greater to at least about 35% or greater, at least about 35% or greater to at least about 40% or greater, at least about 40% or greater to at least about 45% or greater, at least about 45% or greater to at least about 50% or greater, at least about 50% or greater to at least about 55% or greater, at least about 55% or greater to at least about 60% or greater, at least about 60% or greater to at least about 65% or greater, at least about 65% or greater to at least about 70% or greater, at least about 70% or greater to at least about 75% or greater, at least about 75% or greater to at least about 80% or greater, at least about 80% or greater to at least about 85% or greater, at least about 85% or greater to at least about 90% or greater, at least about 90% or greater to at least about 95% or greater, at least about 95% or greater to at least about 100% (meaning that the tumor is completely gone after treatment) compared to the starting size of the tumor.

[0114] In certain embodiments, the in-situ cancer vaccines disclosed herein can improve patient life expectancy compared to the cancer life expectancy of an untreated subject or SOC subject with identical disease condition and predicted outcome. As used herein, “patient life expectancy” is defined as the time at which 50 percent of subjects are alive and 50 percent have passed away. In some embodiments, patient life expectancy can be indefinite following treatment with a composition disclosed herein. In other aspects, patient life expectancy can be increased at least about 5% or greater to at least about 100%, at least about 10% or greater to at least about 95% or greater, at least about 20% or greater to at least about 80% or greater, at least about 40% or greater to at least about 60% or greater compared to an untreated subject or SOC subject or an SOC subject with identical disease condition and predicted outcome. In someembodiments, patient life expectancy can be increased at least about 5% or greater, at least about 10% or greater, at least about 15% or greater, at least about 20% or greater, at least about 25% or greater, at least about 30% or greater, at least about 35% or greater, at least about 40% or greater, at least about 45% or greater, at least about 50% or greater, at least about 55% or greater, at least about 60% or greater, at least about 65% or greater, at least about 70% or greater, at least about 75% or greater, at least about 80% or greater, at least about 85% or greater, at least about 90% or greater, at least about 95% or greater, at least about 100% compared to an untreated subject or SOC subject with identical disease condition and predicted outcome. In some embodiments, patient life expectancy can be increased at least about 5% or greater to at least about 10% or greater, at least about 10% or greater to at least about 15% or greater, at least about 15% or greater to at least about 20% or greater, at least about 20% or greater to at least about 25% or greater, at least about 25% or greater to at least about 30% or greater, at least about 30% or greater to at least about 35% or greater, at least about 35% or greater to at least about 40% or greater, at least about 40% or greater to at least about 45% or greater, at least about 45% or greater to at least about 50% or greater, at least about 50% or greater to at least about 55% or greater, at least about 55% or greater to at least about 60% or greater, at least about 60% or greater to at least about 65% or greater, at least about 65% or greater to at least about 70% or greater, at least about 70% or greater to at least about 75% or greater, at least about 75% or greater to at least about 80% or greater, at least about 80% or greater to at least about 85% or greater, at least about 85% or greater to at least about 90% or greater, at least about 90% or greater to at least about 95% or greater, at least about 95% or greater to at least about 100% compared to an untreated patient with identical disease condition and predicted outcome.

[0115] In some embodiments, the methods of the present disclosure increase anti-tumor activity (e g., reduce cell proliferation, tumor growth, tumor volume, and / or tumor burden or load or reduce the number of metastatic lesions over time) by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to levels prior to treatment or in a control subject (i.e., an untreated subject or an SOC subject). In some embodiments, reduction is measured by comparing cell proliferation, tumor growth, and / or tumor volume in a subject before and after administration of the pharmaceutical composition. In some embodiments, the method of treating or ameliorating a cancer in a subject allows one ormore symptoms of the cancer to improve by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, methods disclosed herein may include administration of the compositions herein to reduce tumor volume, size, load or burden in a subject to an undetectable size, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the subject's tumor volume, size, load or burden prior to treatment. In other embodiments, methods disclosed herein may include administration of the compositions herein to reduce the cell proliferation rate or tumor growth rate in a subject to an undetectable rate, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the rate prior to treatment.

[0116] In some embodiments, a subject to be treated by any of the methods and / or in-situ cancer vaccines disclosed herein can present with one or more cancerous solid tumors, metastatic nodes, or any combination thereof. In some embodiments, a subject herein may have a cancerous tumor cell source that can be less than about 0.2 cm3to at least about 20 cm3or greater, at least about 2 cm3to at least about 18 cm3or greater, at least about 3 cm3to at least about 15 cm3or greater, at least about 4 cm3to at least about 12 cm3or greater, at least about 5 cm3to at least about 10 cm3or greater, or at least about 6 cm3to at least about 8 cm3or greater.

[0117] In certain embodiments, the in-situ cancer vaccines disclosed herein can be effective for treating at least one tumor cell in a solid tumor from a subject in need. In some embodiments, the amount of viable tumor cells may be reduced by at least about 5% or greater, at least about 10% or greater, at least about 15% or greater, at least about 20% or greater, at least about 25% or greater, at least about 30% or greater, at least about 35% or greater, at least about 40% or greater, at least about 45% or greater, at least about 50% or greater, at least about 55% or greater, at least about 60% or greater, at least about 65% or greater, at least about 70% or greater, at least about 75% or greater, at least about 80% or greater, at least about 85% or greater, at least about 90% or greater, at least about 95% or greater, at least about 100% compared to an untreated subject or SOC subject with identical disease condition and predicted outcome.

[0118] Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the in-situ cancer vaccines disclosed herein to a subject (and especially, the BET inhibitors disclosed herein), depending upon the type of disease to be treated or the site ofthe disease. In some embodiments, BET inhibitors disclosed herein can be administered to a subject by intravenous infusion, by subcutaneous administration, by inhalation, by intranasal administration or other mode of administration. In some embodiments, the BET inhibitors or pharmaceutical compositions thereof disclosed herein can be administered to a subject orally.

[0119] In some embodiments, any of the methods disclosed herein for using an in-situ cancer vaccine can further include monitoring occurrence of one or more adverse effects in the subject. Exemplary adverse effects include, but are not limited to, hepatic impairment, toxicity (e.g., hematologic toxicity, neurologic toxicity, cutaneous toxicity, or gastrointestinal toxicity), or any combination thereof. When one or more adverse effects are observed, the method disclosed herein can further include reducing or increasing the dose of one or more of the disclosed active agents (e.g., BET inhibitor), the dose of one or more targeted radiation therapy, or both depending on the adverse effect or effects in the subject.

[0120] In some embodiments, any of the methods disclosed herein for using an in-situ cancer vaccine can further comprise treating a subject with at least one additional therapeutic regimen, for example, chemotherapy, additional radiotherapy, additional immunotherapy, or surgery. The treatment may be administration of one or more immunotherapies and / or chemotherapies. The one or more immunotherapy or chemotherapy may be selected from pembrolizumab, carboplatin, paclitaxel, doxorubicin / epirubicin, cyclophosphamide, or any regulatory approved cancer therapeutic, or combination thereof. The regulatory approved cancer therapeutic may be administered to the subject using dosage and / or treatment regimen as approved by the Food and Drug Administration, European Medicines Agency, or another medical regulatory body. In some embodiments, a subject treated with any of the methods herein can have completed an additional therapeutic regimen, be receiving an additional therapeutic regimen, or can receive an additional therapeutic regimen following treatment according to the methods herein.

[0121] In some aspects, a subject may be treated or have been treated with a chemotherapy. Chemotherapeutic agents may be selected from any one or more of cytotoxic antibiotics, antimetabolites, anti-mitotic agents, alkylating agents, arsenic compounds, DNA topoisomerase inhibitors, taxanes, nucleoside analogues, plant alkaloids, and toxins; and synthetic derivatives thereof. Exemplary compounds include, but are not limited to, alkylating agents: treosulfan,and trofosfamide; plant alkaloids: vinblastine, paclitaxel, docetaxol; DNA topoisomerase inhibitors: doxorubicin, epirubicin, etoposide, camptothecin, topotecan, irinotecan, teniposide, crisnatol, and mitomycin; anti-folates: methotrexate, mycophenolic acid, and hydroxyurea; pyrimidine analogs: 5 -fluorouracil, doxifluridine, and cytosine arabinoside; purine analogs: mercaptopurine and thioguanine; DNA antimetabolites: 2'-deoxy-5-fluorouridine, aphidicolin glycinate, and pyrazoloimidazole; and antimitotic agents: halichondrin, colchicine, and rhizoxin. Compositions comprising one or more chemotherapeutic agents (e.g., FLAG, CHOP) may also be used. FLAG comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF. CHOP comprises cyclophosphamide, vincristine, doxorubicin, and prednisone. In another embodiments, PARP (e.g., PARP-1 and / or PARP-2) inhibitors are used and such inhibitors are well known in the art (e.g., Olaparib, ABT-888, BSL201, BGP-15, INO-lOOl, PJ34, 3- aminobenzamide, 4-amino-l,8-naphthalimide, 6(5H)-phenanthridinone, benzamide, NU1025).

[0122] In an aspect, a subj ect may be treated or have been treated with an immunotherapy. Immunotherapy may comprise, for example, use of a different cancer vaccines and / or sensitized antigen presenting cells. The immunotherapy can involve passive immunity for short-term protection of a host, achieved by the administration of pre-formed antibody directed against a cancer antigen or disease antigen (e.g., administration of a monoclonal antibody, optionally linked to a chemotherapeutic agent or toxin, to a tumor antigen). Immunotherapy can also focus on using the cytotoxic lymphocyte-recognized epitopes of cancer cell lines.

[0123] In an aspect, a subject may be treated or have been treated with hormonal therapy. Hormonal therapeutic treatments can comprise, for example, hormonal agonists, hormonal antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, and steroids (e.g., dexamethasone, retinoids, deltoids, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progestins), vitamin A derivatives (e.g., all-trans retinoic acid (ATRA)); vitamin D3 analogs; antigestagens (e.g., mifepristone, onapristone), or antiandrogens (e.g., cyproterone acetate.

[0124] Those skilled in the art will appreciate that the presently disclosed embodiments teach by way of example and not by limitation. Therefore, the matter contained in this description or shown in the accompanying drawings should be interpreted as illustrative and not in a limitingsense. The following claims are intended to cover all generic and specific features described herein, as well as all statements of the scope of the method and assemblies, which, as a matter of language, might be said to fall there between.IV. Kits

[0125] Also provided in this disclosure are kits. Such kits can include a composition of matter described herein (e.g., one or more components of an in-situ cancer vaccine as described herein), and optionally, in certain embodiments, instructions for performing a method as described herein (e.g., in Section III). Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the in-situ cancer vaccine can be packaged in separate containers). Components of the kit may include, but are not limited to compositions as described herein, representation of methods as described herein, components for therapeutic intervention, systems, assays, reagents, internal standards, or software. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device. The pack may, for example, may comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.

[0126] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline or sterile each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readilyremovable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

[0127] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and / or may be supplied as an electronic-readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit. Said instructions may be derived from any of the methods as described herein (e.g., in Section V).

[0128] In an aspect, a kit comprises (a) a Bromodomain and Extra-Terminal motif (BET) inhibitor and / or a tumor directed radiation therapy (e.g., one or both), and (b) instructions for use according to any one of the methods as described herein (e.g., in Section III).EXAMPLES

[0129] The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.Introduction to Examples

[0130] There are many unmet clinical needs in breast cancer. Specifically, most breast cancers are immunologically cold with minimal or no benefit from existing immunotherapies. For instance, in a recent clinical study, pembrolizumab only slightly improved survival for breast cancer compared to placebo alone (see KEYNOTE-522, NCT03036488; Schmid et at., 2020 NEJM 386:556-567; Schmid et at., 2020 NEIM 382:810-821). Chemotherapy is known for severe toxicity, and there is a goal to achieve long-term remission without the need for a chemotherapy backbone that confers hightoxicity. As a solution, the following examples describe a novel chemotherapy-free, vaccine like immunotherapy therapy that achieve superior tumor control with minimal toxicity.

[0131] Ideal characteristics of a tumor vaccine include the following. First, there is a brief duration of administration (as opposed to current chemo-IO regimens in breast cancer that are administered over 15 cycles over several months). Further there is an ease of administration (for instance, oral administration is preferred over intravenous or intratumoral injections). There should also be minimal toxicity and clear superior tumor control relative to existing standard of care regimens.

[0132] The following examples show data and evidence supporting the innovative approach diagramed in FIG. 1, showing that combining radiation therapy (RT) with a targeted therapeutic enhances immune-stimulatory effects (e.g., activation of cGAS / STING immune activation pathway and / or neoantigen release) and / or reduces immunosuppressive effects (e.g., increase in PD-L1) to promote overall anti -tumor immunity.Example 1: BET Inhibition

[0133] BET inhibitors have potential as targeted therapeutics that enhance immune stimulatory effects. Other studies have shown that pharmacological BET inhibition can overcome both endocrine therapy resistance and radiation resistance in breast cancer (see e.g., JC1 Insight. 2022;7(l 7):el 51851 (e.g., Figure 3B therein) and NPJ Precision Oncology 7: 11 (2023) (e.g., Supplementary Figures 3C-3D), both of which are incorporated herein by reference in their entirety).

[0134] In preliminary studies, it was found that BET inhibition further enhances radiation induced DNA damage (FIG. 2). Additional details on protocols, results, and related experiments are provided in the expanded studies of Example 6.

[0135] In other studies, it was found that BET inhibition silences MYC which is a negative regulator of STING (see . Cancer Immunol Res 2022; 10:829 (e.g., Figure 3G therein), which is incorporated herein by reference in its entirety).

[0136] In preliminary studies, it was found that BET inhibition reverses the silencing of radiation-induced PD-L1 expression (FIG. 3). Additional details on protocols, results, and related experiments are provided in the expanded studies of Example 6. These dataprovided evidence that combining radiation with BET inhibition specifically can modulate several steps involved in immune activation (e.g., activation of cGAS / STING; neoantigen release) and can act as a cancer vaccine to confer anti-tumor immunity (see FIG. 1).Example 2: Efficacy of RT+BET Inhibitor on Tumor Progression and Rechallenge

[0137] A combined radiation therapy -BET inhibition (RT-BET) treatment regimen was developed and tested in comparison to “standard of care” (SOC) chemotherapy plus immunotherapy. As depicted in FIG. 4A, the combination RT+BET inhibitor regimen involved administering a BET inhibitor (e.g., OTX) at day 1, day 2, day 3 and day 4 and administering radiation therapy (e.g., 8 Gy / dose) at day 2 and day 4. This combination therapy was compared to “standard of care” protocol (FIG. 4B) involving (a) administering pembrolizumab and carboplatin every three weeks for 12 weeks while also administering paclitaxel weekly during the same time; followed by (b) administering pembrolizumab, doxorubicin / epirubicin and cyclophosphamide every three weeks for an additional 12 weeks, followed by (c) surgical resection of the tumor and finally (d) administering pembrolizumab every three weeks for an additional 24 weeks.

[0138] These protocols were tested in a triple negative breast cancer (TNBC) syngeneic mouse model (4T1) (see FIG. 5A), with the number of mice being N=8. Additional details on protocols, results, and related experiments are provided in the expanded studies of Example 4. Results for mice injected with 4T1 cells are provided in FIG. 5B, showing that OTX alone performed no better than vehicle in preventing tumor progression. However, a surprising therapeutic efficacy was observed when the BET inhibitor was combined with radiation therapy (e.g., RT+OTX), including a reduction in tumor volume and overall significant suppression of tumor progression compared to SOC or RT alone. In general, the RT+BET inhibitor combination therapy showed superior tumor control than SOC therapy or RT alone. 2 / 8 mice injected with 4T1 cells were tumor-free after RT + OTX therapy.

[0139] A 4T1 rechallenge study (design depicted in FIG. 6A) showed that successfully treated mice (N=2) from previous study demonstrated persistent anti-tumor immunity. Results are provided in FIG. 6B, showing that tumor growth was suppressed in these animals even after 4T1 rechallenge up to 50 days or more after the rechallenge.

[0140] In another experiment, the RT+BET inhibitor combination protocol was tested in a syngeneic ER+ breast cancer mouse model. In this experiment, estradiol sensitive MXT cells (validated in FIG. 7A) were injected into a mouse which was then treated with the protocols outlined in FIGS. 4A and 5A. As shown in FIG. 7B, the RT+BET inhibitor combination also significantly reduced tumor volume as compared to BET alone (e.g., OTX) or RT alone.Example 3: T cell Dependency of RT-BET Inhibitor Therapy

[0141] The T cell dependency of the anti-tumor effect observed with RT+BET inhibitor combination therapy investigated. Additional details on protocols, results, and related experiments are provided in the expanded studies of Example 5. FIG. 8A depicts the experimental design, in which animals were treated to either deplete IgG or CD8+ T cells twice a week after the four-day RT+BET inhibitor protocol. Representative flow cytometry plots indicating successful depletion of CD8 and IgG are shown in FIGS. 8B-8C. Tumor volume was tracked in both animals up to 30 days post treatment. CD8 depleted animals failed to show the expected reduction in tumor volume following the RT+BET inhibitor combination therapy, whereas IgG depleted animals had the expected effect (FIG. 9). Further, CD8 depleted animals also failed to show a reduction in metastasis to either the lung or liver after RT+BET inhibitor combination therapy (FIGS. 10A-10C). Together, these data show that the response to RT+ BET inhibition is cytotoxic CD8 T cell dependent.Example 4: RT+BET Inhibitor Therapy as an Effective Cancer Treatment

[0142] The findings of Example 2 were further developed to demonstrate that RT+BET inhibitor therapy is effective in treating multiple forms of cancer.Comparison of BET Inhibition to Other Targets

[0143] A drug compound screen was performed to confirm that BET inhibitors are more effective for combination with radiation treatment than inhibiting other targets. TABLE 1 shows a list of the tested compounds, different targets recognized, and planned dosing.TABLE 1: Tested Inhibitors

[0144] An in vivo drug screen was developed, in which BALB / c mice (n=l per condition) were injected with 20,000 4T1 cells in the mammary fat pad. When tumors grew to -100 mm3, mice were treated with either RT alone or RT+drug (as the drug compounds are noted in noted on the table. The dosing regimen is provided in FIG. 11 A. Treatment was given over 4 days, with 4 consecutive days of drug and focal, tumor-directed radiation therapy (8 Gy) on days 2 and 4. Two weeks after treatment began, the tumors were measured using digital calipers and tumor volume was calculated using the equation: (long diameter x (short diameter)A2) / 2. Each tumor volume was standardized to that of radiation therapy alone to generate a relative tumor volume (y axis) to show which radiation therapy plus drug combinations were most effective in suppressing tumor growth. Results are provided in FIG. 1 IB, showing that the BET inhibitor (i.e., OTX015) was most effective having the lowest relative tumor volume amongst the inhibitors tested, which validates BET as an inhibitory target. This was believed to be the first in vivo drug screen to identify epigenetic inhibitors that enhance anti-tumor effects of radiation therapy. The combination of radiation therapy with BET inhibitor OTX015 (RT+OTX) was selected for further testing.RT+BET Inhibitor Efficacy on 4T1 and EMT6 Tumors

[0145] For comparison to 4T1 tumor findings (e.g., Example 2 FIGS. 5A-5B), the RT+BET inhibitor treatment regimen was tested with the EMT6 cell line (another triple negative breast cancer (TNBC) cell line). As shown in the treatment regimen of FIG. 12A, for each of the 4T1 and EMT6 cell lines, a 4-day treatment was administered using OTX alone, vehicle alone, RT alone, or RT+OTX. The OTX alone protocol was identical to RT+OTX, except without radiation. Vehicle was administered identically to OTX, except without the active compound. The vehicle was composed of 10% DMSO, 40% PEG300, 5% Tween 80, and 45% saline. FIG. 12B depicts standard-of-care treatment, which was developed to reflect the treatment regimen of the KEYNOTE-522 trial (NCT03036488). In this SOC treatment, mice were administered 200 pg anti-PDl immune checkpoint inhibitor intraperitoneally (I.P.) weekly for 8 weeks. For weeks 1-4, mice were also administered 10 mg / kg paclitaxel chemotherapy I.P. For weeks 5-8, mice were co-administered 2 mg / kg doxorubicin chemotherapy I.P.

[0146] For each cell line, BALB / c mice were injected with either 20,000 4T1 cells or 100,000 EMT6 cells orthotopically in the mammary fat pad. Once tumors reached —100 mm3, mice were randomized into treatment groups. For 4T1 cells, the number of mice was N=8 for VH alone, OTX alone, RT+OTX, and PD1+PTX+DOX, and N=10 for RT alone. For EMT6 cells, the number of mice was N=8 for OTX alone, N=9 for VH, RT alone, and RT+OTX. Tumor diameters were measured twice weekly with digital calipers and volumes were calculated. In 4T1 tumor bearing mice, RT+OTX demonstrated superior efficacy compared to SOC therapy and RT alone (see FIG. 13A, also FIG. 5B). In EMT6 tumor bearing mice, RT+OTX demonstrated superior efficacy compared to RT alone (see FIG. 13B). At the end of the study, 2 / 8 4T1 mice treated with plus OTX were cured while 1 / 9 EMT6 mice were cured. Cured was defined as the mouse ceasing to have a palpable tumor. From these results, it was surprising that only a four-day treatment was sufficient for a complete cure in some mice.RT+BET Inhibitor Efficacy on E0771 Tumors

[0147] Exploratory tests were conducted using E0771 cells to determine the effectiveness of the combination therapy on another syngeneic TNBC mouse model (study design outlined in FIG. 14A). C57BL / 6 mice were injected with 1,000,000 E0771 cells in the mammary fat pad. Both RT and RT+OTX arms were n=6. Once tumors reached -100 mm3, treatment and tumor volume measurements were performed (as described above for 4T1 and EMT6). Tumor masses were weighed on a digital scale 30 days after treatment began (see FIG. 14C). The p-value between RT and RT+OTX was p=0.0340. 3 / 6 mice treated with RT+OTX were cured, demonstrating strong efficacy in the E0771 mouse model.RT+BET Inhibitor Efficacy onMXTl Tumors

[0148] The RT+ BET inhibitor treatment was further tested using a new syngeneic mouse model of ER+ breast cancer, MXT1. This model was chosen as there are not yet well- established syngeneic mouse models for ER+ breast cancer. ER+ breast cancer is the most diagnosed subtype of breast cancer and has historically failed to respond to immunotherapy in the clinical setting. FIG. 15A shows a Western blot comparing expression of estrogen receptor (ER) on MCF7, a human cell line of ER+ BC, to the new mouse cell line of ER+BC, MXT1. Beta-actin was the housekeeping gene used to show that the same amount of protein was loaded into each well, confirming that the high expression of ER in MXT1 was not due to differences in amount of protein loaded. Protein from untreated cell lines was used for this western blot. FIG. 15B depicts a schematic of the treatment regimen. A six-day treatment was utilized, since ER+ BC is generally more resistant to treatment than TNBC. 100 mg / kg OTX or vehicle was administered orally daily for 6 days and 8 Gy radiation therapy was given on days 2, 4, and 6 of treatment 6 hours following OTX.

[0149] BDF1 mice were implanted with an estradiol pellet under the scruff of the neck using a trocar. This was important for increasing estrogen that drives the progression of ER+ BC. The next day, mice were injected with 10,000,000 MXT1 cells in the mammary fat pad. Upon reaching -100 mm3, mice were treated with vehicle, OTX, RT alone, or RT+OTX as described. For the mice, n=4 for RT+OTX, and n=5 for vehicle, OTX alone, and RT alone. Tumor volumes were measured as described previously. Tumor mass for RTand RT+OTX was measured 29 days after treatment began. It was demonstrated that RT+BET inhibitor (e.g., OTX) treatment was effective in treating ER+ breast cancer. As shown in the tumor volume results (FIG. 16A) and tumor mass results (FIG. 16B), the RT+OTX treatment was surprisingly effective at reducing tumor volume and mass in the difficult-to-treat ER+ breast cancer model.RT+BET Inhibitor Efficacy in Sarcoma (MCA205)

[0150] The RT+BET inhibitor treatment was tested beyond breast cancer using a sarcoma model. Sarcoma was selected because it is typically resistant to immunotherapy. C57BL / 6 mice were injected with 500,000 MCA205 fibrosarcoma cells subcutaneously in the flank. Once tumors reached -100 mm3, treatment of the mice was performed, using the same 6-day treatment as described previously (see FIG. 15B). Tumor volumes were measured and mice were euthanized once tumors reached 2000 mm3, or survival was recorded (see FIGS. 1 AUB). It was demonstrated that RT+BET inhibitor treatment was effective in treating sarcoma, having similarly positive results as for TNBC. Between two independent studies, 4 mice were cured by RT+OTX. This result was surprising because sarcomas are poorly immunogenic and there remains a strong need to identify new treatment approaches that minimize toxicity and engage the immune system.RT+BET Inhibitor Efficacy in Comparison to Immunotherapy

[0151] Many immunotherapy approaches use immune checkpoint inhibitors, such as anti- PD1 antibody therapy. As such, anti-PDl therapy was tested in combination with radiation (PD1+RT) for comparison to RT+BET inhibitor therapy. Comparison was performed using the 4T1 cell line, with treatment starting after tumors reached -100 mm3. Results for PDl+RTand RT+OTX therapy are shown in FIG. 18A (mice N=8 for both). For both combinations, a 4-day radiation therapy treatment was utilized, having radiation on days 2 and 4, similar to previous Examples. For the PD1+RT combination, anti-PDl antibody (BioXCell, Cat# BE0146; 200 pg I.P.) was administered on days 1, 2, and 4 of the 4-day treatment and 8 Gy rt was given on days 2 and 4 of treatment.

[0152] An approach combining anti-PDl with BET inhibitor (PD1+OTX) was tested for comparison to RT+OTX (see FIG. 18B). The numbers of mice were N=8 for RT+OTX, andN=10 for PD1+0TX). Treatment started when tumors reached -100 mm3. For PD1+OTX, anti-PDl antibody (200 pig I.P.) and OTX (100 mg / kg oral) were co-administered 3x / week every week until experiment completion. These findings show that RT+OTX is more effective than RT+PD1, which is a combination therapy used in pre-clinical mouse models of TNBC. RT+OTX is also more effective than PD1+OTX, a combination therapy used in leukemia models.

[0153] The toxicity of the RT+ BET inhibitor treatment was measured (see FIGS. 27A- 27C), demonstrating that RT+OTX treatment is minimally toxic. To show that RT+OTX causes minimal toxicity, an experiment was performed on BALB / c mice without tumors. N=5 mice were treated with vehicle or RT+OTX (4-day regimen). Body weight was recorded for the first month following treatment start. No difference between treatments was observed (FIG 27A). Two months after treatment start, blood was collected from the mice and submitted for both CBC (FIG. 27B) and metabolite (FIG. 27C) analysis. No significant differences in CBC values were observed. The only significant difference in metabolites was seen with ALT / ALTV - however, only an elevated ALT is a concern whereas RT+OTX reduces ALT. This data shows the RT+ OTX is advantageously minimally toxic.Example 5: RT+BET Inhibitor Therapy Promotes a Systemic Anti-Tumor Immune Response

[0154] The findings of Example 3 were further developed to demonstrate that a systemic anti-tumor immune response (tumor-specific) follows RT+BET inhibitor treatment.

[0155] FIG. 19A depicts the study design. Mice cured from 4T1 by RT+OTX in Examples 2 and 4 were used. Mice were re-injected with 20,000 4T1 cells on the opposite mammary fat pad from the first injection. Neither mouse regrew the tumor (“4T1 Rechallenge” in FIG. 19B) whereas mice with no prior tumor or treatment injected with the 4T1 cells at the same time did grow tumors (“4T1 Naive” in FIG. 19B). This suggested that the cured mice developed tumor-specific memory against the tumor, preventing its regrowth, as the immune system was seemingly able to fight it off early. To determine if this rejection of tumor growth was specific to the tumor from within the mouse that was cured, or rather a rejection of any tumor, the same mouse that resisted 4T1 rechallenge wasinjected with 100,000 EMT6 cells in the mammary fat pad. The EMT6 tumor grew in the mouse (“EMT6 Rechallenge” in FIG. 19B), suggesting that the rejection of tumor growth in cured mice is specific to the tumor from which the mouse was cured.

[0156] This tumor-specific immune memory was additionally tested with the syngeneic TNBC mouse model, E0771, and sarcoma mouse model, MCA205. For these experiments, mice previously cured (e.g., in Example 4) by RT+OTX were injected in the opposite flank (for MCA205) or mammary fat pad (for E0771) two months after the initial injection of tumor. Cell number injected for rechallenge was the same as for initial tumor injection as described previously for 4T1. The resistance to rechallenge was demonstrated to be present in mice cured of MCA205 (FIG. 20A) and E0771 (FIG. 20B). This data validated generation of tumor-specific immune memory, as rejection occurred irrespective of cell line or cancer type.

[0157] Because of the observed turn or- specific immune memory, RT+BET inhibitor treatment was expected to cause changes in immune populations within the tumor microenvironment. A flow cytometry experiment was performed to explore the immune changes (design depicted in FIG. 21 A). BALB / c mice were injected with 20,000 4T1 cells in the mammary fat pad. Once tumors reached -200 mm3, mice were treated with either vehicle or RT+OTX as previously described using a 4-day treatment. Two weeks after treatment began, the tumors were resected. Tumors were finely minced with a razor and digested with 2 mg / mL collagenase in DMEM. The single-cell digestion was treated with RBC lysis buffer, then stained with fluorescently conjugated antibodies for flow analysis including CD45-PE Vio770, CD3-FITC, CD4-BV421, and CD8-BV786. Data acquisition was performed on a Cytek spectral cytometer. Data analysis was performed using FlowJo. The percentage of CD3+, CD4+, and CD8+ cell populations among CD45+ cells are shown in FIGS. 21B. This experiment revealed that RT+ OTX increases CD3+ cells (T cells). More specifically, both CD4+ (Helper T cells) and CD8+ (Cytotoxic T cells) are enriched. This suggests that T cells are affected by RT+OTX and may play a role in response to treatment.

[0158] To identify if CD8+ T cells were involved in the response to treatment, a CD8+ T cell depletion experiment was performed (see Example 2). BALB / c mice were injected with20,000 4T1 cells in the mammary fat pad. Upon tumors reaching 100 mm3, treatment began. All mice were treated with RT+OTX as previously described (4-day treatment). In addition, mice received either CD8 or IgG (negative control) depletion antibody. The antibodies were given at 200 pg / dose I.P. Administration occurred one day prior to injection of tumor cells, the day of tumor cell injection, and twice weekly throughout the experiment. To validate the depletion was working, blood was collected from the submandibular vein of mice and flow cytometry was performed to determine if CD8+ T cells were present (FIGS. 22A-B, having CD3-FITC on the y-axis and CD8-BV786 on the x-axis). These data confirmed that CD8+ T cell depletion was functioning.

[0159] A tumor growth curve for mice treated with RT+OTX with CD8 or IgG depletion was collected (FIG. 22C). It was observed that depletion of CD8+ T cells reverted response to treatment, suggesting that CD8+ T cells are important for RT+OTX efficacy at the primary tumor. FIG. 22D represents the number of lung metastases observed in the IgG versus CD8 depleted mice. Lungs were excised from mice 24 days after treatment began. Metastases (or nodules) were counted by eye. Images of lungs with (FIG. 22E, bottom) and without (FIG. 22E, top) metastases are shown below the graph on the left. It was observed that depletion of CD8+ T cells resulted in an increase in lung metastatic burden, suggesting that CD8+ T Cells are not only important for controlling primary tumor growth, but also systemic tumor burden.

[0160] This finding supports the view that RT+BET inhibitor therapy is mediated through an immune response, specifically utilizing CD8+ T cells. It also shows that the engagement of this cell population is important for controlling metastatic disease. This supports that the addition of OTX to radiation therapy enhances the elusive abscopal effect (e.g., that local radiation treatment can cause a systemic response).Example 6: RT+OTX Influences Anti-Tumor Immunity through Multiple Mechanisms

[0161] In this example, it is shown that RT+BET inhibitor treatment influences anti-tumor immunity through multiple mechanisms. FIG. 1 depicts aspects of the multi-step processes of cancer cell immunity conferred by the invention. RT+BET inhibitor treatment affects multiple steps of the cycle, resulting in a stronger response. Specifically, an enhancementof anti-tumor immune effects (e.g., activation of cGAS / STING, and neoantigen release) and reduction of pro-tumor or immunosuppressive effect (e.g., reducing PD-L1).

[0162] RT is known to cause DNA damage and double-stranded DNA breaks. This mechanism may cause mutations in the DNA that result in neoantigen generation driving a targeted immune response. To determine how the addition of OTX may be impacting this function of radiation therapy, immunofluorescent staining was performed to visualize yH2AX, a marker of double-stranded DNA breaks. 4T1 cells were seeded in vitro. 24 hours later, cells were treated with 500 pM OTX or DMSO as the vehicle control. 24 hours later, cells were treated with 8 Gy radiation therapy. At the designated timepoint / condition (No radiation therapy, 30 min post-radiation therapy, 120 min post-radiation therapy) cells were fixed with 4% PFA and stained with a yH2AX primary antibody overnight. Cells were then stained with a secondary Alexa Fluor 488 antibody, then mounted on a slide with DAPI-containing antibody to be imaged. Microscopic images were acquired at 63x (FIG. 23 A). Blue represents DAPI (nuclear stain) and green represents yH2AX foci (indicative of double-stranded DNA breaks). Quantification was performed using ImageJ, where yH2AX expression was calculated based on the number of foci (Maxima / high brightness) standardized to the area of the nucleus. It was found that RT+OTX enhanced and prolonged yH2AX expression (FIG. 23B), suggesting that DNA damage is increased and offers a greater potential for immunogenic mutations to develop.

[0163] In addition to measuring yH2AX, the formation of micronuclei following treatment was measured. Micronuclei are another sign of DNA damage and can also be immunogenic. MCF7 cells (human ER+ breast cancer) were seeded in vitro and treated with 1 pM OTX or DMSO with or without radiation therapy. Cells were then stained with DAPI and imaged to count micronuclei (see Udden et al., NPJ Precis Oncol. 2023 Ian 24;7(1): 11).

[0164] . As shown in FIG. 24, an increased prevalence of micronuclei with RT+OTX relative to other conditions was observed, further supporting the idea that RT+OTX promotes DNA damage.

[0165] ESRI mutant human ER+ BC line MCF7 Y537S cells were then explored. These cells were treated with DMSO (Vehicle) or 1 pM OTX with or without 20 Gy radiation therapy. At designated timepoints (no radiation therapy / 0 hr, 4 hr, 8 hr, 10 hr), cells werecollected and lysed. The lysates then underwent chromatin fractionation. This process separates proteins attached to the chromatin from proteins elsewhere in the cells. As shown in FIG. 25, the chromatin-bound fraction was run on a Western blot and probed for proteins associated with DNA repair. A clear reduction in DNA repair proteins bound to the chromatin with RT+OTX was observed, suggesting that the treatment alters the recruitment of DNA repair proteins to damaged DNA. This finding further supported that RT+OTX perpetuates DNA damage and limits repair.

[0166] Next, BRD4, a key BET protein, is known to have broad transcriptional regulatory functions. It was possible that this activity could also contribute to immune response. Prior studies have shown that STING, a key component in the cGAS-STING immune activation pathway, is inversely correlated with the expression of MYC, a major oncogene (Lee Km et al, Cancer Immunol Res, 2022). RT+OTX suppressed MYC expression, as shown in FIG. 26. 4T1 cells were treated with 500 nM OTX and 8 Gy radiation therapy, then collected 24 hours after radiation therapy to analyze on Western blot. It was found that RT+OTX suppressed MYC expression, which is consistent with prior literature. This data provides indirect evidence that RT+OTX may activate the cGAS-STING pathway as another anti-tumor immune mechanism.

[0167] Another mechanism of RT+OTX is suppression of immune checkpoints. RT increases the expression of PDL1, as shown in FIG. 3B. 4T1 cells were seeded in vitro and treated with 8 Gy radiation therapy. Cells were collected at designated timepoints: no radiation therapy / 0 hr, 12 hr, 24 hr, and 48 hr. The lysates were run on Western blot and probed for PD-L1 . It was observed that PD-L1 expression increases over time following radiation therapy, which is an example of an immunosuppressive effect of radiation therapy. To enhance the utility of radiation therapy in an immune setting, there is incentive to counter this effect. It has previously been reported that BET inhibition suppresses PD-L1 expression, which is confirmed in FIG. 3 A. MXT1 cells were plated in vitro and treated with vehicle, radiation therapy (8 Gy), or RT+OTX (500 nM). Cells were collected 48 hours after radiation therapy. It was found that RT+OTX suppresses PD-L1 expression, supporting the idea that OTX can counter immunosuppressive effects of radiation therapy.Conclusion to the Examples

[0168] In conclusion, the data in the foregoing examples demonstrate that the combination of BET inhibition and radiation exerts superior local control over existing SOC treatment and confers overall systemic anti-tumor immunity and immunological memory. Further it is shown that the effects of BET inhibition and radiation are dependent on cytotoxic T cells. Further, the protocol described here is advantageously completely non-invasive and short as it can be accomplished over the course of days vs. months. Further the protocol is chemotherapy -free which gives it the potential for substantially lower toxicity. As several BET inhibitors are in various phases of clinical development (including, for instance, ZEN- 3694), this discovery has the potential for significant clinical translation.

Claims

CLAIMS1. A method of treating a cancer or a tumor in a subj ect in need thereof, the method comprising(a) administering to the subject one or more doses of a tumor directed radiation therapy; and(b) administering to the subject one or more doses of a Bromodomain and ExtraTerminal motif (BET) inhibitor.

2. A method of inducing a cancer specific or tumor specific immune memory response in a subject in need thereof, the method comprising(a) administering to the subject one or more doses of a tumor directed radiation therapy; and(b) administering to the subject one or more doses of a Bromodomain and ExtraTerminal motif (BET) inhibitor.

3. A method of activating host innate immunity against a cancer or tumor in a subject in need thereof, the method comprising(a) administering to the subject one or more doses of a tumor directed radiation therapy; and(b) administering to the subject one or more doses of a Bromodomain and ExtraTerminal motif (BET) inhibitor.

4. The method of claim 3, wherein the activating of host innate immunity comprises activating the cGAS / STING signaling pathway in the subject.

5. A method of increasing neoantigen release in a subject in need thereof, the method comprising(a) administering to the subject one or more doses of a tumor directed radiation therapy; and(b) administering to the subject one or more doses of a Bromodomain and ExtraTerminal motif (BET) inhibitor, wherein the neoantigen is specific for a cancer or tumor in the subject.

6. A method of reducing or preventing immunosuppressive effects of a cancer or tumor in a subject in need thereof, the method comprising(a) administering to the subject one or more doses of a tumor directed radiation therapy; and(b) administering to the subject one or more doses of a Bromodomain and ExtraTerminal motif (BET) inhibitor.

7. The method of claim 6, wherein the reducing or preventing immunosuppressive effects comprises reducing or preventing of PD-L1 at the cancer or tumor.

8. The method of claim 7, wherein the reducing or preventing of PD-L1 comprises reducing PD-L1 expression or activity.

9. The method of any one of claims 1-8, wherein the method comprises administering 2 to 4 doses of tumor directed radiation therapy, optionally 2 to 3 doses of tumor directed radiation therapy.

10. The method of claim 9, wherein each dose of radiation therapy comprises an absorbed dose of about 5 to 10 Gy, optionally wherein the absorbed dose is 8 Gy.

11. The method of claim 2, wherein the method comprises administering 2 doses of radiation therapy.

12. The method of any one of claims 1-11, wherein the tumor directed radiation therapy comprises external-beam radiation therapy, interstitial implantation of radioisotopes (I- 125, palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and / or total abdominal and pelvic radiation therapy.

13. The method of any one of claims 1-12, wherein the BET inhibitor is selected from OTX015, ZEN-3694, JQ1, TEN-010 (JQ2), Molibresib (I-BET762), I-BET 151 (GSK1210151A), I-BET 762 (GSK525762), CPI-203, or CPI-0610, or any combination thereof.

14. The method of claim 13, wherein the BET inhibitor comprises OTX015 (OTX).

15. The method of any one of claims 1-14, wherein the radiation therapy and BET inhibitor are administered concomitantly or concurrently, sequentially, or a combination thereof.

16. The method of any one of claims 1-15, wherein the BET inhibitor is administered daily for the duration of the treatment.

17. The method of claim 16, wherein the BET inhibitor is administered daily for 3 to 5 days.

18. The method of claim 17, wherein the BET inhibitor is administered daily for 4 days.

19. The method of any one of claims 1-18, wherein administering the tumor radiation therapy and the BET inhibitor occurs in less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, or less than 5 days.

20. The method of claim 19, wherein administering the tumor radiation therapy and the BET inhibitor occurs in 4 days.

21. The method of any one of claims 1-20, wherein the radiation therapy is administered twice, every other day, over four days and the BET inhibitor is administered daily for the four days.

22. The method of any one of claims 1-21, wherein the cancer or tumor is a solid tumor.

23. The method of claim 22, wherein the solid tumor is selected from testicular tumor, ovarian tumor, cervical tumor, a kidney tumor, bladder tumor, head-and-neck tumor, skin tumor, bone tumor, brain tumor, thyroid tumor, pancreatic tumor, liver tumor, stomach tumor, lung tumor, endometrial tumor, esophageal tumor, breast tumor, cervical tumor, central nervous system tumor, germ cell tumor, prostate tumor, Hodgkin's lymphoma, non-Hodgkin's lymphoma, neuroblastoma, sarcoma, multiple myeloma, melanoma, mesothelioma, osteogenic sarcoma or any combination thereof.

24. The method of claim 23, wherein the cancer or tumor is a breast cancer or tumor and optionally wherein the breast cancer or tumor is a triple negative breast cancer or an ER+ breast cancer.

25. The method of claim 23, wherein the cancer or tumor is a sarcoma, optionally wherein the sarcoma is a soft tissue sarcoma or bone sarcoma.

26. The method of any one of claims 1-25, further comprising administering one or more immunotherapies and / or chemotherapies, optionally selected from pembrolizumab, carboplatin, paclitaxel, doxorubicin / epirubicin, cyclophosphamide, or any combination thereof.

27. The method of any one of claims 1-26, wherein the subject has improved survival and / or the tumor or cancer has reduced growth and / or metastasis after administering the radiation therapy and the BET inhibitor compared to a subject receiving a standard of care.

28. The method of claim 27, wherein the standard of care comprises administering one or more immunotherapies and / or chemotherapies, optionally selected from pembrolizumab, carboplatin, paclitaxel, doxorubicin / epirubicin, cyclophosphamide, or any combination thereof.

29. The method of claim 28, wherein the standard of care further comprises administering the one or more immunotherapies and / or chemotherapies over the course of 60 weeks.

30. The method of any one of claims 1-29, wherein the subject is human.

31. A kit, comprising(a) a Bromodomain and Extra-Terminal motif (BET) inhibitor and / or a tumor directed radiation therapy; and(b) instructions for use according to any one of claims 1-30.

32. All methods, compositions, products, and kits as shown and disclosed herein.

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