Enhancement of sustained antitumor immunity through multifactorial immunomodulation

Abstract

Methods are provided for enhancing immune responses comprising providing an immunogenic composition comprising a cocktail of an intracellular agonist, an immunosuppressive antagonist, and a cytostatic / damage-inducing agent. Further provided herein are methods for treating primary tumors and preventing metastasis.

Description

[Technical field]

[0001] Claiming priority This application claims the benefit of U.S. Provisional Patent Application No. 63 / 321,205, filed March 18, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Federal Grant Support This invention was made with Government support under Grant No. R01 AI127387 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] 1. Field The present invention relates generally to the fields of molecular biology, immunology, and medicine. More specifically, the present invention relates to methods and compositions for stimulating an anti-tumor immune response. [Background technology]

[0004] 2. Description of Related Technology Triple-negative breast cancer (TNBC) is a highly aggressive subtype of malignant breast cancer that lacks expression of estrogen receptor (ER) and progesterone receptor (PR) and does not show significant amplification or overexpression of human epidermal growth factor receptor 2 (HER2 / ERBB2). + , P.R. + , or HER2 HIIn contrast to tumors, TNBC currently lacks well-characterized molecular targets for treatment and is associated with high rates of recurrence and distant recurrence despite aggressive surgery and adjuvant chemotherapy. Diagnosis of TNBC is often delayed, with the majority of women presenting with advanced disease and a grim 5-year survival rate of 30%. Thus, although TNBC accounts for only 15% of breast cancer diagnoses in the United States, it accounts for half of deaths, an estimated 20,000 per year. Given the absence of effective adjuvant therapies for TNBC, the development of novel therapies that can reduce the incidence of distant recurrence, prevent severe comorbidities, and save lives represents an urgent unmet medical need (Hubalek, Czech et al., 2017; Marra, Viale et al., 2019; Waks & Winer, 2019).

[0005] The concept of immune surveillance in cancer remained controversial for decades, until the advent of immune checkpoint blockade established that tumor-reactive T cells exist and may be functionally relevant by blocking signaling pathways related to tolerance, exhaustion, and / or anergy (Brahmer, Tykodi et al., 2012; Hodi, O'Day et al., 2010; Topalian, Hodi et al., 2012). In some oncological indications, especially melanoma, NSCLC with smoking characteristics, or any dMMR / MSI, +In indications with high mutational burden, such as tumor types, dramatic results can be obtained, as exemplified by the current nearly 50% durable remission rate in stage IV melanoma following combination therapy with inhibitors that block both CTLA-4 and PD-1 signaling pathways (Hellmann, Ciuleanu et al., 2018; Marcus, Lemery et al., 2019; Overman, Lonardi et al., 2018; Wolchok, Chiarion-Sileni et al., 2017). Nevertheless, the majority of tumors still do not respond to checkpoint inhibitor treatment, and even in tumor types that do, they often do not respond if sufficient neoantigen-reactive T cells have not naturally arisen by the time of treatment initiation. To overcome this significant limitation and promote the generation of T cells with higher tumor reactivity, many investigators have experimented with various immune agonist and antagonist compounds, both as single agents and in combination with checkpoint inhibition. One problem with this approach is that a vast number of different agonistic and antagonistic pathways exist, interacting redundantly with each other, with tumors, and with immune effector cells in unknown and unpredictable ways.Therefore, there is an unmet need for the development of novel therapeutics for the treatment of not only TNBC but also other aggressive cancers such as pancreatic ductal adenocarcinoma (PDAC), melanoma, and head and neck cancer. Summary of the Invention

[0006] overview In a first embodiment, an immunogenic composition is provided that includes at least four (e.g., at least five, six, or seven) of a transforming growth factor beta (TGFβ) antagonist, a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist. In some aspects, the composition includes (a) a transforming growth factor beta (TGFβ) antagonist or a 4-1BB agonist, and (b) at least three (e.g., four or five) of a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

[0007] In a first embodiment, an immunogenic composition is provided that includes at least four, at least five, at least six, or all seven of a transforming growth factor beta (TGFβ) antagonist, a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist. In some aspects, the composition includes (a) a transforming growth factor beta (TGFβ) antagonist or a 4-1BB agonist, and (b) at least three, at least four, or all five of a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

[0008] In some embodiments, the composition comprises a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and a proteasome inhibitor. In some embodiments, the composition comprises a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and a proteasome inhibitor. In a specific embodiment, the composition comprises a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and an adenosine A3 receptor agonist. In certain embodiments, the composition comprises a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and an adenosine A3 receptor agonist. In some embodiments, the composition comprises at least five of a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist. In some embodiments, the composition comprises at least five of a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist. In some embodiments, the composition comprises a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.In certain embodiments, the compositions include a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

[0009] In some embodiments, the TGFβ antagonist is galunisertib (LY2157299), travedersen, fresolimumab, LY2382770, lucanix, or PF-03446962. In some embodiments, the STING agonist is a cyclic dinucleotide or a xanthenone analog. For example, the STING agonist is 2'3'-c-di-AM(PS)2(Rp,Rp). In some embodiments, the STING agonist is a cyclic dinucleotide selected from the group consisting of 3'3'-cGAMP, 2'3'-cGAMP, 2'2'-cGAMP, c-di-APM, c-di-GMP, c-di-IMP, and c-di-UMP. In some embodiments, the STING agonist is a xanthenone analog, such as 5,6-dimethylxanthenone-4-acetic acid (DMXAA). In some embodiments, the TLR7 / 8 agonist is resiquimod (R848), CL075, CL097, CL264, CL307, GS-9620, poly(dT), imiquimod, gardquimod, loxoribine, or ssRNA oligonucleotide. In some embodiments, the RIG-I agonist is 5'ppp-dsRNA, MDA5 ligand, LGP2 ligand, ssRNA, dsRNA, poly(dA:dT), or poly(I:C). In some embodiments, the proteasome inhibitor is copper(II) diethyldithiocarbamate, bortezomib, delanzomib, ixazomib, MG132, MG115, IPSI001, ferutamide B, ALLN, leupeptin, epoxomicin, oprozomib, PR-957, carfilzomib, lactacystin, omuralide, salinosporamide A, salinosporamide B, belactosin, cinnabalamide, polyphenol, TMC-95, or PS-519. In certain embodiments, the adenosine A3 receptor agonist is 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (2-Cl-IB-MECA), IB-MECA (CF101), CP-608,039, CP-532,903, MRS5698, MRS5980, LJ-52933, or MRS4322. In some embodiments, the 4-1BB agonist is a 4-1BB agonist antibody, a recombinant 4-1BB ligand (4-1BBL), or a 4-1BB apatamer.For example, the 4-1BB agonist antibody is utomirumab or urelumab.

[0010] In some embodiments, the composition does not include a hydrogel. In other embodiments, the composition includes a biomaterial, such as a hydrogel or a polymer. In some embodiments, the composition does not include an immune checkpoint inhibitor, a cytokine, and / or an antigen.

[0011] Further embodiments provide pharmaceutical compositions comprising the immunogenic composition of the embodiments (e.g., an immunogenic composition comprising at least four (e.g., at least five, six, or seven) of a transforming growth factor beta (TGFβ) antagonist, a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist) and an excipient.

[0012] Further embodiments provide pharmaceutical compositions comprising an immunogenic composition of the embodiments (e.g., an immunogenic composition comprising at least four, at least five, at least six, or all seven of a transforming growth factor beta (TGFβ) antagonist, a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist) and an excipient.

[0013] Another embodiment provides a method of stimulating an anti-tumor immune response in a subject, comprising administering to the subject an effective amount of at least four (e.g., at least five, six, or seven) of a transforming growth factor beta (TGFβ) antagonist, a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist. In some aspects, the method includes administering (a) a transforming growth factor beta (TGFβ) antagonist or a 4-1BB agonist, and (b) at least three (e.g., four or five) of a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

[0014] Another embodiment provides a method of stimulating an anti-tumor immune response in a subject, comprising administering to the subject an effective amount of at least four, at least five, at least six, or all seven of a transforming growth factor beta (TGFβ) antagonist, a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist. In some aspects, the method includes administering (a) a transforming growth factor beta (TGFβ) antagonist or a 4-1BB agonist, and (b) at least three, at least four, or all five of a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

[0015] In some embodiments, the subject is administered a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and a proteasome inhibitor. In some embodiments, the subject is administered a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and a proteasome inhibitor. In some embodiments, the subject is administered a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and an adenosine A3 receptor agonist. In certain embodiments, the subject is administered a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and an adenosine A3 receptor agonist. In specific embodiments, the subject is administered at least five of a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist. In some embodiments, the subject is administered at least five of a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist. In some embodiments, the subject is administered a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.In certain embodiments, the subject is administered a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

[0016] In some embodiments, the TGFβ antagonist is galunisertib (LY2157299), travedersen, fresolimumab, LY2382770, lucanix, or PF-03446962. In some embodiments, the STING agonist is a cyclic dinucleotide or a xanthenone analog. For example, the STING agonist is 2'3'-c-di-AM(PS)2(Rp,Rp). In some embodiments, the STING agonist is a cyclic dinucleotide selected from the group consisting of 3'3'-cGAMP, 2'3'-cGAMP, 2'2'-cGAMP, c-di-APM, c-di-GMP, c-di-IMP, and c-di-UMP. In some embodiments, the STING agonist is a xanthenone analog, such as 5,6-dimethylxanthenone-4-acetic acid (DMXAA). In some embodiments, the TLR7 / 8 agonist is resiquimod (R848), CL075, CL097, CL264, CL307, GS-9620, poly(dT), imiquimod, gardquimod, loxoribine, or ssRNA oligonucleotide. In some embodiments, the RIG-I agonist is 5'ppp-dsRNA, MDA5 ligand, LGP2 ligand, ssRNA, dsRNA, poly(dA:dT), or poly(I:C). In some embodiments, the proteasome inhibitor is copper(II) diethyldithiocarbamate, bortezomib, delanzomib, ixazomib, MG132, MG115, IPSI001, ferutamide B, ALLN, leupeptin, epoxomicin, oprozomib, PR-957, carfilzomib, lactacystin, omuralide, salinosporamide A, salinosporamide B, belactosin, cinnabalamide, polyphenol, TMC-95, or PS-519. In certain embodiments, the adenosine A3 receptor agonist is 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (2-Cl-IB-MECA), IB-MECA (CF101), CP-608,039, CP-532,903, MRS5698, MRS5980, LJ-52933, or MRS4322. In some embodiments, the 4-1BB agonist is a 4-1BB agonist antibody, a recombinant 4-1BB ligand (4-1BBL), or a 4-1BB apatamer.For example, the 4-1BB agonist antibody is utomirumab or urelumab.

[0017] In some embodiments, the subject is a human. In some embodiments, the subject has cancer, such as triple-negative breast cancer (TNBC), pancreatic ductal adenocarcinoma (PDAC), melanoma, or head and neck cancer.

[0018] In some embodiments, the administration occurs prior to surgical intervention. In some embodiments, the administration comprises intratumoral injection. In specific embodiments, the intratumoral injection does not comprise a biomaterial, such as a hydrogel or polymer.

[0019] In some embodiments, the immunogenic composition is administered more than once, hi some embodiments, the immunogenic composition is administered two or more times.

[0020] In certain aspects, administration of the immunogenic composition increases circulating CD8+ T cells, M1 macrophages and / or tumor infiltrating dendritic cells. + IFN-γ + In a particular embodiment, administration of the immunogenic composition results in the production of CD44 cells prior to administration. + CD127 + CD44 compared with T cells + CD127 + A sustained T cell memory response is generated as measured by an increase in T cells. In some embodiments, the method does not include administering a cell therapy to the subject when administering the immunogenic composition, e.g., not within 1 day, 1 week, 2 weeks, 1 month, or 3 months of administering the immunogenic composition. In some embodiments, the method does not include administering an immune checkpoint inhibitor, cytokine, and / or antigen to the subject when administering the immunogenic composition, e.g., not within 1 day, 1 week, 2 weeks, 1 month, or 3 months of administering the immunogenic composition.

[0021] Further embodiments provide methods of treating a subject having cancer, comprising administering to the subject an effective amount of at least four (e.g., at least five, six, or seven) of a transforming growth factor beta (TGFβ) antagonist, a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist. In some aspects, the method includes administering (a) a transforming growth factor beta (TGFβ) antagonist or a 4-1BB agonist, and (b) at least three (e.g., four or five) of a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

[0022] In some embodiments, the subject is administered a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and a proteasome inhibitor. In some embodiments, the subject is administered a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and a proteasome inhibitor. In some embodiments, the subject is administered a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and an adenosine A3 receptor agonist. In certain embodiments, the subject is administered a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and an adenosine A3 receptor agonist. In specific embodiments, the subject is administered at least five of a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist. In some embodiments, the subject is administered at least five of a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist. In some embodiments, the subject is administered a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.In certain embodiments, the subject is administered a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

[0023] In some embodiments, the TGFβ antagonist is galunisertib (LY2157299), travedersen, fresolimumab, LY2382770, lucanix, or PF-03446962. In some embodiments, the STING agonist is a cyclic dinucleotide or a xanthenone analog. For example, the STING agonist is 2'3'-c-di-AM(PS)2(Rp,Rp). In some embodiments, the STING agonist is a cyclic dinucleotide selected from the group consisting of 3'3'-cGAMP, 2'3'-cGAMP, 2'2'-cGAMP, c-di-APM, c-di-GMP, c-di-IMP, and c-di-UMP. In some embodiments, the STING agonist is a xanthenone analog, such as 5,6-dimethylxanthenone-4-acetic acid (DMXAA). In some embodiments, the TLR7 / 8 agonist is resiquimod (R848), CL075, CL097, CL264, CL307, GS-9620, poly(dT), imiquimod, gardquimod, loxoribine, or ssRNA oligonucleotide. In some embodiments, the RIG-I agonist is 5'ppp-dsRNA, MDA5 ligand, LGP2 ligand, ssRNA, dsRNA, poly(dA:dT), or poly(I:C). In some embodiments, the proteasome inhibitor is copper(II) diethyldithiocarbamate, bortezomib, delanzomib, ixazomib, MG132, MG115, IPSI001, ferutamide B, ALLN, leupeptin, epoxomicin, oprozomib, PR-957, carfilzomib, lactacystin, omuralide, salinosporamide A, salinosporamide B, belactosin, cinnabalamide, polyphenol, TMC-95, or PS-519. In certain embodiments, the adenosine A3 receptor agonist is 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (2-Cl-IB-MECA), IB-MECA (CF101), CP-608,039, CP-532,903, MRS5698, MRS5980, LJ-52933, or MRS4322. In some embodiments, the 4-1BB agonist is a 4-1BB agonist antibody, a recombinant 4-1BB ligand (4-1BBL), or a 4-1BB apatamer.For example, the 4-1BB agonist antibody is utomirumab or urelumab.

[0024] In some embodiments, the administration is performed before surgical intervention. In certain embodiments, the subject has not undergone surgical resection of the tumor. In certain embodiments, the administration comprises an injection of the immunogenic composition. In some embodiments, the TGFβ antagonist (or 4-1BB agonist), STING agonist, TLR7 / 8 agonist, RIG-I agonist, proteasome inhibitor, and adenosine A3 receptor agonist are administered as one injection. In some embodiments, the TGFβ antagonist (or 4-1BB agonist), STING agonist, TLR7 / 8 agonist, RIG-I agonist, proteasome inhibitor, and adenosine A3 receptor agonist are administered as two or more injections. In some embodiments, the injection is an intratumoral injection.

[0025] In some embodiments, the cancer is triple-negative breast cancer (TNBC), PDAC, head and neck cancer, or melanoma. In some embodiments, the intratumoral injection does not include a biomaterial such as a hydrogel or polymer. In some embodiments, the intratumoral injection includes a biomaterial such as a hydrogel or polymer.

[0026] In some embodiments, the administration is more than one time, hi some embodiments, the administration is more than one time.

[0027] In some embodiments, administration increases circulating CD8+ T cells, M1 macrophages, and / or tumor infiltrating dendritic cells. In a specific embodiment, the CD8+ T cells are CD8 + IFN-γ + In certain embodiments, administration results in a reduction in the CD44 + CD127 + CD44 compared to T cells + CD127 +A sustained T cell memory response is generated as measured by an increase in T cells. In one embodiment, the method does not include administering a cell therapy to the subject when administering a transforming growth factor beta (TGFβ) antagonist (or 4-1BB agonist), a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist, e.g., not within 1 day, 1 week, 2 weeks, 1 month, or 3 months of administering the immunogenic composition. In one embodiment, the method does not include administering to the subject an immune checkpoint inhibitor, cytokine, and / or antigen when administering the transforming growth factor beta (TGFβ) antagonist (or 4-1BB agonist), stimulator of interferon genes (STING) agonist, Toll-like receptor 7 / 8 (TLR7 / 8) agonist, RIG-I agonist, proteasome inhibitor, adenosine A3 receptor agonist, e.g., not within 1 day, 1 week, 2 weeks, 1 month, or 3 months of administration of the immunogenic composition.

[0028] In some embodiments, the method further comprises administering an anti-cancer therapy, hi some embodiments, the additional anti-cancer therapy comprises chemotherapy, radiation therapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy, or immunotherapy.

[0029] In some embodiments, the patient is a human. In some embodiments, the patient has previously been administered an anti-cancer therapy.

[0030] A further embodiment provides a method of preventing tumor metastasis in a subject having cancer, comprising administering to the subject an effective amount of at least four (e.g., at least five, six, or seven) of a transforming growth factor beta (TGFβ) antagonist, a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist. In some aspects, the method includes administering (a) a transforming growth factor beta (TGFβ) antagonist or a 4-1BB agonist, and (b) at least three (e.g., four or five) of a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

[0031] In some embodiments, the subject is administered a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and a proteasome inhibitor. In some embodiments, the subject is administered a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and a proteasome inhibitor. In some embodiments, the subject is administered a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and an adenosine A3 receptor agonist. In certain embodiments, the subject is administered a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and an adenosine A3 receptor agonist. In specific embodiments, the subject is administered at least five of a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist. In some embodiments, the subject is administered at least five of a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist. In some embodiments, the subject is administered a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.In certain embodiments, the subject is administered a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

[0032] In some embodiments, the TGFβ antagonist is galunisertib (LY2157299), travedersen, fresolimumab, LY2382770, lucanix, or PF-03446962. In some embodiments, the STING agonist is a cyclic dinucleotide or a xanthenone analog. For example, the STING agonist is 2'3'-c-di-AM(PS)2(Rp,Rp). In some embodiments, the STING agonist is a cyclic dinucleotide selected from the group consisting of 3'3'-cGAMP, 2'3'-cGAMP, 2'2'-cGAMP, c-di-APM, c-di-GMP, c-di-IMP, and c-di-UMP. In some embodiments, the STING agonist is a xanthenone analog, such as 5,6-dimethylxanthenone-4-acetic acid (DMXAA). In some embodiments, the TLR7 / 8 agonist is resiquimod (R848), CL075, CL097, CL264, CL307, GS-9620, poly(dT), imiquimod, gardquimod, loxoribine, or ssRNA oligonucleotide. In some embodiments, the RIG-I agonist is 5'ppp-dsRNA, MDA5 ligand, LGP2 ligand, ssRNA, dsRNA, poly(dA:dT), or poly(I:C). In some embodiments, the proteasome inhibitor is copper(II) diethyldithiocarbamate, bortezomib, delanzomib, ixazomib, MG132, MG115, IPSI001, ferutamide B, ALLN, leupeptin, epoxomicin, oprozomib, PR-957, carfilzomib, lactacystin, omuralide, salinosporamide A, salinosporamide B, belactosin, cinnabalamide, polyphenol, TMC-95, or PS-519. In certain embodiments, the adenosine A3 receptor agonist is 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (2-Cl-IB-MECA), IB-MECA (CF101), CP-608,039, CP-532,903, MRS5698, MRS5980, LJ-52933, or MRS4322. In some embodiments, the 4-1BB agonist is a 4-1BB agonist antibody, a recombinant 4-1BB ligand (4-1BBL), or a 4-1BB apatamer.For example, the 4-1BB agonist antibody is utomirumab or urelumab.

[0033] In some embodiments, the administration is performed before surgical intervention. In some embodiments, the subject has not undergone surgical resection of the tumor. In some embodiments, the administration comprises an injection of the immunogenic composition. In certain embodiments, the TGFβ antagonist (or 4-1BB agonist), STING agonist, TLR7 / 8 agonist, RIG-I agonist, proteasome inhibitor, and adenosine A3 receptor agonist are administered as one injection. In other embodiments, the TGFβ antagonist (or 4-1BB agonist), STING agonist, TLR7 / 8 agonist, RIG-I agonist, proteasome inhibitor, and adenosine A3 receptor agonist are administered as two or more injections. In certain embodiments, the injection is an intratumoral injection.

[0034] In some embodiments, the cancer is triple-negative breast cancer (TNBC), PDAC, head and neck cancer, or melanoma. In some embodiments, the intratumoral injection does not include a biomaterial such as a hydrogel or polymer. In other embodiments, the intratumoral injection includes a biomaterial such as a hydrogel or polymer.

[0035] In some embodiments, the administration is performed more than once, while in other embodiments, the administration is performed two or more times.

[0036] In some embodiments, administration increases circulating CD8+ T cells, M1 macrophages, and / or tumor infiltrating dendritic cells. In a specific embodiment, the CD8+ T cells are CD8 + IFN-γ + In one embodiment, the administration results in a reduction in the CD44 + CD127 + CD44 compared to T cells + CD127 + A sustained T cell memory response occurs, as measured by T cell expansion.

[0037] In one aspect, the method does not include administering a cell therapy to the subject when administering a transforming growth factor beta (TGFβ) antagonist (or 4-1BB agonist), a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist, e.g., not within 1 day, 1 week, 2 weeks, 1 month, or 3 months of administration of the immunogenic composition. In certain aspects, the methods do not include administering to the subject an immune checkpoint inhibitor, cytokine, and / or antigen when administering the transforming growth factor beta (TGFβ) antagonist, stimulator of interferon genes (STING) agonist, Toll-like 7 / 8 receptor (TLR7 / 8) agonist, RIG-I agonist, proteasome inhibitor, and adenosine A3 receptor agonist, e.g., not within 1 day, 1 week, 2 weeks, 1 month, or 3 months of administration of the immunogenic composition.

[0038] In some embodiments, the method further comprises administering an anti-cancer therapy, for example, the additional anti-cancer therapy comprises chemotherapy, radiation therapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy, or immunotherapy.

[0039] In some embodiments, the patient is a human. In some embodiments, the patient has previously been administered an anti-cancer therapy.

[0040] Another embodiment provides an immunogenic composition comprising at least four (e.g., at least five, six, or seven) of a transforming growth factor beta (TGFβ) antagonist, a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist. In some aspects, the composition comprises (a) a transforming growth factor beta (TGFβ) antagonist or a 4-1BB agonist, and (b) at least three (e.g., four or five) of a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

[0041] In some embodiments, the composition comprises a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and a proteasome inhibitor. In some embodiments, the composition comprises a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and a proteasome inhibitor. In a specific embodiment, the composition comprises a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and an adenosine A3 receptor agonist. In certain embodiments, the composition comprises a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and an adenosine A3 receptor agonist. In some embodiments, the composition comprises at least five of a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist. In some embodiments, the composition comprises at least five of a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist. In some embodiments, the composition comprises a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.In certain embodiments, the compositions include a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

[0042] In some embodiments, the TGFβ antagonist is galunisertib (LY2157299), travedersen, fresolimumab, LY2382770, lucanix, or PF-03446962. In some embodiments, the STING agonist is a cyclic dinucleotide or a xanthenone analog. For example, the STING agonist is 2'3'-c-di-AM(PS)2(Rp,Rp). In some embodiments, the STING agonist is a cyclic dinucleotide selected from the group consisting of 3'3'-cGAMP, 2'3'-cGAMP, 2'2'-cGAMP, c-di-APM, c-di-GMP, c-di-IMP, and c-di-UMP. In some embodiments, the STING agonist is a xanthenone analog, such as 5,6-dimethylxanthenone-4-acetic acid (DMXAA). In some embodiments, the TLR7 / 8 agonist is resiquimod (R848), CL075, CL097, CL264, CL307, GS-9620, poly(dT), imiquimod, gardquimod, loxoribine, or ssRNA oligonucleotide. In some embodiments, the RIG-I agonist is 5'ppp-dsRNA, MDA5 ligand, LGP2 ligand, ssRNA, dsRNA, poly(dA:dT), or poly(I:C). In some embodiments, the proteasome inhibitor is copper(II) diethyldithiocarbamate, bortezomib, delanzomib, ixazomib, MG132, MG115, IPSI001, ferutamide B, ALLN, leupeptin, epoxomicin, oprozomib, PR-957, carfilzomib, lactacystin, omuralide, salinosporamide A, salinosporamide B, belactosin, cinnabalamide, polyphenol, TMC-95, or PS-519. In certain embodiments, the adenosine A3 receptor agonist is 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (2-Cl-IB-MECA), IB-MECA (CF101), CP-608,039, CP-532,903, MRS5698, MRS5980, LJ-52933, or MRS4322. In some embodiments, the 4-1BB agonist is a 4-1BB agonist antibody, a recombinant 4-1BB ligand (4-1BBL), or a 4-1BB apatamer.For example, the 4-1BB agonist antibody is utomirumab or urelumab.

[0043] In some embodiments, the subject is a human. In some embodiments, the cancer is triple-negative breast cancer (TNBC), PDAC, head and neck cancer, or melanoma.

[0044] In some embodiments, the composition is administered prior to surgical intervention. In some embodiments, the composition is administered by intratumoral injection. In some embodiments, the intratumoral injection does not include a biomaterial, such as a hydrogel or polymer. In other embodiments, the intratumoral injection includes a biomaterial, such as a hydrogel or polymer.

[0045] In certain embodiments, the immunogenic composition is administered more than once, hi some embodiments, the immunogenic composition is administered more than once.

[0046] In some embodiments, administration of the immunogenic composition increases circulating CD8+ T cells, M1 macrophages, and / or tumor infiltrating dendritic cells. + IFN-γ + In some embodiments, the composition inhibits the expression of CD44 cells prior to administration. + CD127 + CD44 compared with T cells + CD127 + A sustained T cell memory response occurs, as measured by T cell expansion.

[0047] In some embodiments, the TGFβ antagonist (or 4-1BB agonist), STING agonist, TLR7 / 8 agonist, RIG-I agonist, proteasome inhibitor, and adenosine A3 receptor agonist are administered as one injection. In other embodiments, the TGFβ antagonist (or 4-1BB agonist), STING agonist, TLR7 / 8 agonist, RIG-I agonist, proteasome inhibitor, and adenosine A3 receptor agonist are administered as two or more injections.

[0048] In some aspects, the intratumoral injection does not include a biomaterial such as a hydrogel or polymer, hi other aspects, the intratumoral injection includes a biomaterial such as a hydrogel or polymer.

[0049] In some embodiments, the immunogenic composition is administered more than once. In some embodiments, the composition is administered two or more times. In certain embodiments, the composition does not include a cell therapy. In specific embodiments, the composition does not include an immune checkpoint inhibitor, a cytokine, and / or an antigen.

[0050] In some embodiments, the composition further comprises an additional anti-cancer therapy, such as chemotherapy, radiation therapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy, or immunotherapy.

[0051] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that this detailed description and the specific examples, while indicating certain embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the detailed description. [Brief description of the drawings]

[0052] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0053] [Figure 1A] Empirical fusion of immunomodulators identifies optimized combination regimens for immunotherapy of TNBC. Cohorts of female Balb / c mice were implanted subcutaneously with 500,000 4T1-luc2 cells and administered one of eight single intratumoral injections of combination immunotherapy regimens shown in Table 1 on day 7. Mice were imaged by IVIS on day 21 (N=5 mice per treatment group). [Figure 1B]We show that empirical integration of immunomodulators identifies optimized combination regimens for immunotherapy of TNBC. Cohorts of female Balb / c mice were implanted subcutaneously with 500,000 4T1-luc2 cells and received one of eight single intratumoral injections of combination immunotherapy regimens shown in Table 1 on day 7. We show that luciferase photon intensity was cross-analyzed between groups using Living Image software to estimate total tumor burden as determined by luciferase activity. [Figure 1C] We show that empirical fusion of immunomodulators identifies optimized combination regimens for immunotherapy of TNBC. Cohorts of female Balb / c mice were implanted subcutaneously with 500,000 4T1-luc2 cells and administered one of eight single intratumoral injections of combination immunotherapy regimens shown in Table 1 on day 7. Images are shown 24 days post-treatment for selected treatment arms inoculated with the parental 4T-1luc2neg cell line. [Figure 1D] We show that empirical integration of immunomodulatory agents identifies optimized combination regimens for immunotherapy of TNBC. Cohorts of female Balb / c mice were implanted subcutaneously with 500,000 4T1-luc2 cells and administered one of eight single intratumoral injections of the combination immunotherapy regimens shown in Table 1 on day 7. Tumor growth, as determined by biweekly caliper measurements over a 35-day period, of the parental 4T-1luc2neg cell line is shown. N=5 mice per group for each experiment shown. A representative experiment of five is shown. [Figure 1E] We show that empirical fusion of immunomodulators identifies optimized combination regimens for immunotherapy of TNBC. Cohorts of female Balb / c mice were implanted subcutaneously with 500,000 4T1-luc2 cells and administered one of eight single intratumoral injections of the combination immunotherapy regimens shown in Table 1 on day 7. As shown by Kaplan-Meier survival analysis, only treatment group #13 (red circle) had all animals still alive at day 47 after treatment. N=5 mice per group. Representative experiment of 3 is shown. Error bars=+ / -SEM. *p<0.05, **p<0.01 by one-way ANOVA with Tukey's post-hoc test. [Figure 2A]We show that TGF-β receptor antagonism, but not repeated dosing, restores efficacy of optimized treatment regimen #13 after removal of 4-1BBL. Treatment group #15 (#13 without 4-1BBL) and treatment group #16 (#15 + TGF-βR antagonist galunisertib) were introduced as rational modifications. Cohorts of female Balb / c mice were subcutaneously implanted with 500,000 4T1-luc2 cells and administered intratumoral injections of treatment #15 on day 7 only, days 7 and 10, or days 7, 10, and 14. Mice were imaged by IVIS 16 days after inoculation. [Figure 2B] TGF-β receptor antagonism, but not repeated dosing, restores efficacy of optimized treatment regimen #13 after removal of 4-1BBL. Treatment group #15 (#13 without 4-1BBL) and treatment group #16 (#15 + TGF-βR antagonist galunisertib) show tumors introduced as rational modifications and tumor growth was also measured biweekly with calipers and plotted. Increasing dosing frequency contributed to growth delay but ultimately did not result in durable tumor control. Error bars = + / - SEM. *p<0.05, **p<0.01 by unpaired two-tailed Student's t-test with correction for multiple comparisons. [Figure 2C] Figure 1 shows that TGF-β receptor antagonism, but not repeated dosing, restores efficacy of optimized treatment regimen #13 after removal of 4-1BBL. Treatment group #15 (#13 without 4-1BBL) and treatment group #16 (#15 + TGF-βR antagonist galunisertib) were introduced as rational modifications. A single intratumoral injection of treatment #15 is compared to a single intratumoral injection of treatment #16 (#15 + TGF-βR antagonism) and tumors were measured with calipers every other week. N=5 animals per group for each experiment. One of three representative experiments is shown. Error bars = + / - SEM. *p<0.05, **p<0.01 by unpaired two-tailed Student's t-test with correction for multiple comparisons. [Figure 2D]We show that TGF-β receptor antagonism, but not repeated dosing, restores efficacy of optimized treatment regimen #13 after removal of 4-1BBL. Treatment group #15 (#13 without 4-1BBL) and treatment group #16 (#15 + TGF-βR antagonist galunisertib) were introduced as rational modifications. We show that repeated dosing of treatment #16 was able to confer sustained antitumor effects in a dose-response fashion. Cohorts of female Balb / c mice were subcutaneously implanted with 500,000 4-T1TNBC cells and injected intratumorally with treatment #16 on days 9, 12, and 15. Each cohort received the indicated percentage of 1.00x (100%) of treatment #16 dose. Error bars = + / - SEM. *p<0.05, **p<0.01 by Student's unpaired two-tailed t-test with correction for multiple comparisons. [Figure 2E] We show that TGF-β receptor antagonism, but not repeated dosing, restores efficacy of optimized treatment regimen #13 after removal of 4-1BBL. Treatment group #15 (#13 without 4-1BBL) and treatment group #16 (#15 + TGF-βR antagonist galunisertib) were introduced as rational modifications. Kaplan-Meier survival analysis of D is shown. N=5 animals per group for each experiment D / E. One representative experiment out of three is shown. [Figure 2F] Figure 1 shows that TGF-β receptor antagonism, but not repeated dosing, restores efficacy of optimized treatment regimen #13 after removal of 4-1BBL. Treatment group #15 (#13 without 4-1BBL) and treatment group #16 (#15 + TGF-βR antagonist galunisertib) were introduced as rational modifications. 500,000 4T-1luc-2+ cells were orthotopically implanted under the fourth nipple in the mammary fat pad. Animals were treated intratumorally with vehicle control, galunisertib at a concentration equal to that used in the optimized regimen, galunisertib at a concentration 3x that used in the optimized regimen, or the full optimized regimen 16 itself. Error bars = + / - SEM. *p<0.05, **p<0.01 by one-way ANOVA with Tukey's post-hoc test. [Figure 3-1]Tumor treatment with regimen #16 increases levels of circulating CD8+IFN-γ+ T cells and memory T cells, and significantly reduces exhausted regulatory CD4+ T cells. Cohorts of female Balb / c mice were implanted subcutaneously with 500,000 4T-1 cells, groups were treated intratumorally with treatments 12-16 (all at dose level 1.00) on days 9 and 12, and circulating PBMCs were analyzed by retro-orbital blood collection and flow cytometry on day 15. (Figure 3A) Absolute circulating CD8+ T cells per 106 total cells are shown. (Figure 3B) Fold increase in IFN-γ+CD8+ T cells compared to untreated controls are shown. (Figure 3C) Percentage of circulating exhausted CD4+ T cells (defined as CTLA-4+, PD-1+) are shown. (Figure 3D) Fold decrease in circulating regulatory CD4+ T cells (defined as CTLA-4+, Foxp3+) compared to untreated controls are shown. For each experiment AD, red lines = group mean, N = 5 mice per group. One of three representative experiments is shown. Error bars = + / - SEM. *p<0.05, ***p<0.005 by one-way ANOVA with Tukey's post-hoc test. For memory characterization, circulating PBMCs were collected on days 20 and 35 and analyzed by flow cytometry. (Fig. 3E) Comparison of circulating CD8+CD44+CD127+ cells between vehicle control and treatment #16 on days 20 and 35 after treatment. (Fig. 3F / G) Visualization of individual cohort members in vehicle control and treatment groups. (Fig. 3H) Comparison of circulating CD4+CD44+CD127+ cells between vehicle control and treatment #16 on days 20 and 35 after treatment. (Fig. 3I / J) Visualization of individual cohort members in vehicle control and treatment groups. For each experiment shown, red line = group mean, N = 5 mice per group. One representative experiment out of three is shown. For EJ: error bars = + / - SEM. *p<0.05, **p<0.01 by Student's unpaired two-tailed t-test. [Figure 3-2] See description of Figure 3-1. [Figure 3-3] See description of Figure 3-1. [Diagram 3-4] See description of Figure 3-1. [Figure 3-5]See description of Figure 3-1. [Figure 4-1] We show that TNBC intratumoral injection with scheme #16 generates a transferable memory and propagates the abscopal effect. (Figure 4A) Schematic diagram shows that adoptive transfer experiments were performed. Briefly, mice inoculated with palpable TNBC tumors were treated three times with scheme #16. Thirty days after the last treatment, whole spleen cells were harvested and 100,000 whole spleen cells were adoptively transferred into naïve recipients. A control cohort received 100,000 spleen cells derived from uninoculated / untreated animals. Three days after adoptive transfer, recipient mice were intraperitoneally injected with 500,000 4T-1-luc2 cells and (Figure 4B) imaged by IVIS after an additional 7 days. To characterize a demonstrable abscopal effect, a cohort of female Balb / c mice was implanted subcutaneously with 500,000 4T-1 parental (luc2neg) cells and injected intratumorally with Regimen #16 or vehicle control on days 13, 17, and 20. 250,000 4T1-luc2 cells were implanted subcutaneously at a distal site on day 11 and imaged by IVIS on days 13 and 23, but without direct treatment. (Figure 4C) Primary tumor growth was assessed by caliper measurement. (Figure 4D) Growth of untreated satellite foci was assessed by IVIS luminescence. (Figure 4E) IVIS images of satellite tumor burden after treating primary tumors with vehicle control or Regimen #16 2 and 12 days after implantation. Representative of 3 experiments shown, N=5 mice per group. Error bars=+ / -SEM. *p<0.05 by Student's unpaired two-tailed t-test. [Figure 4-2] See description of Figure 4-1. [Figure 4-3] See description of Figure 4-1. [Figure 5-1]We show that optimized treatment Regimen 16 remodels the tumor microenvironment and ameliorates aggressive MOC-2 HNSCC. (Figure 5A) Cohorts of C57BL / 6 mice were subcutaneously implanted with 100,000 MOC-2 HNSCC cells and administered intratumoral injections of Regimen 16 or vehicle control on days 7, 10, and 14. Tumor volumes were determined by caliper measurements every other week. In subsequent experiments, animals were treated twice with vehicle or Regimen 16, after which tumors were excised, digested, and analyzed by flow cytometry to measure the impact of Regimen 16 on the tumor microenvironment. (Figure 5B) We show that a massive influx of CD8+ T cells was observed among MOC-2 MHSCC tumors treated with Regimen 16. (Figure 5C) Representative CD4 / CD8 flow plots of vehicle-treated MOC-2 tumor microenvironments. (Figure 5D) Representative CD4 / CD8 flow plots of Regimen 16-treated MOC-2 tumor microenvironments. All major populations of MDSCs, including (Fig. 5E) Gr-1+CD11b+, (Fig. 5F) Ly6c+CD11b+, and (Fig. 5G) Ly6c+Gr-1+, were substantially reduced among tumors treated with Regimen 16. (Fig. 5H) Tumor-associated F4 / 80+CD11b+ macrophages were also substantially eliminated among tumors treated with Regimen 16. (Fig. 5I) Representative F4 / 80CD11b flow plots of vehicle-treated MOC-2 tumor microenvironments. (Fig. 5J) Representative F4 / 80CD11b flow plots of Regimen 16-treated MOC-2 tumor microenvironments. (Fig. 5K) The optimized antitumor regimen also induced a one-third reduction in the number of all CD45+ cells that co-express PD-L1. A representative example of three experiments is shown. N=5 mice per group. For A, error bars = + / - SEM, for bar graphs, error bars = + / - SD. *p<0.05, **p<0.01, ***p<0.005 by Student's unpaired two-tailed t-test. [Figure 5-2] See description of Figure 5-1. [Figure 5-3] See description of Figure 5-1. [Figure 5-4] See description of Figure 5-1. [Figure 6-1]We show that optimized treatment Regimen 16 resolves MYCN-amplified neuroblastoma and induces dedifferentiation of tolerogenic myeloid cells. (Figure 6A) Cohorts of 129X1 / SvJ mice were subcutaneously implanted with single cell suspensions derived from Th-MYNC tumors and received intratumoral injections of Regimen 16 or vehicle control on days 7, 10, and 14. Tumor volumes were determined by caliper measurements. Circulating CD8+ cell content was characterized by flow analysis after retro-orbital bleeding. (Figure 6B) Representative plots of PBMCs treated with vehicle are shown. (Figure 6C) Representative plots of PBMCs treated with Regimen 16 are shown. In subsequent experiments, animals were treated twice with vehicle or Regimen 16, after which tumors were excised, digested, and analyzed by flow cytometry to measure the effect of Regimen 16 on the tumor microenvironment. Analysis revealed a myeloid cell population in the process of converting from a tolerogenic to an inflammatory phenotype following treatment with Regimen 16. (FIG. 6D) Representative F4 / 80+ macrophage intracellular arginase analysis of a Th-MYCN tumor microenvironment treated with vehicle. (FIG. 6E) Representative F4 / 80+ macrophage intracellular arginase analysis of a Th-MYCN tumor microenvironment treated with Regimen 16. (FIG. 6F) Representative CD11b+ macrophage intracellular arginase analysis of a Th-MYCN tumor microenvironment treated with vehicle. (FIG. 6G) Representative CD11b+ macrophage intracellular arginase analysis of a Th-MYCN tumor microenvironment treated with Regimen 16. (FIG. 6H) Representative CD11c+ / PD-L1+ cell analysis of a Th-MYCN tumor microenvironment treated with vehicle. (FIG. 6I) Representative CD11c+ / PD-L1+ cell analysis of a Th-MYCN tumor microenvironment treated with Regimen 16. (FIG. 6J) Representative CD11c+ / CTLA-4+ cell analysis of Th-MYCN tumor microenvironments treated with vehicle. (FIG. 6K) Representative CD11c+ / CTLA-4+ cell analysis of Th-MYCN tumor microenvironments treated with Regimen 16. Representative of 3 experiments shown. N=3 mice per group. Error bars=+ / -SEM. ****p<0.001 by unpaired two-tailed Student's t-test. [Figure 6-2] See description of Figure 6-1. [Figure 6-3]See description of Figure 6-1. [Figure 7] This shows that antagonizing tumor immunosuppressive mechanisms alone does not result in antitumor effects. (Figure 7A) Combinations of reagents injected intratumorally on day 12. (Figure 7B) Female Balb / c mice were subcutaneously implanted with 250,000 4T-1TNBC cells in the hind limbs and intratumorally injected with one of the four combinations described in (A) on day 12. Tumor volumes were measured with a caliper every 2-3 days. (Figure 7C) Quantification of results on day 21. For both B and C, n=5. One representative experiment out of three is shown. Error bars=+ / -SEM. *p<0.05 by Student's unpaired two-tailed t-test with correction for multiple comparisons. [Figure 8] We show that combined agonism of three major intracellular PRR pathways (TLR, RIG-I, STING) results in a transient antitumor effect. (Figure 8A) Reagent combinations injected intratumorally on day 12. (Figure 8B) Female Balb / c mice were subcutaneously implanted with 250,000 4T-1TNBC cells in the hind limbs and intratumorally injected with one of the four combinations described in (A) on day 12. Tumor volumes were measured with a caliper every 2-3 days. (Figure 8C) Quantification of results on day 21. For both B and C, n=5. One representative experiment out of three is shown. Error bars=+ / -SEM. *p<0.05 by Student's unpaired two-tailed t-test with correction for multiple comparisons. [Figure 9] Optimized Regimen 16 is shown to resolve orthotopic TNBC. 500,000 4T-1luc-2+ cells were orthotopically implanted under the 4th nipple in the mammary fat pad. Animals were treated intratumorally with vehicle control, galnesitive at a concentration equal to that used in the optimized regimen, galnesitive at 3x the concentration used in the optimized regimen, or the full optimized regimen 16 24 itself. As shown: IVIS imaging of individual animals with data shown in Figure 2F. N=6 mice per group. [Figure 10]Tumor treatment with formulation #16 increases the levels of tumor-resident CD8+IFN-g+ T cells and significantly reduces exhausted regulatory CD4+ T cells. Cohorts of female Balb / c mice were implanted subcutaneously with 500,000 4T-1 cells, groups were treated intratumorally with treatments 9-16 (all at dose level 1.00) on days 9 and 12, and circulating PBMCs were analyzed on day 15 by retroorbital blood sampling and flow cytometry. Tumors were resected on day 15 and the tumor microenvironment was analyzed. (Figure 10A) Absolute tumor-infiltrating CD8+ T cell counts per 106 total cells (left axis) and percent change in tumor-infiltrating CD8+ T cells (right axis) are shown. (Figure 10B) Absolute tumor-infiltrating regulatory CD4+ T-cell counts (defined as CTLA-4+, Foxp3+) per 10,000 CD45+ cells are shown. (FIG. 10C) Percentage of tumor-infiltrating exhausted CD4+ T cells (defined as total CD4+CTLA-4+PD-1+). For each experiment, red line = group mean, n=5 mice per group. One representative experiment out of four is shown. Error bars = + / - SEM. *p<0.05 by one-way ANOVA with Tukey's post-hoc test. [Figure 11] Figure 11 shows that intratumoral injection of optimized plan 16 biases the tumor microenvironment towards a pro-inflammatory phenotype in TNBC. Cohorts of female Balb / c mice were inoculated subcutaneously with 500,000 4T-1 cells and each group was injected intratumorally with #16 on days 9 and 12. Tumors were excised on day 15 and the microenvironment was analyzed as described. (Figure 11A) Tumor-infiltrating dendritic cells are shown. (Figure 11B) M1 macrophages are shown. (Figure 11C) Myeloid-derived suppressor cells (MDSCs) are shown. For each experiment shown, n=5 mice per group. One of four representative experiments47 is shown. Error bars=+ / -SEM. *p<0.05 by unpaired two-tailed Student's t-test. [Figure 12] Figure 5: Optimized Regimen 16 Resolves Aggressive MOC-2 HNSCs. Cohorts of C57BL / 6J mice were implanted subcutaneously with 100,000 MOC-2 HNSCC cells. Animals were treated intratumorally with vehicle control or optimized regimen 16 and tumor volumes were determined by caliper measurements. As shown: Individual animals with data shown in Figure 5A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] Description of exemplary embodiments To account for biological redundancy and unpredictability, this study aimed to optimize combinatorial approaches to immune modulation in cancer using both rational and empirical strategies. The results demonstrated that optimal combinations of intracellular pattern recognition receptor (PRR) agonists, immune inhibitory antagonists, and cytostatic / damage inducers can generate effective and durable antitumor responses in TNBC model systems that are generally considered to be non-immunogenic (Brockstedt, Diagana et al., 2002; Feola, Capasso et al., 2018; Liu, Wang et al., 2018).

[0055] Tumors typically lack the canonical danger signals required to activate adaptive immunity and, moreover, frequently employ a variety of immunomodulatory mechanisms that downregulate adaptive responses and contribute to evasion from immune surveillance. Given the variety of mechanisms involved in tumor evasion from immune recognition, it is not surprising that single-agent immunomodulatory approaches have been largely unsuccessful in generating sustained antitumor responses.

[0056] Thus, in certain embodiments, methods and compositions are provided herein for a unique combination of immunomodulatory and cytostatic agents that realign the tumor microenvironment and improve or eliminate complex and / or poor prognosis tumor types, including the non-immunogenic 4T-1 model of TNBC, the aggressive MOC-2 model of HNSCC, and the high-risk MYCN-amplified model of neuroblastoma. A course of treatment optimized for TNBC resulted in a 50% complete response rate and eliminated metastatic spread in all animals tested at the highest dose. Immune responses were transferable between treatment recipients and naive donors through adoptive transfer, and a significant abscopal effect against distant untreated lesions was experimentally demonstrated. Similar results were observed in HNSCC and neuroblastoma models, with substantial remodeling of the tumor microenvironment documented in all model systems. Data indicate that targeting multiple pathways and mechanisms of action may result in substantial synergistic effects. These results were also shown in other cancers, such as PDAC and melanoma, and therefore the method may be used to treat other non-immunogenic cancers.

[0057] I. Definition As used herein, "essentially free" with respect to a particular component is used herein to mean that none of the components are intentionally formulated into the composition in a particular respect and / or are present only as contaminants or in trace amounts.Thus, the total amount of the particular component resulting from any unintentional contamination of the composition is well below 0.05%, preferably below 0.01%.Most preferred is a composition in which the amount of the particular component cannot be detected by standard analytical methods.

[0058] As used herein, "a" or "an" may mean one or more. As used herein in the claims, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one.

[0059] Use of the term "or" in the claims is used to mean "and / or," unless expressly indicated to refer only to alternatives or where the alternatives are mutually exclusive, but the present disclosure also supports a definition that refers only to alternatives and only to "and / or." As used herein, "another" can mean at least a second, or more.

[0060] The term "essentially" should be understood to mean that the method or composition includes only the specified steps or materials and does not materially affect the basic and novel characteristics of those methods and compositions.

[0061] As used herein, a composition that is "substantially free" of a particular substance or ingredient contains ≦30%, ≦20%, ≦15%, more preferably ≦10%, even more preferably ≦5%, or most preferably ≦1% of that substance or ingredient.

[0062] As used herein, the terms "substantially" or "approximately" may be applied to modify any quantitative comparison, value, measurement, or other representation that can be acceptably varied without resulting in a change in the fundamental function with which it is associated.

[0063] The term "about" generally means within the standard deviation of the stated value, as determined using standard analytical techniques to measure the stated value. These terms may also be used to refer to plus or minus 5% of the stated value.

[0064] As used herein, "essentially free" with respect to a particular component is used herein to mean that none of the components are intentionally formulated into the composition in a particular respect and / or are present only as contaminants or present in trace amounts.Thus, the total amount of the particular component resulting from any unintentional contamination of the composition is well below 0.05%, preferably below 0.01%.Most preferred is a composition in which the amount of the particular component cannot be detected by standard analytical methods.

[0065] The term "substantially free" is used to refer to 98% of the listed component and less than 2% of the component being substantially free from the composition or particle.

[0066] "Treatment" or "treating" includes (1) inhibiting a disease in a subject or patient experiencing or exhibiting the pathology or symptomology of a disease (e.g., halting further development of the pathology and / or symptomology), (2) ameliorating a disease in a subject or patient experiencing or exhibiting the pathology or symptomology of a disease (e.g., reversing the pathology and / or symptomology), and / or (3) causing a measurable reduction in either the disease or the pathology of a disease in a subject or patient experiencing or exhibiting the pathology or symptomology of a disease.

[0067] "Prevention" or "preventing" includes (1) inhibiting the onset of a disease in a subject or patient, who may be at risk and / or predisposed to the disease, but who has not yet experienced or exhibited any or all of the disease's pathology or symptomology, and / or (2) delaying the onset of a disease in a subject or patient, who may be at risk and / or predisposed to the disease, but who has not yet experienced or exhibited any or all of the disease's pathology or symptomology.

[0068] In some embodiments, all compounds of the present disclosure may be used for the prevention and treatment of one or more diseases or disorders discussed herein or elsewhere. In some embodiments, one or more of the compounds characterized or exemplified herein as intermediates, metabolites, and / or prodrugs may nevertheless be useful in the prevention and treatment of one or more diseases or disorders. Thus, unless expressly stated to the contrary, all compounds of the present invention are considered "active compounds" and "therapeutic compounds" intended for use as active pharmaceutical ingredients (APIs). Actual suitability for human or veterinary use is typically determined using a combination of clinical trial protocols and regulatory procedures, such as those administered by the Food and Drug Administration (FDA). In the United States, the FDA is responsible for protecting the public health by ensuring the safety, efficacy, quality, and security of human and veterinary drugs, vaccines, and other biological products, as well as medical devices.

[0069] "Prophylactically treating" includes (1) reducing or alleviating the risk of developing a disease in a subject or patient, where the subject or patient may be at risk and / or predisposed to the disease, but has not yet experienced or exhibited any or all of the pathology or symptomology of the disease, and / or (2) delaying the onset of a disease in a subject or patient, where the subject or patient may be at risk and / or predisposed to the disease, but has not yet experienced or exhibited any or all of the pathology or symptomology of the disease.

[0070] As used herein, the term "patient" or "subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human patients are adults, juveniles, infants, and fetuses.

[0071] The term "effective," as used in the specification and / or claims, means adequate to achieve a desired, expected, or intended result. An "effective amount," "therapeutically effective amount," or "pharmacologically effective amount," when used in the context of treating a patient or subject with a compound, means the amount of the compound that, when administered to a subject or patient for treating or preventing a disease, is sufficient to effect such treatment or prevention.

[0072] As used herein, "IC 50 The term "inhibitory dose" refers to an inhibitory dose that is 50% of the maximal response obtained. This quantitative measure indicates the amount of a particular drug or other substance (an inhibitor) that is needed to inhibit a given biological, biochemical, or chemical process (or a component of the process, i.e., an enzyme, a cell, a cell receptor, or a microorganism) by half.

[0073] An "anti-cancer" agent can adversely affect the cancer cells / tumor of a subject, for example, by promoting the death of cancer cells, inducing apoptosis in cancer cells, decreasing the rate of proliferation of cancer cells, reducing the incidence or number of metastases, shrinking tumor size, inhibiting tumor growth, decreasing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or tumors, preventing or inhibiting the progression of cancer, or extending the lifespan of a cancer patient.

[0074] "Aggressive cancers" as referred to herein are cancers that grow and metastasize more aggressively, presenting challenges that make them more difficult to treat than common tumor types. Cancer cells often become resistant to standard treatment options, and patients may quickly exhaust these options.

[0075] As used generally herein, "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues, organs, and / or body fluids of human beings and animals, without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0076] "Pharmaceutically acceptable salt" refers to a salt of a compound of the present invention that is pharma- ceutically acceptable as defined above and has the desired pharmacological activity. Non-limiting examples of such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; or with 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4' methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hex ... Examples of suitable pharmaceutical bases include acid addition salts formed with organic acids such as sanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acid, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiary butyl acetic acid, trimethyl acetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts, which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Non-limiting examples of acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, and N-methylglucamine. It will be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable.Additional examples of pharma- ceutically acceptable salts and their methods for preparation and use are provided in Handbook of Pharmaceutical Salts: Properties, and Use (P.H. Stahl & C.G. Wermuth, eds., Verlag Helvetica Chimica Acta, 2002).

[0077] As used herein, a "durable" anti-tumor response refers to a continuous response that is still capable of preventing the formation of new tumors at least 3 months (e.g., at least 6 months, 12 months, 2 years, 3 years, 4 years, or 5 years or more) after formation or that is still capable of adoptive transfer between subjects at least 3 months (e.g., at least 6 months, 12 months, 2 years, 3 years, 4 years, or 5 years or more) after formation.

[0078] II. Immunomodulators Certain aspects of the embodiments relate to immunomodulators that include inhibitors and agonists of various pathways, such as the TGFβ pathway and the PRR pathway. In a specific aspect, a therapeutic cocktail is provided herein that includes a combination of an intracellular agonist, an immune inhibitory antagonist, and a cytostatic / damage-inducing agent. The PRR agonist may include a STING agonist, a TLR7 / 8 agonist, and a RIG-I agonist. In a particular aspect, the cocktail includes a STING agonist, a TLR7 / 8 agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

[0079] A. PRR agonists a. STING agonist In some embodiments, the methods and compositions include a stimulator of interferon genes (STING) pathway activator, such as DDX41, STING, cGAS, IRF3, TBK1 or STAT6, or a fragment or variant thereof. STING is responsible for sensing cytoplasmic DNA and inducing proinflammatory mediators. After binding DNA in the cytoplasm, STING activates signaling through the TANK-binding kinase 1 (TBK-1) / IRF-3 axis, resulting in the production of IFN-β. This pathway has been shown to play an important role in sensing DNA viruses as well as some autoimmune diseases. Recent data have identified that the STING pathway is absolutely required to induce spontaneous T cell priming against tumor antigens in vivo. Tumor DNA was detected within tumor-infiltrating DCs, which led to IFN-β production and T cell activation.

[0080] The STING agonist of the present invention may be selected from chemical activators of the STING pathway selected from cyclic dinucleotides and xanthenone analogs. Cyclic dinucleotides include, but are not limited to, 3'3'-cGAMP, 2'3'-cGAMP, 2'2'-cGAMP, c-di-APM, c-di-GMP, c-di-IMP, and c-di-UMP. Xanthenone analogs include, but are not limited to, 5,6-dimethylxanthenone-4-acetic acid (DMXAA; Vadimezan or ASA404), which is a tumor vascular disrupting agent. In some embodiments, the STING agonist is 2'3'-c-di-AM(PS)2(Rp,Rp), which is a c-di-AMP bisphosphorothioate analog and Rp isomer (also called ADU-S100 or MIW815) (CAS number: 1638750-95-4) (sodium salt).

[0081] b. TLR7 / 8 agonists In some embodiments, the methods and compositions include an agonist of an immune stimulatory Toll-like receptor (TLR), such as a TLR3, TLR7, TLR8, or TLR9 agonist, such as a TLR7 agonist and / or a TLR8 agonist (TLR7 / 8). The TLR agonist may be BCG, a TLR7 agonist (e.g., poly-OICLC, imiquimod), a TLR8 agonist (e.g., resiquimod (R848), or a TLR9 agonist (e.g., CPG7909). In some embodiments, the TLR7 agonist is CL075, CL097, CL264, CL307, GS-9620, poly(dT), imiquimod, gardquimod, resiquimod (R848), loxoribine, or a ssRNA oligonucleotide. Exemplary TLR-9 agonists include CpG oligodeoxynucleotides (CpG Oligodeoxynucleotides). Other TLR agonists are described, for example, in U.S. Patent Publication No. 2014 / 0005255, incorporated herein by reference.

[0082] c. RIG-I agonist In certain aspects, the methods and compositions of the invention include a retinoic acid-inducible gene-1 (RIG-1)-like receptor ligand. In some aspects, the RIG-1-like receptor ligand is further defined as a RIG-1, MDA5, LGP2, or IPS-1 ligand.

[0083] RIG-I-like receptor (RLR) ligands, as known in the art, refer to activators of RIG-I, Mda5, and LGP2 signaling. These ligands include, but are not limited to, single-stranded RNA, double-stranded RNA, and 5'-triphosphate RNA. RIG-I-like receptor ligands also refer to modifications introduced into RNA molecules that can cause binding and activation of RIG-I, Mda5, and LGP2, resulting in RLR-like biological activity. In some embodiments, the RLR ligand may be a modulator of a general adaptor protein, such as IPS-1, also known as MAVS, VISA, or CARDIF. For example, the RIG-I-like receptor ligand is selected from the group consisting of MDA5 ligand, LGP2 ligand, ssRNA, dsRNA, 5'ppp-dsRNA, poly(dA:dT), and poly(I:C).

[0084] B. TGFβ antagonist Transforming growth factor beta (TGFβ) is a secreted protein that controls the growth, differentiation, and other functions of most cells. It is a type of cytokine involved in immunity, cancer, bronchial asthma, pulmonary fibrosis, heart disease, diabetes, and multiple sclerosis. TGF-y exists in at least three isoforms called TGF-β1, TGF-β2, and TGF-β3. The TGF-β family is part of a superfamily of proteins known as the transforming growth factor beta superfamily, which includes inhibin, activin, anti-Müllerian hormone, bone morphogenetic protein, decapentaplegic, and Vg-1.

[0085] In some embodiments, the TGFβ inhibitor is galunisertib (LY2157299), travedersen, fresolimumab, LY2382770, lucanix, or PF-03446962.

[0086] C. Proteasome Inhibitors Proteasomes are protease complexes that mediate many cellular mechanisms, including cell cycle progression, cellular apoptosis, and maintaining optimal levels of intracellular proteins required for normal cellular processes, through ubiquitin-dependent or ubiquitin-independent protein degradation. Inhibition of proteasomes induces cell cycle arrest and apoptosis through the regulation of several pathways, such as stabilization of p53, activation of C-Jun NH2-terminal kinase (JNK), inactivation of nuclear factor kappa B (NFκB), and activation of both intrinsic and extrinsic caspase cascades. Furthermore, inhibition of proteasomes can lead to accumulation of unfolded proteins in the endoplasmic reticulum (ER), followed by activation of the unfolded protein response (UPR) pathway, which can lead to apoptosis. The proteasome inhibitor of the present invention may be any proteasome inhibitor known in the art. In particular, it is one of the proteasome inhibitors described in more detail in the following paragraphs.

[0087] Proteasome inhibitors that may be used in the methods and compositions of the invention include copper(II) diethyldithiocarbamate, (a) peptide boronates such as bortezomib (also known as Velcade™ and PS341), delanzomib (also known as CEP-18770), ixazomib (also known as MLN9708) or ixazomib citrate; (b) peptide aldehydes such as MG132 (Z-Leu-Leu-Leu-H), MG115 (Z-Leu-Leu-Nva-H), IPSI 001, ferutamide B, ALLN (Ac-Leu-Leu-N1e-H, also known as calpain inhibitor I), and leupeptin (Ac-Leu-Leu-Arg-al); (c) peptide vinyl sulfones, (d) epoxomicin, oprozomib (PR-047 or ONX (e) β-lactones such as lactacystin, omuralide, salinosporamide A (also known as NPI-0052 and marizomib), salinosporamide B, belactosin, cinnabalamide, polyphenols, TMC-95, and PS-519.

[0088] D. Adenosine A3 receptor agonists Adenosine receptors (ARs) are G protein-coupled receptors (GPCRs) that sense imbalances between energy / oxygen / nutrient demand and supply. Extracellular adenosine concentrations increase in response to hypoxia and other stresses, and four subtypes of ARs (A, B, C, D, E ... 1 A.R., A. 2A A.R., A. 2B A.R. and A. 3 A 1 A.R., A. 2A A.R. and A. 3 Agonists of the AR are the subject of preclinical and clinical evaluation (Jacobson et al., Front. Cell. Neurosci., 2019). The adenosine A(3) receptor (A3AR) is coupled to G proteins involved in a variety of intracellular signaling pathways and physiological functions. Exemplary A(3) receptors include3 AR agonists include, but are not limited to, 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (2-Cl-IB-MECA; CF102, namodenoson), IB-MECA (CF101), CP-608,039, CP-532,903, MRS5698, MRS5980, LJ-52933 (4'-thio-Cl-IB-MECA), and MRS4322.

[0089] E.4-1BB agonist In some embodiments, the immunogenic composition comprises a costimulatory receptor 4-1BB agonist. 4-1BB belongs to the TNF receptor family and includes multiple T cell costimulatory receptors that have been targeted with agonistic antibodies, such as GITR, CD40, CD27, HVEM, LIGHT, APRIL, TWEAK, etc. Exemplary 4-1BB agonists include, but are not limited to, 4-1BB agonist antibodies (e.g., utomilumab (PF-05082566) or urelumab (BMS-663513)), recombinant 4-1BBL (including, but not limited to, soluble, matrix-bound, scaffold-bound), and 4-1BB aptamers.

[0090] III.How to use Further provided herein is a method for treating or delaying the progression of cancer in an individual, e.g., preventing metastasis, comprising administering to the individual an immunogenic composition of the present embodiments. The composition may be administered as an intratumoral injection.

[0091] Examples of cancers that may be treated include lung cancer, head and neck cancer, breast cancer, brain cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, lymphoma, preneoplastic lesions, colon cancer, melanoma, bladder cancer, etc. In certain aspects, the cancer is triple negative breast cancer.

[0092] The cancer may specifically be of the following histological types, but is not limited to: malignant neoplasms; carcinomas; non-small cell lung cancer; kidney cancer; renal cell carcinoma; clear cell renal cell carcinoma; lymphoma; blastoma; sarcoma; undifferentiated carcinoma; meningioma; brain cancer; oropharyngeal cancer; nasopharyngeal cancer; cholangiocarcinoma; pheochromocytoma; islet cell carcinoma; Li-Fraumeni tumor; thyroid cancer; parathyroid cancer; pituitary tumor; adrenal tumor; osteogenic sarcoma tumor; neuroendocrine tumor; breast cancer; lung cancer; head and neck cancer; prostate cancer; esophageal cancer; tracheal cancer; liver cancer; bladder cancer; gastric cancer; pancreatic cancer; ovarian cancer; uterine cancer; cervical cancer; sperm Focal carcinoma;Colon cancer;Rectal cancer;Skin cancer;Giant cell and spindle cell carcinoma;Small cell carcinoma;Small cell carcinoma of the lung;Papillary carcinoma;Oral cancer;Oropharyngeal cancer;Nasopharyngeal cancer;Respiratory cancer;Genitourinary cancer;Squamous cell carcinoma;Lymphoepithelial carcinoma;Basal cell carcinoma;Piloma carcinoma;Transitional cell carcinoma;Papillary transitional cell carcinoma;Adenocarcinoma;Gastrointestinal cancer;Malignant gastrinoma;Choledochocarcinoma;Hepatocellular carcinoma;Combined hepatocellular carcinoma and cholangiocarcinoma;Trabecular adenocarcinoma;Adenoid cystic carcinoma;Adenocarcinoma in adenomatous polyps;Adenocarcinoma, familial adenomatous polyposis;Solid tumors;Malignant carcinoid tumors;Branchial alveolar adenocarcinoma;Papillary gland Breast cancer;chromophobe carcinoma;eosinophilic carcinoma;eosinophilic adenocarcinoma;basophilic carcinoma;clear cell adenocarcinoma;granular cell carcinoma;follicular adenocarcinoma;papillary and follicular adenocarcinoma;non-encapsulating sclerosing carcinoma;adrenal cortical carcinoma;endometrial carcinoma;skin adnexal carcinoma;apocrine adenocarcinoma;sebaceous adenocarcinoma;selene adenocarcinoma;mucoepidermoid carcinoma;cystadenocarcinoma;papillary cystadenocarcinoma;papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma;mucinous adenocarcinoma;signet ring cell carcinoma;invasive ductal carcinoma;medullary carcinoma;lobular carcinoma;inflammatory carcinoma;Paget's disease of the breast;acinic cell carcinoma;adenosquamous carcinoma;adenocarcinoma with squamous metaplasia;malignant thymoma;malignant ovarian stromal tumor;malignant Sheath tumor;Malignant granulosa cell tumor;Malignant androblastoma;Sertoli cell carcinoma;Malignant Leydig cell tumor;Malignant lipid cell tumor;Malignant paraganglioma;Malignant extramammary paraganglioma;Pheochromocytoma;Glomocytoma;Malignant melanoma;Amelanomatous melanoma;Superficial spreading melanoma;Malignant melanoma in giant pigmented nevus;Lentigo maligna melanoma;Acral lentigo melanoma;Nodular melanoma;Epithelioid cell melanoma;Blue nevus, malignant;Sarcoma;Fibrosarcoma;Malignant fibrous histiocytoma;Myxosarcoma;Liposarcoma;Leiomyosarcoma;Rhabdomyosarcoma;Embryonal rhabdomyosarcoma;Alveolar rhabdomyosarcoma;Stromatous sarcoma;Malignant mixed tumor;Mullerian mixed tumor;Nephroblastoma;Hepatoblastoma;Carcinosarcoma;Malignant mesenchymoma;Malignant Brenner tumor;Malignant phyllodes tumor;Synovial sarcoma;Malignant mesothelioma;Germinal dysplasia;Embryonal carcinoma;Malignant teratoma;Malignant ovarian goiter;Choriocarcinoma;Malignant mesonephroma;Angiosarcoma;Malignant hemangioendothelioma;Kaposi's sarcoma;Malignant hemangiopericytoma;Lymphangiosarcoma;Osteosarcoma;Juxtamoxifen osteosarcoma;Chondrosarcoma;Malignant chondroblastoma;Mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Malignant odontogenic tumor;Ameloblast odontoma;malignant ameloblastoma;ameloblastic fibrosarcoma;endocrine, neuroendocrine, or hematopoietic carcinoma;malignant pinealoma;chordoma;cancer of central or peripheral nervous system tissue;malignant glioma;ependymoma;astrocytoma;protoplasmic astrocytoma;fibrous astrocytoma;astroblastoma;glioblastoma;oligodendroglioma;oligodendroglioma;primitive neuroectodermal;cerebellar sarcoma;ganglioneuroblastoma;neuroblastoma;retinoblastoma;olfactory tumor;malignant myeloma Membranomas;Neurofibrosarcomas;Malignant neurilemmomas;Malignant granular cell tumors;B-cell lymphomas;Malignant lymphomas;Hodgkin's disease;Hodgkins;Low-grade / follicular non-Hodgkin's lymphoma;Paragranulomas;Small lymphocytic malignant lymphoma;Large cell diffuse malignant lymphoma;Follicular malignant lymphoma;Mycosis fungoides;Mantle cell lymphoma;Waldenstrom's macroglobulinemia;Other specified non-Hodgkin's lymphomas;Malignant histiocytosis; Multiple myeloma;mast cell sarcoma;immunoproliferative small intestinal disease;leukemia;lymphocytic leukemia;plasma cell leukemia;erythroleukemia;lymphoblastic leukemia;myeloid leukemia;basophilic leukemia;eosinophilic leukemia;monocytic leukemia;mast cell leukemia;megakaryoblastic leukemia;myelosarcoma;chronic lymphocytic leukemia (CLL);acute lymphoblastic leukemia (ALL);hairy cell leukemia;chronic myeloblastic leukemia;and / or hairy cell leukemia.;

[0093] In some embodiments, the subject is a mammal, e.g., a primate, preferably a higher primate, e.g., a human (e.g., a patient having or at risk of having a condition described herein). In one embodiment, the subject is in need of an enhanced immune response. In certain embodiments, the subject is immunocompromised or at risk of becoming immunocompromised. For example, the subject is undergoing or has undergone chemotherapy and / or radiation therapy. Alternatively, or in combination, the subject is immunocompromised or at risk of becoming immunocompromised as a result of an infection. In some aspects, the subject is a mammal. Mammals include, but are not limited to, goats, cows, pigs, dogs, cats, donkeys, monkeys, apes, mice, hamsters, rabbits, and other rodents.

[0094] A. Formulation and Administration The present disclosure provides pharmaceutical compositions comprising immunomodulatory agents. When clinical applications are contemplated, pharmaceutical compositions must be prepared in a form suitable for the intended application. Generally, this requires preparing compositions that are essentially free of pyrogens and other impurities that may be harmful to humans or animals. In general, it is desirable to use appropriate salts, buffers, and lipids to deliver oligonucleotides and allow uptake by target cells. Such methods and compositions are well known in the art, for example, as disclosed in U.S. Patent Nos. 6,747,014 and 6,753,423, which are incorporated herein by reference. The compositions of the present disclosure comprise an effective amount of oligonucleotides for cells, dissolved or dispersed in a pharma-ceutically acceptable carrier or vehicle.

[0095] The active compositions of the present disclosure may include classical pharmaceutical preparations.The administration of these compositions according to the present disclosure is carried out via any common route, as long as the target tissue is available via that route.The administration may be carried out by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or by introduction into the CNS, such as spinal fluid.These compositions will usually be administered as pharma-ceutically acceptable compositions as described above.

[0096] In certain embodiments, administration is by injection, such as intratumoral injection. Suitable pharmaceutical forms for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form should be sterile and fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent, for example, sugar, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0097] Sterile injectable solution is prepared by incorporating the active compound in the required amount in a suitable solvent containing various other ingredients as listed above, as necessary, and then filtering and sterilizing.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other ingredients as listed above.In the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is high vacuum drying and freeze-drying technology, which obtains a powder of active ingredient and any additional desired ingredient from its previously sterile filtered solution.

[0098] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, lipids, nanoparticles, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. The use of such media and agents for pharma-ceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated. Supplementary active ingredients can also be incorporated into the composition.

[0099] For oral administration, the compositions of the present disclosure may be combined with excipients. The compositions of the present disclosure may be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins), formed with inorganic acids such as hydrochloric acid or phosphoric acid, or with organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0100] Upon formulation, solutions are administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulations are readily administered in a variety of dosage forms, including injectable solutions, drug-release capsules, and the like. For parenteral administration in aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, employable sterile aqueous media will be known to those of skill in the art in view of the present disclosure. For example, a dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous injection fluid or injected at the proposed injection site (see, for example, "Remington's Pharmaceutical Sciences," 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations must meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biologics Standards. In some embodiments, the dose of each component in the immunogenic composition may be between 1 and 100 times the dose shown in Table 2. For example, the dose of a proteasome inhibitor (e.g., copper(II) diethyldithiocarbamate) may be 120 μg-12 mg, the dose of an adenosine A3 receptor agonist (e.g., 2-Cl-IB-MECA) may be 28 μg-2.8 mg, the dose of a STING agonist (e.g., 2'3'-c-di-AM(PS))2(Rp,Rp)) may be 0.50 μg-0.05 mg, the dose of a RIG-I agonist (e.g., Iyovec) may be 0.53 μg-0.053 mg, the dose of a TLR7 / 8 agonist (e.g., resiquimod, R848) may be 21 μg-2.1 mg, the dose of a TGFβ inhibitor (e.g., galunisertib) may be 42 μg-4.2 mg, or any range derivable therein.

[0101] B. Combination Therapy In order to enhance the effectiveness of the immunogenic compositions of the present invention, it may be desirable to combine these compositions with other drugs that are effective in treating the disease of interest.In some embodiments, the subject may be administered with an immune checkpoint inhibitor or vaccine.In some embodiments, the subject is administered with radiation therapy or chemotherapy.In some embodiments, the subject is administered with a targeted kinase inhibitor, such as a BRAF inhibitor.

[0102] As a non-limiting example, the treatment of cancer may be performed using the immunogenic composition of the present embodiment together with other anti-cancer drugs. An "anti-cancer drug" can adversely affect a subject's cancer, for example, by promoting the death of cancer cells, inducing apoptosis of cancer cells, reducing the rate of proliferation of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to a tumor or cancer cells, promoting an immune response to a cancer cell or tumor, preventing or inhibiting the progression of cancer, or extending the life span of a subject with cancer. More generally, these other compositions are provided in a combined amount effective to kill or inhibit the proliferation of cells. This process may include contacting the cells with the anti-cancer peptide or nanoparticle complex and the drug(s) or multiple factor(s) simultaneously. This can be accomplished by contacting the cells with a single composition or pharmacological formulation that includes both drugs, or by contacting the cells with two different compositions or formulations simultaneously, one composition including the immunogenic composition and the other composition including the second drug(s).

[0103] Treatment with the immunogenic composition may precede or follow treatment with the other agent by intervals ranging from minutes to weeks. In embodiments in which the other agent and the immunogenic composition are applied separately to the subject, generally no significant time will elapse between the times of administration so that the agent and the immunogenic composition can still effectively exert their combined effect on the cells. In such cases, it is contemplated that both modalities may contact the cells within about 12-24 hours of each other, more preferably within about 6-12 hours of each other. In some circumstances, it may be desirable to significantly extend the treatment period such that several days (e.g., 2, 3, 4, 5, 6, or 7 days) to several weeks (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 weeks) elapse between each administration.

[0104] If the immunogenic composition is "A" and a secondary agent such as radiotherapy, chemotherapy or an anti-inflammatory agent is "B", various combinations can be employed: TIFF2025509713000001.tif22159

[0105] In an embodiment, the administration of the immunogenic composition of this embodiment to patients follows the general protocol for the administration of chemotherapy drugs, taking into account the toxicity (if any) of the vector. It is expected that the treatment cycle will be repeated as necessary. It is also contemplated that various standard therapies, as well as surgical intervention, will be applied in combination with the described hyperproliferative cell therapy.

[0106] a.Chemotherapy Cancer treatment also includes various combination therapies.In some aspects, the immunogenic composition of the embodiment is administered (or formulated) with chemotherapeutic agent.For example, in some aspects, the chemotherapeutic agent is a protein kinase inhibitor such as EGFR, VEGFR, AKT, Erb1, Erb2, ErbB, Syk, Bcr-Abl, JAK, Src, GSK-3, PI3K, Ras, Raf, MAPK, MAPKK, mTOR, c-Kit, eph receptor or BRAF inhibitor. Non-limiting examples of protein kinase inhibitors include afatinib, axitinib, bevacizumab, bosutinib, cetuximab, crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab, ruxolitinib, saracatinib, sorafenib, sunitinib, trastuzumab, vandetanib, AP23451, bem These include rafenib, MK-2206, GSK690693, A-443654, VQD-002, miltefosine, perifosine, CAL101, PX-866, LY294002, rapamycin, temsirolimus, everolimus, ridaforolimus, alvocidib, genistein, selumetinib, AZD-6244, vatalanib, P1446A-05, AG-024322, ZD1839, P276-00, GW572016, or mixtures thereof.

[0107] Further combination chemotherapy includes, for example, alkylating agents such as thiotepa and cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including the synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins; and duocarmycins. (including synthetic analogs, KW-2189 and CB1-TM1); ezoucovin; pancratistatin; sarcodictin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, clophosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitroureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma I and calicheamicin omega I1); dynemicins (including dynemicin A); bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, autranycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromicin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, epinephrine, Mitomycins such as sorbicin, idarubicin, marcelomycin, and mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, keramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin, and trimetrexate; fludarabine, 6-mercaptopurine, thiamiprine, and thiamine. Purine analogues such as aguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal drugs such as mitotane and trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; enyluracil; amsacrine; bestravulic acid; bisantrene; eda Traxat;Defoamin;Demecolcine;Diaziquone;Elformitin;Elliptinium acetate;Epothilone;Etoglucide;Gallium nitrate;Hydroxyurea;Lentinan;Lonidynin;Maytansinoids such as maytansine and ansamitocins;Mitoguazone;Mitoxantrone;Mopidammol;Nitraerin;Pentostatin;Fenamet;Pirarubicin;Rosoxantrone;Podophyllic acid;2-Ethylhydrazide;Procarbazine;PSK polysaccharide complex;Razoxane;Rhizoxin;Schizofiran;Spirogermanium;Tenuazonic acid;Triazicon;2,2',2"-trichlorotriethylamine;trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidin);urethane;vindesine;dacarbazine;mannomustine;mitobronitol;mitolactol;pipobroman;gacitosine;arabinosides ("Ara-C");cyclophosphamide;taxoids, such as paclitaxel and docetaxel gemcitabine;6-thioguanine;mercaptopurine;platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin;vinblastine;platinum;etoposide (VP-16);ifosphatase amide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethyllornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl protein tansferase inhibitors, transplatinum, and pharma- ceutical acceptable salts, acids, or derivatives of any of the above. In certain embodiments, the compositions provided herein may be used in combination with gefitinib. In other embodiments, the present embodiments may be practiced in combination with Glivac (e.g., about 400 to about 800 mg / day of Glivac may be administered to the patient). In certain embodiments, one or more chemotherapeutic agents may be used in combination with the compositions provided herein.;

[0108] B. Radiation therapy Other agents that cause DNA damage and are widely used include what are commonly known as gamma radiation, X-rays, and / or direct delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents such as microwave and UV radiation are also contemplated. All of these agents most likely cause widespread damage to DNA, the precursors of DNA, DNA replication and repair, and chromosome assembly and maintenance. X-ray dosage ranges from daily doses of 50-200 roentgens over an extended period (3-4 weeks) to single doses of 2000-6000 roentgens. Dosage ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by tumor cells.

[0109] The terms "contacted" and "exposed," when applied to a cell, are used herein to describe the process by which a therapeutic composition and a chemotherapeutic or radiotherapeutic agent are delivered to or directly juxtaposed with a target cell. To achieve cell death or stasis, both agents are delivered to the cell in a combined amount effective to kill the cell or prevent it from dividing.

[0110] c gene therapy In yet another embodiment, the secondary treatment is gene therapy, in which a therapeutic polynucleotide is administered before, after, or simultaneously with the therapeutic composition. Viral vectors for expression of gene products are well known in the art and include eukaryotic expression systems such as adenoviruses, adeno-associated viruses, retroviruses, herpes viruses, lentiviruses, poxviruses including vaccinia viruses, and papilloma viruses including SV40. Alternatively, administration of the expression construct can be achieved using lipid-based vectors such as liposomes or DOTAP:cholesterol vesicles. All of these methods are well known in the art (see, e.g., Sambrook et al., 1989; Ausubel et al. 1998; Ausubel, 1996).

[0111] d.Surgery Approximately 60% of cancer patients undergo some type of surgery, including preventative, diagnostic or staging, curative and palliative surgery. Curative surgery is a cancer treatment that can be used in conjunction with other therapies, such as the treatments provided herein, chemotherapy, radiation therapy, hormonal therapy, gene therapy, immunotherapy and / or alternative therapies.

[0112] Curative surgery includes resection, where all or part of the cancerous tissue is physically removed, excised, and / or destroyed. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and misoscopically controlled surgery (Mohs surgery). It is further contemplated that the present embodiments can be used in conjunction with the removal of superficial cancer, pre-cancer, or incidental amounts of normal tissue. In some aspects, tumor resection is performed after the immunogenic composition of the embodiments is administered. In some aspects, tumor resection is performed 2, 3, 4, or 5 weeks after the tumor is treated with the immunogenic composition of the present invention. EXAMPLES

[0113] IV. Working Examples The following examples are included to demonstrate preferred embodiments of the present invention. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the present invention, and therefore can be considered to constitute preferred modes for its practice. However, those skilled in the art should understand in light of this disclosure that many changes can be made in the specific embodiments disclosed and still obtain similar or similar results without departing from the spirit and scope of the present invention.

[0114] Example 1 - Multifactorial immunomodulation for antitumor immunity Empirical generation of optimized antitumor combination therapy. The literature describes a vast number of signaling pathways and mechanisms, the agonism or antagonism of which may modulate the immune system and affect the growth of various tumors. However, the combination therapy with clodronate (M2 macrophage depletion), epacadostat (indoleamine 2,3-dioxygenase inhibition), Nω-hydroxy-nor-L-arginine (selective arginase inhibition) and LN acetate is well-established. G Administration of a number of compounds reported to eliminate or inhibit key components of tumor-associated immune suppression, including l-monomethylarginine (L-NMMA, nitric oxide synthase inhibitor), showed no beneficial effect on tumor growth when injected intratumorally in the 4T-1 model of TNBC (designated as schemes 2-4 in Figures 7A-C). Furthermore, combinations including clodronate resulted in an observable acceleration of tumor growth 9 days after administration into established tumors (also Figures 7A-C). These results were somewhat surprising given that the importance of both M2 macrophages and MDSCs to tumorigenesis in the 4T-1 model system has been well characterized (Hamilton, Bosiljcic et al., 2014, Simpson, Templeton et al., 2012, Sinha, Clements et al., 2007, Sinha, Clements et al., 2005a, Sinha, Clements et al., 2005b). Another approach using a combination of immune agonist agents yielded slightly better results (designated Regimen 5-7 in Figure 8A-8C). Although single-agent administration of individual PRR agonists did not significantly slow tumor growth, a combination of agonists stimulating each of the three major intracellular PRR signaling pathways (STING, TLR, and RLR) (Krieg, 2007; Zhang, Wang et al., 2019; Zhao, Du et al., 2019) reduced tumor size by 50% 9 days after intratumoral administration into established tumors (also Figure 8A-8C). Although these results were promising, the antitumor effects observed at day 21 were not maintained at later time points, and no long-term benefit was observed from these treatment regimens.

[0115] Tumors are believed to evade immune recognition through a variety of mechanisms related to the lack of critical danger signals, as well as active immunosuppression through recruitment of MDSCs, polarization of macrophages, and expression of immunosuppressive ligands and cytokines (Beatty & Gladney, 2015; Vinay, Ryan et al., 2015). It was therefore reasoned that a viable tumor therapy would need to address most of these mechanisms and would also need to use some sort of cytocidal agent to induce cell death, thereby generating the necessary antigens that might prime the subsequent adaptive immune response (Galluzzi, Buque et al., 2017; Yatim, Cullen et al., 2017). To this end, three cocktails of different reagents were created, each corresponding to one of three broad categories of MoA (providing danger, inhibiting immunosuppression, cytostatic / cytotoxic), and these cocktails were combined in a variety of different ways (Table 1) prior to treatment of established 4T-1 tumors (designated as plans 8–14 in Table 1). This rational, empirical process identified four different combinations of drugs (designs 10, 12, 13, and 14) that significantly reduced 4T-1 tumor cell luciferase expression 2 weeks after administration (Figure 1A-B), but not the parental 4T-1luc2 neg When using the luc2 cell line, only two treatment combinations (regimen 12 and 13) were identified that still significantly reduced tumor size 4 weeks after treatment (Figure 1C-D). negThe use of the parenteral was important to validate that an immune response could be generated against the native 4T-1 antigen as well as the heterologous luciferase antigen. Of these two formulations, only one (Plan 13) had 100% of the cohort members still alive at 6 weeks post-treatment (Figure 1E). This combination of reagents, consisting of the three PRR agonists identified in Figure 8, plus a 4-1BB agonist, (Chester, Sanmamed et al., 2018) an adenosine A3 receptor agonist (Jafari, Panjehpour et al., 2017), and a proteasome inhibitor alone, did not have any immunosuppressive inhibitors, consistent with previous data (Mitchell, 2003, Skrott & Cvek, 2012).

[0116] The inclusion of 4-1BBL in the most effective regimen was unexpected given the data in Figure 8, which show that the presence of 4-1BBL antagonizes the antitumor effects of TLR, STRING, and RLH agents. Furthermore, 4-1BB agonism has been shown to be toxic at therapeutic doses in preclinical and early-stage human clinical trials (Lee, Salek-Ardakani et al., 2009; Segal, Logan et al., 2017). Thus, the 4-1BB agonist was removed from regimen 13 (subsequently designated regimen 15, i.e., 15 = 13 without the addition of 4-1BBL) in an attempt to reproduce comparable efficacy by alternative rational modifications. Repeated dosing regimens were attempted, demonstrating greater efficacy with three doses than two and with two doses than one (Figures 2A-B). However, three doses of regimen 15 could not reproduce the efficacy of the original regimen 13 formulation with 4-1BBL. TGF-β is one of the most important immunosuppressive cytokines in the tumor microenvironment and is known to mediate tumor escape from immune surveillance (Batlle & Massague, 2019). Inhibition of TGF-β signaling has previously been shown to be active against TNBC in mouse xenograft models (Bhola, Balko et al., 2013) and has also been shown to have clinical benefit in early-phase human trials (Rodon, Carducci et al., 2015). Given these observations, we next compared a single dose of a formulation lacking 4-1BB agonism (Regimen 15) to a single dose of a regimen containing the TGF-β kinase signaling inhibitor galunisertib (Regimen 16). This modification resulted in a substantial increase in efficacy (Figure 2C).

[0117] Finally, a boosting regimen was combined with regimen 16 and a dose-response analysis was performed at concentrations of 1x, 0.5x, 0.125x, 0.0625x, and 0.03125x. As shown in Figures 2D-E, both the 1x and 0.5x doses of regimen 16 resulted in significant inhibition of tumor growth, resulting in sustained cures in 50% of mice at the 1x dose level and 25% of mice at the 0.5x dose level. The 0.125x and 0.0625x doses resulted in intermediate levels of tumor control, while the 0.03125x dose produced an effect indistinguishable from that of the vehicle control. Similar to the experiment outlined in Figures 1C-D, this experiment used 4T-1luc2 to verify that the protective immune response was not dependent on the presence of heterologous luciferase. neg This was done using the parental cell line. The therapeutic model was then validated in an orthotopic setting (Zhang et al., 2019). In this set of experiments, 500,000 4T-1luc-2 + Cells were implanted under the fourth nipple in the mammary fat pad. Seven days after implantation, animals were randomized to be treated intratumorally with vehicle control, galnesitib at a concentration equal to that used in the optimized regimen, galnesitib at a concentration three times that used in the optimized regimen, or the full optimized regimen 16 itself. As shown in Figure 9 and Figure 2F, treatment regimen 16 produced results in the orthotopic setting identical to those observed ectopically. Furthermore, galnesitib alone was shown to have no effect on tumor growth. Treatment regimen 16 (components listed in Figure 2C), given intratumorally as a three-dose regimen, is considered the final optimized regimen for subsequent characterization in multiple model systems in the future.

[0118] Impact of the optimized treatment regimen on the 4T-1TNBC tumor microenvironment. Next, it was sought to characterize the impact of the optimized treatment regimen on the immune system and tumor microenvironment. Established 4T-1 tumors were treated with optimized regimen 16, re-treated 3 days later, and analyzed an additional 3 days after the second treatment. Animals were bled retro-orbitally and optimized regimen 16 was compared to vehicle control, as well as to the suboptimal regimens 12-15. Compared to vehicle control, the optimized regimen significantly increased circulating CD8 +Regimen 16 induced a 7-fold increase in circulating CD8 T cells, whereas the next best regimens (13 and 15) induced only a 3-4-fold increase (Figure 3A), and the other regimens were completely ineffective. + The increase in the number of CD8 + IFN-γ + This was accompanied by a significant 2.5-fold increase in circulating exhausted CD4 + and virtually eliminated regulatory T cells (Figures 3C-3D). Results were similar within the tumor microenvironment, with Plan 16 demonstrating the highest number of tumor-infiltrating CD8 + Induce tumor-infiltrating exhausted CD4 cells + Circulating PBMC from mice receiving optimized regimen 16 also significantly reduced CD8 + (Figure 3E to Figure 3G) and CD4 + (Figure 3H-J) In both compartments, CD44 + CD127 + They showed a significant and prolonged proliferation of T cells, indicating the development of a sustained T cell memory response (Boettler, Panther et al., 2006; Huster, Busch et al., 2004). The expression of this memory phenotype was observed in the CD4 + CD25 + Foxp3 + Regulatory T cells and PD-1 + CD4 + This was expected by the decline of exhausted T cells in both the tumor microenvironment and peripheral circulation (Boettler et al., 2006; Liu, Putnam et al., 2006). Comparison of the myeloid cell compartment between the optimized treatment and vehicle control also yielded significant results in the resolution of 4T-1 tumors. In this analysis, CD11c + CD80 + CD86 + Tumor-infiltrating dendritic cells and CD11b + CD68 + CD80 + CD86 +There was a significant increase in M1 macrophages and CD11b + Gr-1 + Arginase + The MDSC population, defined as MDSC+, was significantly reduced (Figures 11A to 11C).

[0119] Effect of the optimized treatment regimen on memory development. To verify the presence of persistent memory, mice were implanted with 4T-1 tumors and treated with the optimized regimen 16 on days 12, 16, and 20 after implantation. Thirty days later (50 days after implantation), spleen cells were harvested from treated mice and adoptively transferred into the naïve cohort. The control group was adoptively transferred with spleen cells harvested from tumor-inoculated mice that had been injected intratumorally with vehicle control alone. Three days later, mice were injected intraperitoneally with 500,000 4T-1-luc2 cells, and both cohorts were imaged 7 days later. As shown in Figure 4A-B, mice adoptively transferred with cells derived from treated animals were able to eliminate a large intraperitoneal bolus of tumor. Meanwhile, mice adoptively transferred with naïve spleen cells grew large abdominal masses. The ability of antitumor immunity to be adoptively transferred from the T cell spleen cell population suggested the possibility of an abscopal effect within treated animals. To determine whether treatment with the optimized tumor formulation was able to confer an abscopal effect, mice were treated with 'primary' luc2 neg 4T-1 tumors and "secondary" luc2 + This experimental design was important to demonstrate that the immune response controlling the growth of untreated metastases was derived from the native 4T-1 antigen and not against the xenogeneic luciferase antigen. Furthermore, the luc2 antigen, which acts as a metastatic nidus, was not induced by the luc2 antigen. neg The implantation of tumors allowed the experimental dynamics to proceed with uniform temporal kinetics in all mice, without relying on the randomness of spontaneous metastatic spread. neg Tumors were treated with the optimized treatment regimen on days 13, 17, and 20 after implantation. + The "metastases" of primary luc2 were imaged at baseline on day 13 and again on day 23. As shown in Figure 4C-E, primary luc2 neg Among tumor-treated mice, untreated luc2+ Lesions were luc2 in mice treated with vehicle control primary tumors. + Compared to the lesion, it showed a six-fold reduction in size, indicating the production of a demonstrable abscopal effect.

[0120] Impact of the optimized regimen in other hard-to-treat model systems. To determine whether the optimized treatment regimen could be applied to hard-to-treat or non-immunogenic model systems other than TNBC, a series of experiments was repeated in a cohort of animals subcutaneously implanted with the aggressive HNSCC MOC-2 cell line. As shown in Figure 5A and Figure 11, treatment of MOC-2 HNSCC with optimized regimen 16 resulted in a dramatic reduction in the volume or complete disappearance of these tumors. In subsequent experiments, animals were treated twice with vehicle or regimen 16, after which tumors were excised, digested, and analyzed by flow cytometry to measure the impact of regimen 16 on the tumor microenvironment. In these experiments, CD8 + A massive influx of T cells was observed (Figures 5B-5D). Moreover, all major populations of MDSCs were substantially reduced (Figures 5E-5G), as were tumor-associated macrophages, which were virtually eliminated among tumors treated with Regimen 16 (Figures 5H-5J). The optimized antitumor regimen resulted in the elimination of all CD45 T cells co-expressing PD-L1. + It also induced a three-fold decrease in cell number (Fig. 5K).

[0121] The study was then transferred to the second most difficult-to-treat model system, high-risk MYCN-amplified neuroblastoma. Single cell suspensions were generated from 7-week-old Th-MYCN homozygous tumors and ectopically implanted into the flanks of syngeneic wild-type mice. Tumors were then treated with three consecutive injections of Plan 16 or vehicle control, and growth was monitored by caliper measurements. As shown in Figure 6A, treatment with Plan 16 was sufficient to completely eliminate MYCN-amplified neuroblastoma, with total CD8+ cells expressing MYCN-amplified neuroblastoma several weeks after tumor resolution. +Extensive systemic proliferation of peripheral blood spleen cells was observed (i.e., Figure 6B-6C). Similar to the HNSCC MOC-2 model, subsequent experiments were performed, treating tumors with two doses of Plan 16 or vehicle, and then analyzing the microenvironment of the resolved tumors. In contrast to the MOC-2 model, in which the myeloid cell compartment was substantially resolved after two treatments, the resolution of the myeloid cell compartment in the Th-MYCN model was slower in time, providing an opportunity to observe and characterize the resolution process of myeloid cells in response to treatment with Plan 16. Importantly, characterization of this intermediate stage indicates that myeloid cells first dedifferentiate from their pro-tumorigenic and immunosuppressive phenotypes before resolution. CD11c + , CD11b + , F4 / 80 + , Gr-1 + , or Ly6C + No significant differences in the number of cells were observed between treated and mock-treated tumor cell populations (not shown), but F4 / 80 + (Figure 6D to Figure 6E) and CD11b + In both cases (Figure 6F-G), macrophage populations stopped expressing arginase, which is an immunosuppressant T H 2 phenotype. + The APC population stopped expressing both PD-L1 (Figures 6H-I) and CTLA-4 (Figures 6J-K), again indicating a transition from a tolerogenic to an inflammatory phenotype.

[0122] In this study, a formulation composed of a natural intracellular signaling ligand, a cytocidal / cytostatic drug, and a TGF-β signaling inhibitor was shown to produce highly effective antitumor responses in a TNBC model system that is generally considered non-immunogenic, as well as in other difficult-to-treat model systems, including MYCN-amplified neuroblastoma and MOC-2 HNSCC. In an aggressive 4T-1 TNBC model, treatment effectively prevented metastasis, cured primary tumors in 50% of animals receiving the highest dose, and clearly involved the generation of cellular memory as determined by adoptive transfer experiments. The components of the formulation were derived from a rational and empirical development process aimed at optimizing synergy between various agents that function through three broad mechanisms believed to be important in the generation of antitumor immunity. These three mechanisms are 1) upregulating interferon signaling and inhibiting T H1) providing danger signals via ligation of intracellular pattern recognition receptors that promote immune responses (Carroll, Jin et al., 2016; Kawai & Akira, 2009; Shi, Vistica et al., 2013; Spranger, Javorovic et al., 2010; Stetson & Medzhitov, 2006; Sun, Wu et al., 2013; Xagorari & Chlichlia, 2008); 2) inhibiting perhaps the most important mediator of tumor-associated immune suppression (i.e., TGF-β) (Beatty & Gladney, 2015; Sinha et al., 2005a; Sinha et al., 2005b; Vinay et al., 2015); and 3) endogenous DAMP signaling (Galluzzi et al., 2017; Yatim et al., 2016). and the introduction of cytocidal agents to generate cytotoxicity (e.g., 2017) and, through cellular damage, provide antigenic material from which downstream adaptive immune responses are primed. The literature suggests a very wide variety of different agents that may be used to achieve these broad goals, but how the groups of individual agents interact could not have been predicted prior to the critical proof-of-concept experiments documented here. Indeed, certain combinations of promising agents conferred no antitumor effect whatsoever. This seemed especially true for formulations combining agents aimed at inhibiting multiple mechanisms of tumor-associated immune suppression, at least in the non-immunogenic 4T-1 TNBC model system in which optimization was performed. Given the recent high-profile failure of the IDO inhibitor epacadostat in a randomized Phase III trial in combination with pembrolizumab, these observations were perhaps not entirely unpredictable (Garber, 2018). The broad applicability of the optimized scheme to TNBC was subsequently validated in the Th-MYCN and MOC-2 HNSCC model systems.

[0123] It is known that successful clinical responses to anti-PD-1 checkpoint blockade are significantly correlated with tumor mutational burden (Cristecu et al., 2018; Mandal et al., 2019). It is believed that a higher number of coding mutations increases the likelihood of sufficient generation of high-affinity T cell epitopes from which productive T cell responses can be generated spontaneously. However, in cancer patients, this process occurs under steady-state homeostatic conditions or in the context of cell damage and DAMP release after cytotoxic chemotherapy and radiotherapy. In contrast, combining multiple PRR signaling ligands with inhibition of immunosuppressive TGF-β signaling initiates interferon signaling, which subsequently mediates increased expression of MHC, upregulation of the immunoproteasome, maturation of myeloid DCs, activation of plasmacytoid DCs, and many other immune-enhancing effects not usually induced by standard-of-care chemotherapy or radiotherapy, altering homeostasis during treatment with optimized regimens (Hervas-Stubbs et al., 2011). These effects may shift the balance between tolerance and immunity, allowing the generation of T cell responses to high affinity neoepitopes, but also the persistence of responses to intermediate and low affinity T cell clones that may be reactive not only to neoepitopes, but also to upregulated developmental and differentiation antigens, as well as post-translational modifications that are relatively specific to tumors (Ilyas et al., 2015).

[0124] Characterization of the microenvironment of resolved tumors in all three model systems provided an intriguing window into at least one of the mechanisms by which optimized treatment regimens generate substantial antitumor effects. In all model systems, mid-treatment analysis of MDSCs showed either a substantial reduction in myeloid cell mass or a snapshot in time capturing the dedifferentiation of MDSCs as they downregulate immunosuppressive markers such as PD-L1. The same was true for M2 macrophages, where arginase expression was reduced or lost, indicative of a conversion to an M1 inflammatory phenotype. CD11c +Cells were similarly affected, with downregulation of both CDLA-4 and PD-L1 expression levels, and changes observed in various myeloid compartments included the upregulation of CD8 + Influx of cells, plus circulating CD8 + This was accompanied by a systemic upregulation of cells.

[0125] In summary, this study demonstrated that a unique combination of immunomodulatory and cytostatic agents, designed and optimized through both rational and empirical approaches, realigns the tumor microenvironment and improves poor prognosis tumor types, including the 4T-1 TNBC model, which is generally considered non-immunogenic (Brockstedt et al., 2002; Feola et al., 2018; Liu et al., 2018), as well as difficult-to-treat HNSCC and high-risk neuroblastoma models. The course of treatment optimized for 4T-1 TNBC resulted in a 50% complete response rate and eliminated metastatic spread in all animals tested at the highest dose. Results were similar in the HNSCC model, but more robust in high-risk neuroblastoma, where all tumors resolved after treatment. Immune responses were transferable between treatment recipients and naive donors through adoptive transfer, and a substantial abscopal effect was also demonstrated. The results demonstrate that targeting multiple immune stimulatory and immune suppressive pathways can result in dramatic synergistic effects.

[0126] Example 2 - Materials and Methods Mice. 6-10 week old female Balb / c mice or C57BL / 6 animals were obtained from Baylor College of Medicine or Jackson Laboratory (Bar Harbor, ME). Th-MYCN mice on a 129X1 / SvJ background were a kind gift from Dr. William Weiss (UCSF, San Francisco, CA) and were bred in-house. All mice were maintained according to specific IACUC requirements at Baylor College of Medicine and in accordance with animal protocol AN-8375.

[0127] Tumor implantation and measurement. 500,000 4T-1TNBC cells (+ / -luc2 transduced) were injected subcutaneously into the hind limb / back of each mouse and palpated (approximately 50–100 mm) before treatment. 3 For orthotopic 4T-1 experiments, 0.5 million 4T-1 cells were transplanted into the mammary fat pad according to the method of Zhang et al. 34 After tumor establishment, experimental cohorts were assembled by stratified randomization. Simple randomization attempted to minimize confounding variables such as order of cohort treatment, order of tumor measurements, and cage position. Tumors were treated with either the indicated antitumor regimen or vehicle control and were measured by IVIS imaging (if applicable) or calculated by caliper measurements. 100,000 MOC-2 HNSCC cells were injected subcutaneously into the hind limb / back of each mouse and were palpable (approximately 50–100 mm) prior to treatment. 3 ) Tumor size and progression were determined by caliper measurements. Single-cell suspensions were prepared from Th-MYCN tumors according to the method of Kroesen et al. 12 The suspension was injected subcutaneously into the hind leg / back of each mouse and palpated (approximately 50–100 mm) before treatment. 3 Tumor size and progression were determined by caliper measurements. Tumor volume was calculated according to the formula (longest diameter x y). 2 ) x Calculated by measuring in two dimensions along the shortest diameter.

[0128] IVIS imaging. After tumor implantation, mice were injected with 100 μl of 10 mg / ml D-luciferin (Regis Technologies, Morton Grove, IL), incubated for 7 min, and bioluminescence was measured after 60 s of illumination using an IVIS imaging system (Caliper Life Sciences, Waltham, MA). Imaging was performed at 2–4 day intervals.

[0129] Analysis of the tumor microenvironment. Tumor microenvironment leukocytes were analyzed using a mouse tumor dissociation kit and dissociation agent from Miltenyi Biotec (Gaithersburg, MD) according to the manufacturer's instructions. Briefly, tumors were excised, cut into smaller 2-4 mm fragments, and placed into individual GentleMACS C tubes containing 2.5 ml of enzyme mixture (2.35 ml RPMI-1640, 100 μl Enzyme D, 50 μl Enzyme R, and 12.5 μl Enzyme A). The tubes were then inverted and secured onto a heated GentleMACS Octo dissociator, and the appropriate program was run to disaggregate the tumor into a single cell suspension. After program completion, the suspension was filtered through a 70 nm cell filter to remove remaining debris, and cells were pelleted, washed twice with PBS, and then stained for analysis by flow cytometry. Myeloid cell markers analyzed included Gr-1, Ly6C, CTLA-4, PD-L1, CD11b, CD11c, F4 / 80, CD45, CD68, CD80, CD86, intracellular arginase, intracellular TGF-β, and / or intracellular IL-12.

[0130] Analysis of circulating PBMCs. Mice were bled retro-orbitally at designated intervals with samples appropriately mixed with EDTA to prevent clotting. Erythrocytes were lysed by treatment with ammonium chloride (Sigma-Aldrich) according to the manufacturer's instructions. Leukocyte pellets were washed once with PBS, resuspended in 1% PBS, 0.01% normal goat serum + sodium azide, and kept at 4°C for staining. Cells were then stained for surface CD45, CD3, CD8, CD4, CD25, CTLA-4, PD-1, CD44, CD127, intracellular Foxp3, and / or IFN-γ.

[0131] Antitumor Reagents. For optimization in the TNBC model, all reagents were dissolved in DMSO or sterile water based on solubility requirements and were diluted with 100 mM NaCl, 0.1% NaCl, 0.1% DMSO, or 0.1% DMSO. For optimization in the TNBC model, all reagents were dissolved in DMSO or sterile water based on solubility requirements and diluted with 100 mM NaCl, 0.1% DMSO, or 0.1% DMSO. For optimization in the TNBC model, all reagents were dissolved in DMSO or sterile water based on solubility requirements and were diluted with 100 mM NaCl, 0.1% DMSO, or 0.1% DMSO. 375ng / 50μl 5'ppp-dsRNA / Lyovec (Invivogen, San Diego, CA), 15μg / 50μl R848 (Invivogen), 30μg / 50μl galunisertib (Selleck Chemicals, Houston, TX), 5μg / 50μl epicadostat (Selleck Chemicals), 6μg / 50μl N w The 100-ng / 50μl 4-hydroxy-nor-L-arginine (Sigma-Aldrich, St. Louis, MO), 100ng / 50μl L-NMMA (Abcam, Boston, MA), 80μg / 50μl clodronate (Abcam), and 1μg / 50μl 4-1BB ligand (Novas Bio). Combination treatments were reconstituted as 10x stocks, combined as needed, and diluted to 1x concentration in the appropriate diluent. Treatments were injected intratumorally as a 50μl bolus using a 28-gauge needle. In HNSCC and neuroblastoma models, the mouse-specific STING agonist DMXAA replaced the broader species-reactive STING agonist 2'3'-c-di-AM(PS)2(Rp,Rp) at 715ng / 50μl. Other reagent concentrations in the HNSCC and neuroblastoma models included copper(II) diethyldithiocarbamate at 172 μg / 50 μl, 2-C1-IB-MECA at 40 μg / 50 μl, 5′ppp-dsRNA / Lyovec at 755 ng / 50 μl, R848 at 30 μg / 50 μl, and galunisertib at 60 μg / 50 μl.

[0132] Adoptive transfer. Spleens were harvested, physically dissociated, and made into a single cell suspension by passing through a 70 μM cell strainer (Corning, Glendale, AZ). After lysing red blood cells with ammonium chloride (Thermo Fisher Scientific, Waltham, MA), leukocytes were washed, pelleted, and resuspended in PBS before being injected intraperitoneally into naive recipient mice. After 48 hours, adoptively transferred animals were injected intraperitoneally with 500,000 4T1-luc2 tumor cells followed by IVIS imaging.

[0133] Flow cytometry analysis. All flow cytometry analyses were performed using an LSRII flow cytometer (BD Biosciences) and analyzed with FlowJo version 10.0.00003 for MacIntosh (Tree Star, Ashland, OR).

[0134] Statistical analysis. Significant differences were determined by one-way analysis of variance (ANOVA) with Tukey's honestly significant difference (HSD) post-hoc test unless otherwise stated. Pairwise comparisons were performed by Student's two-tailed t-test. Kaplan-Meier survival significance was determined by the log-rank (Mantel-Cox) test. All data are presented as mean ± SEM or SD, as stated. All analyses were performed using Prism software version 8.3 for iOS (GraphPad Software Inc.) or Microsoft Excel version 16.30 for Mac. Statistical significance was defined as p ≤ 0.05. Power analysis showed that experimental differences as small as + / - 36% of μ were statistically significant with a sample size of n = 5, assuming σ = 20% of μ, α = 0.05, and a power of 0.8. Therefore, we typically used 5 mice per each cohort unless otherwise stated. Normality and variance of the data were assessed graphically.

[0135] (Table 1) TIFF2025509713000002.tif229132TIFF2025509713000003.tif23275

[0136] (Table 2) TIFF2025509713000004.tif24256

[0137] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes may be applied to the methods, and to the steps or sequence of steps of the methods described herein, without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be obtained. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0138] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF2025509713000005.tif187146TIFF2025509713000006.tif230146TIFF2025509713000007.tif238146TIFF2025509713 000008.tif245146TIFF2025509713000009.tif231146TIFF2025509713000010.tif245146TIFF2025509713000011.tif4128

Claims

1. An immunogenic composition comprising at least four of a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, an adenosine A3 receptor agonist, and a 4-1BB agonist.

2. 10. The composition of claim 1, comprising a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and a proteasome inhibitor.

3. 10. The composition of claim 1, comprising a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and an adenosine A3 receptor agonist.

4. 10. The composition of claim 1, comprising a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and a proteasome inhibitor.

5. 2. The composition of claim 1, comprising a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and an adenosine A3 receptor agonist.

6. 2. The composition of claim 1, comprising at least five of a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

7. 2. The composition of claim 1, comprising at least five of a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

8. 10. The composition of claim 1, comprising a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

9. 10. The composition of claim 1, comprising a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

10. 10. The composition of any one of claims 1 to 9, wherein the TGFβ antagonist is galunisertib (LY2157299), travedersen, fresolimumab, LY2382770, lucanix, or PF-03446962.

11. The composition of any one of claims 1 to 10, wherein the STING agonist is a cyclic dinucleotide or a xanthenone analog.

12. The composition of any one of claims 1 to 11, wherein the STING agonist is 2'3'-c-di-AM(PS)2(Rp,Rp).

13. The composition of claim 11, wherein the STING agonist is a cyclic dinucleotide selected from the group consisting of 3'3'-cGAMP, 2'3'-cGAMP, 2'2'-cGAMP, c-di-APM, c-di-GMP, c-di-IMP, and c-di-UMP.

14. The composition of claim 13 , wherein the STING agonist is a xanthenone analog.

15. The composition of claim 14, wherein the xanthenone analog is 5,6-dimethylxanthenone-4-acetic acid (DMXAA).

16. 16. The composition of any one of claims 1 to 15, wherein the TLR7 / 8 agonist is resiquimod (R848), CL075, CL097, CL264, CL307, GS-9620, poly(dT), imiquimod, gardquimod, loxoribine, or a ssRNA oligonucleotide.

17. The composition of any one of claims 1 to 16, wherein the RIG-I agonist is 5'ppp-dsRNA, MDA5 ligand, LGP2 ligand, ssRNA, dsRNA, poly(dA:dT), or poly(I:C).

18. 18. The composition of any one of claims 1 to 17, wherein the proteasome inhibitor is copper(II) diethyldithiocarbamate, bortezomib, delanzomib, ixazomib, MG132, MG115, IPSI001, felutamide B, ALLN, leupeptin, epoxomicin, oprozomib, PR-957, carfilzomib, lactacystin, omuralide, salinosporamide A, salinosporamide B, belactosin, cinnabalamide, polyphenol, TMC-95, or PS-519.

19. 19. The composition of any one of claims 1 to 18, wherein the adenosine A3 receptor agonist is 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (2-Cl-IB-MECA), IB-MECA (CF101), CP-608,039, CP-532,903, MRS5698, MRS5980, LJ-52933, or MRS4322.

20. The composition of any one of claims 1 to 19, wherein the 4-1BB agonist is a 4-1BB agonist antibody, a recombinant 4-1BB ligand (4-1BBL), or a 4-1BB apatamer.

21. The composition of claim 20, wherein the 4-1BB agonist antibody is utomirumab or urelumab.

22. The composition of any one of claims 1 to 21, which does not comprise a hydrogel.

23. The composition of any one of claims 1 to 22, which does not comprise an immune checkpoint inhibitor, a cytokine, and / or an antigen.

24. A pharmaceutical composition comprising the immunogenic composition of any one of claims 1 to 23 and an excipient.

25. 1. A method of stimulating an anti-tumor immune response in a subject, comprising administering to the subject effective amounts of at least four of a transforming growth factor beta (TGFβ) antagonist, a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

26. 26. The method of claim 25, wherein the subject is administered a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and a proteasome inhibitor.

27. 26. The method of claim 25, wherein the subject is administered a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and a proteasome inhibitor.

28. 26. The method of claim 25, wherein the subject is administered a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and an adenosine A3 receptor agonist.

29. 26. The method of claim 25, wherein the subject is administered a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and an adenosine A3 receptor agonist.

30. 26. The method of claim 25, wherein the subject is administered at least five of the following: a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

31. 26. The method of claim 25, wherein the subject is administered at least five of the following: a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

32. 26. The method of claim 25, wherein the subject is administered a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

33. 26. The method of claim 25, wherein the subject is administered a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

34. 34. The method of any one of claims 25 to 33, wherein the TGFβ antagonist is galunisertib (LY2157299), travedersen, fresolimumab, LY2382770, lucanix, or PF-03446962.

35. The method of any one of claims 25 to 34, wherein the STING agonist is a cyclic dinucleotide or a xanthenone analog.

36. The method of any one of claims 25 to 35, wherein the STING agonist is 2'3'-c-di-AM(PS)2(Rp,Rp).

37. 36. The method of claim 35, wherein the STING agonist is a cyclic dinucleotide selected from the group consisting of 3'3'-cGAMP, 2'3'-cGAMP, 2'2'-cGAMP, c-di-APM, c-di-GMP, c-di-IMP, and c-di-UMP.

38. 36. The method of claim 35, wherein the STING agonist is a xanthenone analog.

39. 39. The method of claim 38, wherein the xanthenone analog is 5,6-dimethylxanthenone-4-acetic acid (DMXAA).

40. 40. The method of any one of claims 25 to 39, wherein the TLR7 / 8 agonist is resiquimod (R848), CL075, CL097, CL264, CL307, GS-9620, poly(dT), imiquimod, gardquimod, loxoribine, or a ssRNA oligonucleotide.

41. The method of any one of claims 25 to 40, wherein the RIG-I agonist is 5'ppp-dsRNA, MDA5 ligand, LGP2 ligand, ssRNA, dsRNA, poly(dA:dT), or poly(I:C).

42. 42. The method of any one of claims 25 to 41, wherein the proteasome inhibitor is copper(II) diethyldithiocarbamate, bortezomib, delanzomib, ixazomib, MG132, MG115, IPSI001, ferutamide B, ALLN, leupeptin, epoxomicin, oprozomib, PR-957, carfilzomib, lactacystin, omuralide, salinosporamide A, salinosporamide B, belactosin, cinnabalamide, polyphenol, TMC-95, or PS-519.

43. 43. The method of any one of claims 25 to 42, wherein the adenosine A3 receptor agonist is 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (2-Cl-IB-MECA), IB-MECA (CF101), CP-608,039, CP-532,903, MRS5698, MRS5980, LJ-52933, or MRS4322.

44. The method of any one of claims 25 to 43, wherein the 4-1BB agonist is a 4-1BB agonist antibody, a recombinant 4-1BB ligand (4-1BBL), or a 4-1BB apatamer.

45. The method of claim 44, wherein the 4-1BB agonist antibody is utomirumab or urelumab.

46. The method of any one of claims 25 to 43, wherein the subject is a human.

47. The method of any one of claims 25 to 46, wherein the subject has cancer.

48. 48. The method of any one of claims 25 to 47, wherein the cancer is triple-negative breast cancer (TNBC), pancreatic ductal adenocarcinoma (PDAC), melanoma, or head and neck cancer.

49. The method according to any one of claims 47 to 48, wherein said administration is performed prior to a surgical intervention.

50. 50. The method of any one of claims 47 to 49, wherein the administration comprises intratumoral injection.

51. 51. The method of claim 50, wherein the intratumoral injection does not include a hydrogel.

52. The method of any one of claims 25 to 51, wherein the immunogenic composition is administered more than once.

53. The method of any one of claims 25 to 52, wherein the immunogenic composition is administered two or more times.

54. The method of any one of claims 47 to 54, wherein administering the immunogenic composition increases circulating CD8+ T cells, M1 macrophages and / or tumor-infiltrating dendritic cells.

55. The CD8+ T cells are CD8+ T cells. ＋ IFN-γ ＋ 55. The method of claim 54, which is a cell.

56. By administering the immunogenic composition, CD44 ＋ CD127 ＋ CD44 compared to T cells ＋ CD127 ＋ 56. The method of any one of claims 25 to 55, which results in a sustained T cell memory response as measured by an expansion of T cells.

57. The method of any one of claims 25 to 56, which does not include administering a cell therapy to the subject at the time the immunogenic composition is administered.

58. 58. The method of any one of claims 25 to 57, which does not include administering to the subject an immune checkpoint inhibitor, a cytokine, and / or an antigen at the time the immunogenic composition is administered.

59. 1. A method of treating a subject having cancer, comprising administering to the subject effective amounts of at least four of a transforming growth factor beta (TGFβ) antagonist, a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

60. 60. The method of claim 59, wherein the subject is administered a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and a proteasome inhibitor.

61. 60. The method of claim 59, wherein the subject is administered a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and a proteasome inhibitor.

62. 60. The method of claim 59, wherein the subject is administered a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and an adenosine A3 receptor agonist.

63. 60. The method of claim 59, wherein the subject is administered a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and an adenosine A3 receptor agonist.

64. 60. The method of claim 59, wherein the subject is administered at least five of the following: a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

65. 60. The method of claim 59, wherein the subject is administered at least five of the following: a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

66. 60. The method of claim 59, wherein the subject is administered a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

67. 60. The method of claim 59, wherein the subject is administered a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

68. 68. The method of any one of claims 59 to 67, wherein the TGFβ antagonist is galunisertib (LY2157299), travedersen, fresolimumab, LY2382770, lucanix, or PF-03446962.

69. The method of any one of claims 59 to 68, wherein the STING agonist is a cyclic dinucleotide or a xanthenone analog.

70. The method of any one of claims 59 to 69, wherein the STING agonist is 2'3'-c-di-AM(PS)2(Rp,Rp).

71. 70. The method of claim 69, wherein the STING agonist is a cyclic dinucleotide selected from the group consisting of 3'3'-cGAMP, 2'3'-cGAMP, 2'2'-cGAMP, c-di-APM, c-di-GMP, c-di-IMP, and c-di-UMP.

72. 70. The method of claim 69, wherein the STING agonist is a xanthenone analog.

73. 73. The method of claim 72, wherein the xanthenone analog is 5,6-dimethylxanthenone-4-acetic acid (DMXAA).

74. 74. The method of any one of claims 59 to 73, wherein the TLR7 / 8 agonist is resiquimod (R848), CL075, CL097, CL264, CL307, GS-9620, poly(dT), imiquimod, gardquimod, loxoribine, or a ssRNA oligonucleotide.

75. 75. The method of any one of claims 59 to 74, wherein the RIG-I agonist is 5'ppp-dsRNA, MDA5 ligand, LGP2 ligand, ssRNA, dsRNA, poly(dA:dT), or poly(I:C).

76. 76. The method of any one of claims 59 to 75, wherein the proteasome inhibitor is copper(II) diethyldithiocarbamate, bortezomib, delanzomib, ixazomib, MG132, MG115, IPSI001, felutamide B, ALLN, leupeptin, epoxomicin, oprozomib, PR-957, carfilzomib, lactacystin, omuralide, salinosporamide A, salinosporamide B, belactosin, cinnabalamide, polyphenol, TMC-95, or PS-519.

77. 77. The method of any one of claims 59 to 76, wherein the adenosine A3 receptor agonist is 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (2-Cl-IB-MECA), IB-MECA (CF101), CP-608,039, CP-532,903, MRS5698, MRS5980, LJ-52933, or MRS4322.

78. The method of any one of claims 59 to 77, wherein the 4-1BB agonist is a 4-1BB agonist antibody, a recombinant 4-1BB ligand (4-1BBL), or a 4-1BB apatamer.

79. The method of claim 78, wherein the 4-1BB agonist antibody is utomirumab or urelumab.

80. 78. The method of any one of claims 59 to 77, wherein said administration occurs prior to a surgical intervention.

81. The method of any one of claims 59 to 80, wherein the subject has not undergone surgical resection of a tumor.

82. The method of any one of claims 59 to 81, wherein administering comprises injection of the immunogenic composition.

83. 83. The method of claim 82, wherein the TGFβ antagonist, STING agonist, TLR7 / 8 agonist, RIG-I agonist, proteasome inhibitor, and adenosine A3 receptor agonist are administered as a single injection.

84. 83. The method of claim 82, wherein the TGFβ antagonist, STING agonist, TLR7 / 8 agonist, RIG-I agonist, proteasome inhibitor, and adenosine A3 receptor agonist are administered as two or more injections.

85. The method of any one of claims 82 to 84, wherein the injection is an intratumoral injection.

86. 87. The method of any one of claims 59-86, wherein the cancer is triple-negative breast cancer (TNBC), PDAC, head and neck cancer, or melanoma.

87. 87. The method of claim 85 or 86, wherein the intratumoral injection does not include a hydrogel.

88. 86. The method of claim 85, wherein the intratumoral injection comprises a hydrogel.

89. 88. The method of any one of claims 59 to 87, wherein the administration is more than one time.

90. 90. The method of any one of claims 59 to 89, wherein the administration is two or more times.

91. The method of any one of claims 42 to 91, wherein the administration increases circulating CD8+ T cells, M1 macrophages and / or tumor-infiltrating dendritic cells.

92. The CD8+ T cells are CD8+ T cells. ＋ IFN-γ ＋ 92. The method of claim 91, which is a cell.

93. By administration, CD44 before administration ＋ CD127 ＋ CD44 compared to T cells ＋ CD127 ＋ 93. The method of any one of claims 59 to 92, wherein a sustained T cell memory response is generated as measured by an expansion of T cells.

94. 95. The method of any one of claims 59-94, which does not include administering a cell therapy to the subject at the time that a transforming growth factor beta (TGFβ) antagonist, a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist are administered.

95. 96. The method of any one of claims 59 to 95, which does not include administering to the subject an immune checkpoint inhibitor, a cytokine, and / or an antigen at the time the transforming growth factor beta (TGFβ) antagonist, the stimulator of interferon genes (STING) agonist, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist, the RIG-I agonist, the proteasome inhibitor, and the adenosine A3 receptor agonist are administered.

96. 96. The method of any one of claims 59 to 95, further comprising administering an additional anti-cancer therapy.

97. 97. The method of claim 96, wherein the additional anti-cancer therapy comprises chemotherapy, radiation therapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy, or immunotherapy.

98. The method of any one of claims 59 to 97, wherein the patient is a human.

99. 99. The method of any one of claims 59-98, wherein the patient has previously been administered an anti-cancer therapy.

100. 1. A method of preventing tumor metastasis in a subject having cancer, comprising administering to the subject effective amounts of at least four of a transforming growth factor beta (TGFβ) antagonist, a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

101. 101. The method of claim 100, wherein the subject is administered a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and a proteasome inhibitor.

102. 101. The method of claim 100, wherein the subject is administered a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and a proteasome inhibitor.

103. 101. The method of claim 100, wherein the subject is administered a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and an adenosine A3 receptor agonist.

104. 101. The method of claim 100, wherein the subject is administered a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and an adenosine A3 receptor agonist.

105. 101. The method of claim 100, wherein the subject is administered at least five of the following: a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

106. 101. The method of claim 100, wherein the subject is administered at least five of the following: a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

107. 101. The method of claim 100, wherein the subject is administered a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

108. 101. The method of claim 100, wherein the subject is administered a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

109. 109. The method of any one of claims 100-108, wherein the TGFβ antagonist is galunisertib (LY2157299), travedersen, fresolimumab, LY2382770, lucanix, or PF-03446962.

110. The method of any one of claims 100 to 109, wherein the STING agonist is a cyclic dinucleotide or a xanthenone analog.

111. The method of any one of claims 100 to 110, wherein the STING agonist is 2'3'-c-di-AM(PS)2(Rp,Rp).

112. The method of claim 110, wherein the STING agonist is a cyclic dinucleotide selected from the group consisting of 3'3'-cGAMP, 2'3'-cGAMP, 2'2'-cGAMP, c-di-APM, c-di-GMP, c-di-IMP, and c-di-UMP.

113. The method of claim 110, wherein the STING agonist is a xanthenone analog.

114. 114. The method of claim 113, wherein the xanthenone analog is 5,6-dimethylxanthenone-4-acetic acid (DMXAA).

115. 115. The method of any one of claims 100 to 114, wherein the TLR7 / 8 agonist is resiquimod (R848), CL075, CL097, CL264, CL307, GS-9620, poly(dT), imiquimod, gardquimod, loxoribine, or a ssRNA oligonucleotide.

116. 116. The method of any one of claims 100 to 115, wherein the RIG-I agonist is 5'ppp-dsRNA, MDA5 ligand, LGP2 ligand, ssRNA, dsRNA, poly(dA:dT), or poly(I:C).

117. 117. The method of any one of claims 100 to 116, wherein the proteasome inhibitor is copper(II) diethyldithiocarbamate, bortezomib, delanzomib, ixazomib, MG132, MG115, IPSI001, ferutamide B, ALLN, leupeptin, epoxomicin, oprozomib, PR-957, carfilzomib, lactacystin, omuralide, salinosporamide A, salinosporamide B, belactosin, cinnabalamide, polyphenol, TMC-95, or PS-519.

118. The method of any one of claims 100 to 117, wherein the adenosine A3 receptor agonist is 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (2-Cl-IB-MECA), IB-MECA (CF101), CP-608,039, CP-532,903, MRS5698, MRS5980, LJ-52933, or MRS4322.

119. The method of any one of claims 100 to 118, wherein the 4-1BB agonist is a 4-1BB agonist antibody, a recombinant 4-1BB ligand (4-1BBL), or a 4-1BB apatamer.

120. The method of claim 119, wherein the 4-1BB agonist antibody is utomirumab or urelumab.

121. 119. The method of any one of claims 100 to 118, wherein said administration occurs prior to a surgical intervention.

122. The method of any one of claims 100 to 121, wherein the subject has not undergone surgical resection of a tumor.

123. The method of any one of claims 100 to 122, wherein the administration comprises injection of the immunogenic composition.

124. 124. The method of claim 123, wherein the TGFβ antagonist, STING agonist, TLR7 / 8 agonist, RIG-I agonist, proteasome inhibitor, and adenosine A3 receptor agonist are administered as a single injection.

125. 124. The method of claim 123, wherein the TGFβ antagonist, STING agonist, TLR7 / 8 agonist, RIG-I agonist, proteasome inhibitor, and adenosine A3 receptor agonist are administered as more than one injection.

126. 126. The method of any one of claims 123 to 125, wherein the injection is an intratumoral injection.

127. 128. The method of any one of claims 100-127, wherein the cancer is triple-negative breast cancer (TNBC), PDAC, head and neck cancer, or melanoma.

128. 128. The method of claim 126 or 127, wherein the intratumoral injection does not include a hydrogel.

129. 129. The method of any one of claims 100 to 128, wherein the administration is more than one time.

130. 130. The method of any one of claims 100 to 129, wherein the administration is two or more times.

131. The method of any one of claims 100 to 131, wherein the administration increases circulating CD8+ T cells, M1 macrophages and / or tumor-infiltrating dendritic cells.

132. The CD8+ T cells are CD8+ T cells. ＋ IFN-γ ＋ The method of claim 131, which is a cell.

133. By administration, CD44 before administration ＋ CD127 ＋ CD44 compared to T cells ＋ CD127 ＋ 133. The method of any one of claims 100-132, wherein a sustained T cell memory response is generated as measured by an expansion of T cells.

134. 135. The method of any one of claims 100-134, which does not include administering a cell therapy to the subject at the time a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist are administered.

135. 136. The method of any one of claims 100 to 135, which does not include administering to the subject an immune checkpoint inhibitor, a cytokine, and / or an antigen at the time the transforming growth factor beta (TGFβ) antagonist, the stimulator of interferon genes (STING) agonist, the Toll-like receptor 7 / 8 (TLR7 / 8) agonist, the RIG-I agonist, the proteasome inhibitor, and the adenosine A3 receptor agonist are administered.

136. 136. The method of any one of claims 100-135, further comprising administering an additional anti-cancer therapy.

137. 137. The method of claim 136, wherein the additional anti-cancer therapy comprises chemotherapy, radiation therapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy, or immunotherapy.

138. The method of any one of claims 100 to 137, wherein the patient is a human.

139. 139. The method of any one of claims 100-138, wherein the patient has previously been administered an anti-cancer therapy.

140. 1. An immunogenic composition comprising at least four of a transforming growth factor beta (TGFβ) antagonist, a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist, for use in treating cancer by administering the composition to a subject.

141. The composition for use according to claim 140, wherein the composition comprises a 4-1BB agonist, a Stimulator of Interferon Genes (STING) agonist, a Toll-like Receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and a proteasome inhibitor.

142. The composition for use according to claim 140, wherein the composition comprises a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and a proteasome inhibitor.

143. The composition for use according to claim 140, wherein the composition comprises a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and an adenosine A3 receptor agonist.

144. The composition for use according to claim 140, wherein the composition comprises a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, and an adenosine A3 receptor agonist.

145. 141. The composition for use of claim 140, wherein the composition comprises at least five of a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

146. The composition for use according to claim 140, wherein the composition comprises at least five of a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

147. The composition for use according to claim 140, wherein the composition comprises a 4-1BB agonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

148. The composition for use according to claim 140, wherein the composition comprises a transforming growth factor beta (TGFβ) antagonist, a stimulator of interferon genes (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

149. 149. The composition for use according to any one of claims 140 to 148, wherein the TGFβ antagonist is galunisertib (LY2157299), travedersen, fresolimumab, LY2382770, lucanix, or PF-03446962.

150. A composition for use according to any one of claims 140 to 149, wherein the STING agonist is a cyclic dinucleotide or a xanthenone analog.

151. A composition for use according to any one of claims 140 to 150, wherein the STING agonist is 2'3'-c-di-AM(PS)2(Rp,Rp).

152. The composition for use of claim 150, wherein the STING agonist is a cyclic dinucleotide selected from the group consisting of 3'3'-cGAMP, 2'3'-cGAMP, 2'2'-cGAMP, c-di-APM, c-di-GMP, c-di-IMP, and c-di-UMP.

153. The composition for use of claim 152, wherein the STING agonist is a xanthenone analog.

154. The composition for use of claim 153, wherein the xanthenone analog is 5,6-dimethylxanthenone-4-acetic acid (DMXAA).

155. 155. The composition for use according to any one of claims 140 to 154, wherein the TLR7 / 8 agonist is resiquimod (R848), CL075, CL097, CL264, CL307, GS-9620, poly(dT), imiquimod, gardquimod, loxoribine, or a ssRNA oligonucleotide.

156. The composition for use according to any one of claims 140 to 155, wherein the RIG-I agonist is 5'ppp-dsRNA, MDA5 ligand, LGP2 ligand, ssRNA, dsRNA, poly(dA:dT), or poly(I:C).

157. 157. The composition for use of any one of claims 140 to 156, wherein the proteasome inhibitor is copper(II) diethyldithiocarbamate, bortezomib, delanzomib, ixazomib, MG132, MG115, IPSI001, ferutamide B, ALLN, leupeptin, epoxomicin, oprozomib, PR-957, carfilzomib, lactacystin, omuralide, salinosporamide A, salinosporamide B, belactosin, cinnabalamide, polyphenol, TMC-95, or PS-519.

158. 158. The composition for use according to any one of claims 140 to 157, wherein the adenosine A3 receptor agonist is 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (2-Cl-IB-MECA), IB-MECA (CF101), CP-608,039, CP-532,903, MRS5698, MRS5980, LJ-52933, or MRS4322.

159. The composition for use according to any one of claims 140 to 158, wherein the 4-1BB agonist is a 4-1BB agonist antibody, a recombinant 4-1BB ligand (4-1BBL), or a 4-1BB apatamer.

160. The composition for use according to claim 159, wherein the 4-1BB agonist antibody is utomirumab or urelumab.

161. The composition for use according to any one of claims 140 to 158, wherein the subject is a human.

162. The composition for use according to any one of claims 140 to 161, wherein the cancer is triple-negative breast cancer (TNBC), PDAC, head and neck cancer, or melanoma.

163. The composition for use according to any one of claims 140 to 162, wherein said composition is administered prior to a surgical intervention.

164. The composition for use according to any one of claims 140 to 163, wherein the composition is administered by intratumoral injection.

165. 165. The composition for use according to any one of claims 140 to 164, wherein the intratumoral injection does not comprise a hydrogel.

166. 166. The composition for use of claim 165, wherein the immunogenic composition is administered more than once.

167. The composition for use of claim 165, wherein the immunogenic composition is administered more than once.

168. The composition for use of claim 165, wherein administering the immunogenic composition increases circulating CD8+ T cells, M1 macrophages and / or tumor infiltrating dendritic cells.

169. The CD8+ T cells are CD8+ T cells. ＋ IFN-γ ＋ The composition for use according to claim 168, which is a cell.

170. The composition reduces CD44 ＋ CD127 ＋ CD44 compared to T cells ＋ CD127 ＋ 170. A composition for use according to any one of claims 140 to 169, which generates a sustained T cell memory response as measured by an expansion of T cells.

171. The composition for use of claim 140, wherein the TGFβ antagonist, STING agonist, TLR7 / 8 agonist, RIG-I agonist, proteasome inhibitor, and adenosine A3 receptor agonist are administered as a single injection.

172. The composition for use of claim 140, wherein the TGFβ antagonist, STING agonist, TLR7 / 8 agonist, RIG-I agonist, proteasome inhibitor, and adenosine A3 receptor agonist are administered as more than one injection.

173. 173. The composition for use according to any one of claims 140 to 172, wherein the intratumoral injection does not comprise a hydrogel.

174. The composition for use according to any one of claims 140 to 173, wherein the immunogenic composition is administered more than once.

175. The composition for use according to any one of claims 140 to 174, wherein the composition is administered two or more times.

176. The composition for use according to any one of claims 140 to 175, wherein the composition does not comprise a cell therapy.

177. The composition for use according to any one of claims 140 to 176, wherein the composition does not comprise an immune checkpoint inhibitor, a cytokine, and / or an antigen.

178. 178. The composition for use according to any one of claims 140 to 177, wherein the composition further comprises an additional anti-cancer therapy.

179. The composition for use of claim 178, wherein the additional anti-cancer therapy comprises chemotherapy, gene therapy, surgery, hormonal therapy, anti-angiogenic therapy or immunotherapy.

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