Enhancement of persistent anti-tumor immunity by multi-factor immunomodulation

Abstract

Provided herein are methods for enhancing an immune response comprising providing an immunogenic composition comprising a mixture of an intracellular agonist, an immunosuppressive antagonist, and a cell inhibitor / damage inducer. Further provided herein are methods of treating a primary tumor and preventing metastasis.

Description

[0001] Priority claim

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 321,205, filed on March 18, 2022, which is incorporated herein by reference in its entirety.

[0003] Federal Grant Support

[0004] 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. background 1. Technical Field

[0006] The present invention relates generally to the fields of molecular biology, immunology and medicine. More specifically, it relates to methods and compositions for stimulating anti-tumor immune responses.

[0007] 2. Description of Related Technology

[0008] Triple-negative breast cancer (TNBC) is a highly aggressive and malignant subtype of 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). + , PR + or HER2 HI Compared to other tumors, TNBC currently lacks clear molecular targets for therapy and is associated with high recurrence rates and distant recurrences despite aggressive surgery and adjuvant chemotherapy. The diagnosis of TNBC is often delayed, and most women are already in advanced disease, with a poor five-year survival rate of 30%. Therefore, although TNBC accounts for only 15% of breast cancer diagnoses in the United States, its deaths account for half of all deaths, an estimated 20,000 per year. Given the lack of effective adjuvant therapy for TNBC, there is an urgent unmet medical need for the development of new treatment options that can reduce the incidence of distant recurrences, prevent serious comorbidities, and save lives. (Hubalek, Czech et al., 2017, Marra, Viale et al., 2019, Waks & Winer, 2019)

[0009] The concept of cancer immune surveillance remained controversial for decades until the advent of immune checkpoint inhibition, which demonstrated that tumor-reactive T cells do exist and may be rendered functionally relevant by blocking signaling pathways associated with tolerance, exhaustion, and / or anergy (Brahmer, Tykodi et al., 2012, Hodi, O'Day et al., 2010, Topalian, Hodi et al., 2012). This is particularly true for certain tumor indications, particularly those with a high mutational burden, such as melanoma, smoking-prone NSCLC, or any dMMR / MSI tumors. + Tumor type, the results can be significant, as exemplified by the nearly 50% durable remission rate of stage IV melanoma following combination therapy with inhibitors that block both the CTLA-4 signaling pathway and the PD-1 signaling pathway. (Hellmann, Ciuleanu et al., 2018, Marcus, Lemery et al., 2019, Overman, Lonardi et al., 2018, Wolchok, Chiarion-Sileni et al., 2017) Despite this, most tumors still fail to respond to checkpoint inhibitor therapy, and even tumor types that can respond often do not respond if they have not spontaneously generated enough tumor antigen-reactive T cells at the start of therapy. To overcome this significant limitation and induce the production of more tumor-reactive T cells, many researchers have tried a variety of different immune agonist and antagonist compounds, both as single agents and in combination with checkpoint inhibition. One of the problems with this approach is that there are a large number of different agonist and antagonist pathways that interact with each other, with tumors, and with immune effector cells in redundant and unknown and unpredictable ways. Therefore, there is an unmet need to develop new therapeutic options for treating TNBC as well as other aggressive cancers such as pancreatic ductal adenocarcinoma (PDAC), melanoma, and head and neck cancer. Summary of the invention

[0010] In a first embodiment, an immunogenic composition is provided, comprising at least 4 (e.g., at least 5, 6, or 7) of the following: transforming growth factor β (TGFβ) antagonists, 4-1BB agonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors, and adenosine A3 receptor agonists. In some aspects, the composition comprises (a) a transforming growth factor β (TGFβ) antagonist or a 4-1BB agonist and (b) at least 3 (e.g., 4 or 5) of the following: interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors, and adenosine A3 receptor agonists.

[0011] In a first embodiment, an immunogenic composition is provided, comprising at least 4, at least 5, at least 6 or all 7 of the following: transforming growth factor β (TGFβ) antagonists, 4-1BB agonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors and adenosine A3 receptor agonists. In some aspects, the composition comprises (a) a transforming growth factor β (TGFβ) antagonist or a 4-1BB agonist and (b) at least 3, at least 4 or all 5 of the following: interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors and adenosine A3 receptor agonists.

[0012] In some aspects, the composition comprises a 4-1BB agonist, an interferon gene stimulator (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist and a proteasome inhibitor. In certain aspects, the composition comprises a transforming growth factor β (TGFβ) antagonist, an interferon gene stimulator (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist and a proteasome inhibitor. In a specific aspect, the composition comprises a 4-1BB agonist, an interferon gene stimulator (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist and a adenosine A3 receptor agonist. In a particular aspect, the composition comprises a transforming growth factor β (TGFβ) antagonist, an interferon gene stimulator (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist and adenosine A3 receptor agonist. In some aspects, the composition comprises at least 5 of the following: transforming growth factor β (TGFβ) antagonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors and adenosine A3 receptor agonists. In some aspects, the composition comprises at least 5 of the following: 4-1BB agonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors and adenosine A3 receptor agonists. In some aspects, the composition comprises transforming growth factor β (TGFβ) antagonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors and adenosine A3 receptor agonists. In certain aspects, 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.

[0013] In some aspects, the TGFβ antagonist is galusertib (LY2157299), trabedersen, fresolimumab, LY2382770, Lucanix, or PF-03446962. In some aspects, the STING agonist is a cyclic dinucleotide or a xanthone analog. For example, the STING agonist is 2'3'-c-di-AM(PS)2(Rp,Rp). In some aspects, 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 aspects, the STING agonist is a xanthone analog, such as 5,6-dimethylxanthone-4-acetic acid (DMXAA). In some aspects, the TLR7 / 8 agonist is resiquimod (R848), CL075, CL097, CL264, CL307, GS-9620, poly (dT), imiquimod, gardiquimod, loxoribine or ssRNA oligonucleotide. In some aspects, the RIG-I agonist is 5'ppp-dsRNA, MDA5 ligand, LGP2 ligand, ssRNA, dsRNA, poly (dA: dT) or poly (I: C). In certain aspects, the proteasome inhibitor is copper (II) diethyldithiocarbamate, bortezomib, delanzomib, ixazomib, MG132, MG115, IPSI 001, fellutamide B, ALLN, leupeptin, epoxomicin, oprozomib, PR-957, carfilzomib, lactacystin, Omuralide, Salinosporamide A, Salinosporamide B, Belactosine, Cinnabaramide, polyphenols, TMC-95 or PS-519. In certain aspects, the adenosine A3 receptor agonist is 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylformamide (2-Cl-IB-MECA), IB-MECA (CF101), CP-608,039, CP-532,903, MRS5698, MRS5980, LJ-529 33 or MRS4322. In some aspects, the 4-1BB agonist is a 4-1BB agonist antibody, a recombinant 4-1BB ligand (4-1BBL) or a 4-1BB aptamer.For example, the 4-1BB agonist antibody is utomilumab or urelumab.

[0014] In some aspects, the composition does not include a hydrogel. In other aspects, the composition does include a biomaterial, such as a hydrogel or a polymer. In some aspects, the composition does not include an immune checkpoint inhibitor, a cytokine and / or an antigen.

[0015] Another embodiment provides a pharmaceutical composition comprising the immunogenic composition of the present embodiment (e.g., an immunogenic composition comprising at least 4 (e.g., at least 5, 6, or 7) of the following: a transforming growth factor β (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.

[0016] Another embodiment provides a pharmaceutical composition comprising the immunogenic composition of the present embodiment (e.g., an immunogenic composition comprising at least 4, at least 5, at least 6, or all 7 of the following: a transforming growth factor β (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.

[0017] Another embodiment provides a method for stimulating an anti-tumor immune response in a subject, the method comprising administering to the subject an effective amount of at least 4 of the following (e.g., at least 5, 6, or 7): transforming growth factor β (TGFβ) antagonists, 4-1BB agonists, interferon gene stimulators (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors, and adenosine A3 receptor agonists. In some aspects, the method comprises administering (a) a transforming growth factor β (TGFβ) antagonist or a 4-1BB agonist and (b) at least 3 of the following (e.g., 4 or 5): interferon gene stimulators (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors, and adenosine A3 receptor agonists.

[0018] Another embodiment provides a method for stimulating an anti-tumor immune response in a subject, the method comprising administering to the subject an effective amount of at least 4, at least 5, at least 6 or all 7 of the following: transforming growth factor β (TGFβ) antagonists, 4-1BB agonists, interferon gene stimulators (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors and adenosine A3 receptor agonists. In some aspects, the method comprises administering (a) a transforming growth factor β (TGFβ) antagonist or a 4-1BB agonist and (b) at least 3, at least 4 or all 5 of the following: interferon gene stimulators (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors and adenosine A3 receptor agonists.

[0019] In some aspects, 4-1BB agonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists and proteasome inhibitors are applied to the subject. In some aspects, transforming growth factor β (TGFβ) antagonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists and proteasome inhibitors are applied to the subject. In some aspects, 4-1BB agonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists and adenosine A3 receptor agonists are applied to the subject. In specific aspects, transforming growth factor β (TGFβ) antagonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists and adenosine A3 receptor agonists are applied to the subject. In a specific aspect, at least 5 of the following items are applied to the subject: 4-1BB agonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors and adenosine A3 receptor agonists. In some aspects, at least 5 of the following items are applied to the subject: transforming growth factor β (TGFβ) antagonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors and adenosine A3 receptor agonists. In some aspects, 4-1BB agonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors and adenosine A3 receptor agonists are applied to the subject. In certain aspects, the subject is administered a transforming growth factor β (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.

[0020] In some aspects, the TGFβ antagonist is galusertib (LY2157299), trabedersen, fresolimumab, LY2382770, Lucanix, or PF-03446962. In some aspects, the STING agonist is a cyclic dinucleotide or a xanthone analog. For example, the STING agonist is 2'3'-c-di-AM(PS)2(Rp,Rp). In some aspects, 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 aspects, the STING agonist is a xanthone analog, such as 5,6-dimethylxanthone-4-acetic acid (DMXAA). In some aspects, the TLR7 / 8 agonist is resiquimod (R848), CL075, CL097, CL264, CL307, GS-9620, poly (dT), imiquimod, gardiquimod, loxoribine or ssRNA oligonucleotide. In some aspects, the RIG-I agonist is 5'ppp-dsRNA, MDA5 ligand, LGP2 ligand, ssRNA, dsRNA, poly (dA: dT) or poly (I: C). In certain aspects, the proteasome inhibitor is copper (II) diethyldithiocarbamate, bortezomib, delanzomib, ixazomib, MG132, MG115, IPSI 001, fellutamide B, ALLN, leupeptin, epoxomicin, oprozomib, PR-957, carfilzomib, lactacystin, Omuralide, Salinosporamide A, Salinosporamide B, Belactosine, Cinnabaramide, polyphenols, TMC-95 or PS-519. In certain aspects, the adenosine A3 receptor agonist is 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylformamide (2-Cl-IB-MECA), IB-MECA (CF101), CP-608,039, CP-532,903, MRS5698, MRS5980, LJ-529 33 or MRS4322. In some aspects, the 4-1BB agonist is a 4-1BB agonist antibody, a recombinant 4-1BB ligand (4-1BBL) or a 4-1BB aptamer.For example, the 4-1BB agonist antibody is utomilumab or urelumab.

[0021] In some aspects, the subject is a human. In certain aspects, the subject has a cancer, such as triple negative breast cancer (TNBC), pancreatic ductal adenocarcinoma (PDAC), melanoma, or head and neck cancer.

[0022] In certain aspects, administration is performed prior to surgical intervention. In some aspects, administration comprises intratumoral injection. In specific aspects, the intratumoral injection does not contain a biomaterial, such as a hydrogel or a polymer.

[0023] In some aspects, the immunogenic composition is administered more than once. In certain aspects, the immunogenic composition is administered two or more times.

[0024] In certain aspects, administration of the immunogenic composition results in an increase in circulating CD8+ T cells, M1 macrophages, and / or tumor infiltrating dendritic cells. In specific aspects, the CD8+ T cells are CD8 + IFN-γ + In certain aspects, administration of the immunogenic composition results in a persistent T cell memory response, such as by binding to CD44 + CD127 + T cells compared to CD44 + CD127 + The increase of T cells is measured. In some aspects, the method does not include applying cell therapy to the subject when the immunogenic composition is applied, such as not applying within 1 day, 1 week, 2 weeks, 1 month or 3 months of the immunogenic composition. In some aspects, the method does not include applying immune checkpoint inhibitors, cytokines and / or antigens to the subject when the immunogenic composition is applied, such as not applying within 1 day, 1 week, 2 weeks, 1 month or 3 months of the immunogenic composition.

[0025] Another embodiment provides a method for treating a subject with cancer, the method comprising administering to the subject an effective amount of at least 4 of the following (e.g., at least 5, 6, or 7): transforming growth factor β (TGFβ) antagonists, 4-1BB agonists, interferon gene stimulators (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors, and adenosine A3 receptor agonists. In some aspects, the method comprises administering (a) a transforming growth factor β (TGFβ) antagonist or a 4-1BB agonist and (b) at least 3 of the following (e.g., 4 or 5): interferon gene stimulators (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors, and adenosine A3 receptor agonists.

[0026] In some aspects, 4-1BB agonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists and proteasome inhibitors are applied to the subject. In some aspects, transforming growth factor β (TGFβ) antagonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists and proteasome inhibitors are applied to the subject. In some aspects, 4-1BB agonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists and adenosine A3 receptor agonists are applied to the subject. In specific aspects, transforming growth factor β (TGFβ) antagonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists and adenosine A3 receptor agonists are applied to the subject. In a specific aspect, at least 5 of the following items are applied to the subject: 4-1BB agonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors and adenosine A3 receptor agonists. In some aspects, at least 5 of the following items are applied to the subject: transforming growth factor β (TGFβ) antagonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors and adenosine A3 receptor agonists. In some aspects, 4-1BB agonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors and adenosine A3 receptor agonists are applied to the subject. In certain aspects, the subject is administered a transforming growth factor β (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.

[0027] In some aspects, the TGFβ antagonist is galusertib (LY2157299), trabedersen, fresolimumab, LY2382770, Lucanix, or PF-03446962. In some aspects, the STING agonist is a cyclic dinucleotide or a xanthone analog. For example, the STING agonist is 2'3'-c-di-AM(PS)2(Rp,Rp). In some aspects, 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 aspects, the STING agonist is a xanthone analog, such as 5,6-dimethylxanthone-4-acetic acid (DMXAA). In some aspects, the TLR7 / 8 agonist is resiquimod (R848), CL075, CL097, CL264, CL307, GS-9620, poly (dT), imiquimod, gardiquimod, loxoribine or ssRNA oligonucleotide. In some aspects, the RIG-I agonist is 5'ppp-dsRNA, MDA5 ligand, LGP2 ligand, ssRNA, dsRNA, poly (dA: dT) or poly (I: C). In certain aspects, the proteasome inhibitor is copper (II) diethyldithiocarbamate, bortezomib, delanzomib, ixazomib, MG132, MG115, IPSI 001, fellutamide B, ALLN, leupeptin, epoxomicin, oprozomib, PR-957, carfilzomib, lactacystin, Omuralide, Salinosporamide A, Salinosporamide B, Belactosine, Cinnabaramide, polyphenols, TMC-95 or PS-519. In certain aspects, the adenosine A3 receptor agonist is 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylformamide (2-Cl-IB-MECA), IB-MECA (CF101), CP-608,039, CP-532,903, MRS5698, MRS5980, LJ-529 33 or MRS4322. In some aspects, the 4-1BB agonist is a 4-1BB agonist antibody, a recombinant 4-1BB ligand (4-1BBL) or a 4-1BB aptamer.For example, the 4-1BB agonist antibody is utomilumab or urelumab.

[0028] In some aspects, administration is performed before surgical intervention. In certain aspects, the subject has not undergone surgical resection of the tumor. In certain aspects, administration includes injection of the immunogenic composition. In some aspects, 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 a single injection. In certain aspects, 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 more than one injection. In certain aspects, the injection is intratumoral injection.

[0029] In some aspects, the cancer is triple negative breast cancer (TNBC), PDAC, head and neck cancer or melanoma. In some aspects, the intratumoral injection does not include biomaterials, such as hydrogels or polymers. In some aspects, the intratumoral injection includes biomaterials, such as hydrogels or polymers.

[0030] In some aspects, the administration is more than one time. In some aspects, the administration is two or more times.

[0031] In some aspects, administration results in an increase in circulating CD8+ T cells, M1 macrophages, and / or tumor infiltrating dendritic cells. In specific aspects, the CD8+ T cells are CD8 + IFN-γ + In certain aspects, administration results in a persistent T cell memory response, such as by binding to CD44 + CD127 + T cells compared to CD44 + CD127 +The increase of T cells is measured. In some aspects, the method does not include applying the transforming growth factor β (TGFβ) antagonist (or 4-1BB agonist), interferon gene stimulator (STING) agonist, Toll-like receptor 7 / 8 (TLR7 / 8) agonist, RIG-I agonist, proteasome inhibitor and adenosine A3 receptor agonist to the subject. Cell therapy is applied to the subject, such as 1 day, 1 week, 2 weeks, 1 month or 3 months of the immunogenic composition. In some aspects, the method does not include applying the transforming growth factor β (TGFβ) antagonist (or 4-1BB agonist), interferon gene stimulator (STING) agonist, Toll-like receptor 7 / 8 (TLR7 / 8) agonist, RIG-I agonist, proteasome inhibitor and adenosine A3 receptor agonist to the subject. Immune checkpoint inhibitors, cytokines and / or antigens are applied to the subject, such as 1 day, 1 week, 2 weeks, 1 month or 3 months of the immunogenic composition.

[0032] In some aspects, the method further comprises administering an additional anti-cancer therapy. In certain aspects, the additional anti-cancer therapy comprises chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy or immunotherapy.

[0033] In some aspects, the patient is a human. In some aspects, the patient has previously been administered anti-cancer therapy.

[0034] Another embodiment provides a method for preventing tumor metastasis in a subject with cancer, the method comprising administering to the subject an effective amount of at least 4 of the following (e.g., at least 5, 6, or 7): transforming growth factor β (TGFβ) antagonists, 4-1BB agonists, interferon gene stimulators (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors, and adenosine A3 receptor agonists. In some aspects, the method comprises administering (a) a transforming growth factor β (TGFβ) antagonist or a 4-1BB agonist and (b) at least 3 of the following (e.g., 4 or 5): interferon gene stimulators (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors, and adenosine A3 receptor agonists.

[0035] In some aspects, 4-1BB agonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists and proteasome inhibitors are applied to the subject. In some aspects, transforming growth factor β (TGFβ) antagonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists and proteasome inhibitors are applied to the subject. In some aspects, 4-1BB agonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists and adenosine A3 receptor agonists are applied to the subject. In specific aspects, transforming growth factor β (TGFβ) antagonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists and adenosine A3 receptor agonists are applied to the subject. In a specific aspect, at least 5 of the following items are applied to the subject: 4-1BB agonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors and adenosine A3 receptor agonists. In some aspects, at least 5 of the following items are applied to the subject: transforming growth factor β (TGFβ) antagonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors and adenosine A3 receptor agonists. In some aspects, 4-1BB agonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors and adenosine A3 receptor agonists are applied to the subject. In certain aspects, the subject is administered a transforming growth factor β (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.

[0036] In some aspects, the TGFβ antagonist is galusertib (LY2157299), trabedersen, fresolimumab, LY2382770, Lucanix, or PF-03446962. In some aspects, the STING agonist is a cyclic dinucleotide or a xanthone analog. For example, the STING agonist is 2'3'-c-di-AM(PS)2(Rp,Rp). In some aspects, 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 aspects, the STING agonist is a xanthone analog, such as 5,6-dimethylxanthone-4-acetic acid (DMXAA). In some aspects, the TLR7 / 8 agonist is resiquimod (R848), CL075, CL097, CL264, CL307, GS-9620, poly (dT), imiquimod, gardiquimod, loxoribine or ssRNA oligonucleotide. In some aspects, the RIG-I agonist is 5'ppp-dsRNA, MDA5 ligand, LGP2 ligand, ssRNA, dsRNA, poly (dA: dT) or poly (I: C). In certain aspects, the proteasome inhibitor is copper (II) diethyldithiocarbamate, bortezomib, delanzomib, ixazomib, MG132, MG115, IPSI 001, fellutamide B, ALLN, leupeptin, epoxomicin, oprozomib, PR-957, carfilzomib, lactacystin, Omuralide, Salinosporamide A, Salinosporamide B, Belactosine, Cinnabaramide, polyphenols, TMC-95 or PS-519. In certain aspects, the adenosine A3 receptor agonist is 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylformamide (2-Cl-IB-MECA), IB-MECA (CF101), CP-608,039, CP-532,903, MRS5698, MRS5980, LJ-529 33 or MRS4322. In some aspects, the 4-1BB agonist is a 4-1BB agonist antibody, a recombinant 4-1BB ligand (4-1BBL) or a 4-1BB aptamer.For example, the 4-1BB agonist antibody is utomilumab or urelumab.

[0037] In some aspects, administration is performed before surgical intervention. In some aspects, the subject has not undergone surgical resection of the tumor. In some aspects, administration includes injection of the immunogenic composition. In certain aspects, 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 a single injection. In other aspects, 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 more than one injection. In certain aspects, the injection is intratumoral injection.

[0038] In some aspects, the cancer is triple negative breast cancer (TNBC), PDAC, head and neck cancer or melanoma. In some aspects, the intratumoral injection does not include a biomaterial, such as a hydrogel or a polymer. In other aspects, the intratumoral injection does include a biomaterial, such as a hydrogel or a polymer.

[0039] In certain aspects, the administration is more than one time. In other aspects, the administration is two or more times.

[0040] In some aspects, administration results in an increase in circulating CD8+ T cells, M1 macrophages, and / or tumor infiltrating dendritic cells. In specific aspects, the CD8+ T cells are CD8 + IFN-γ + In certain aspects, administration results in a persistent T cell memory response, such as by binding to CD44 + CD127 + T cells compared to CD44 + CD127 + Measured by the increase in T cells.

[0041] In certain aspects, the method does not include administering cell therapy to the subject when administering the transforming growth factor β (TGFβ) antagonist (or 4-1BB agonist), interferon gene stimulator (STING) agonist, Toll-like receptor 7 / 8 (TLR7 / 8) agonist, RIG-I agonist, proteasome inhibitor and adenosine A3 receptor agonist, such as 1 day, 1 week, 2 weeks, 1 month or 3 months of the immunogenic composition. In specific aspects, the method does not include administering immune checkpoint inhibitors, cytokines and / or antigens to the subject when administering the transforming growth factor β (TGFβ) antagonist, interferon gene stimulator (STING) agonist, Toll-like receptor 7 / 8 (TLR7 / 8) agonist, RIG-I agonist, proteasome inhibitor and adenosine A3 receptor agonist, such as 1 day, 1 week, 2 weeks, 1 month or 3 months of the immunogenic composition.

[0042] In some aspects, the method further comprises administering an additional anti-cancer therapy. For example, the additional anti-cancer therapy comprises chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy or immunotherapy.

[0043] In some aspects, the patient is a human. In some aspects, the patient has previously been administered anti-cancer therapy.

[0044] Another embodiment provides an immunogenic composition comprising at least 4 (e.g., at least 5, 6, or 7) of the following: transforming growth factor β (TGFβ) antagonists, 4-1BB agonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors, and adenosine A3 receptor agonists. In some aspects, the composition comprises (a) a transforming growth factor β (TGFβ) antagonist or a 4-1BB agonist and (b) at least 3 (e.g., 4 or 5) of the following: interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors, and adenosine A3 receptor agonists.

[0045] In some aspects, the composition comprises a 4-1BB agonist, an interferon gene stimulator (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist and a proteasome inhibitor. In certain aspects, the composition comprises a transforming growth factor β (TGFβ) antagonist, an interferon gene stimulator (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist and a proteasome inhibitor. In a specific aspect, the composition comprises a 4-1BB agonist, an interferon gene stimulator (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist and a adenosine A3 receptor agonist. In a particular aspect, the composition comprises a transforming growth factor β (TGFβ) antagonist, an interferon gene stimulator (STING) agonist, a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, a RIG-I agonist and adenosine A3 receptor agonist. In some aspects, the composition comprises at least 5 of the following: transforming growth factor β (TGFβ) antagonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors and adenosine A3 receptor agonists. In some aspects, the composition comprises at least 5 of the following: 4-1BB agonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors and adenosine A3 receptor agonists. In some aspects, the composition comprises transforming growth factor β (TGFβ) antagonists, interferon gene stimulator (STING) agonists, Toll-like receptor 7 / 8 (TLR7 / 8) agonists, RIG-I agonists, proteasome inhibitors and adenosine A3 receptor agonists. In certain aspects, 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.

[0046] In some aspects, the TGFβ antagonist is galusertib (LY2157299), trabedersen, fresolimumab, LY2382770, Lucanix, or PF-03446962. In some aspects, the STING agonist is a cyclic dinucleotide or a xanthone analog. For example, the STING agonist is 2'3'-c-di-AM(PS)2(Rp,Rp). In some aspects, 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 aspects, the STING agonist is a xanthone analog, such as 5,6-dimethylxanthone-4-acetic acid (DMXAA). In some aspects, the TLR7 / 8 agonist is resiquimod (R848), CL075, CL097, CL264, CL307, GS-9620, poly (dT), imiquimod, gardiquimod, loxoribine or ssRNA oligonucleotide. In some aspects, the RIG-I agonist is 5'ppp-dsRNA, MDA5 ligand, LGP2 ligand, ssRNA, dsRNA, poly (dA: dT) or poly (I: C). In certain aspects, the proteasome inhibitor is copper (II) diethyldithiocarbamate, bortezomib, delanzomib, ixazomib, MG132, MG115, IPSI 001, fellutamide B, ALLN, leupeptin, epoxomicin, oprozomib, PR-957, carfilzomib, lactacystin, Omuralide, Salinosporamide A, Salinosporamide B, Belactosine, Cinnabaramide, polyphenols, TMC-95 or PS-519. In certain aspects, the adenosine A3 receptor agonist is 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylformamide (2-Cl-IB-MECA), IB-MECA (CF101), CP-608,039, CP-532,903, MRS5698, MRS5980, LJ-529 33 or MRS4322. In some aspects, the 4-1BB agonist is a 4-1BB agonist antibody, a recombinant 4-1BB ligand (4-1BBL) or a 4-1BB aptamer.For example, the 4-1BB agonist antibody is utomilumab or urelumab.

[0047] In some aspects, the subject is a human. In certain aspects, the cancer is triple negative breast cancer (TNBC), PDAC, head and neck cancer, or melanoma.

[0048] In some aspects, the composition is administered prior to surgical intervention. In some aspects, the composition is administered by intratumoral injection. In some aspects, the intratumoral injection does not contain a biomaterial, such as a hydrogel or a polymer. In other aspects, the intratumoral injection does contain a biomaterial, such as a hydrogel or a polymer.

[0049] In certain aspects, the immunogenic composition is administered more than once. In some aspects, the immunogenic composition is administered two or more times.

[0050] In some aspects, administration of the immunogenic composition results in an increase in circulating CD8+ T cells, M1 macrophages, and / or tumor infiltrating dendritic cells. In certain aspects, the CD8+ T cells are CD8 + IFN-γ + In some aspects, the composition results in a persistent T cell memory response, such as by binding to CD44 + CD127 + T cells compared to CD44 + CD127 + Measured by the increase in T cells.

[0051] In some aspects, the TGF β antagonist (or 4-1BB agonist), STING agonist, TLR7 / 8 agonist, RIG-I agonist, proteasome inhibitor and adenosine A3 receptor agonist are used as a single injection. In other aspects, the TGF β antagonist (or 4-1BB agonist), STING agonist, TLR7 / 8 agonist, RIG-I agonist, proteasome inhibitor and adenosine A3 receptor agonist are used as more than one injection.

[0052] In certain aspects, the intratumoral injection does not contain a biological material, such as a hydrogel or a polymer. In other aspects, the intratumoral injection does contain a biological material, such as a hydrogel or a polymer.

[0053] In some aspects, the immunogenic composition is administered more than once. In certain aspects, the composition is administered twice or more. In particular aspects, the composition does not include cell therapy. In specific aspects, the composition does not include immune checkpoint inhibitors, cytokines and / or antigens.

[0054] In some aspects, the composition further comprises an additional anti-cancer therapy, such as chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenic therapy, or immunotherapy.

[0055] Other objects, features and advantages of the present invention will become clear from the following detailed description. However, it should be understood that the detailed description and specific examples, although indicating preferred embodiments of the present invention, are only given by way of illustration, because those skilled in the art will become apparent from these embodiments and various changes and modifications within the spirit and scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The following drawings form part of this specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood by referring to one or more of these drawings in combination with the detailed description of specific embodiments given herein.

[0057] Figure 1A to Figure 1E : An empirical cocktail of immunomodulators determines an optimized combination regimen for TNBC immunotherapy. 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 treatment regimens for combination immunotherapy listed in Table 1 on day 7. (Figure 1A) On day 21, mice were imaged by IVIS (N = 5 mice per treatment group). ( Figure 1B ) The luciferase photon intensity between groups was cross-analyzed using Living Image software to estimate the total tumor burden determined by luciferase activity. (Figure 1C) Inoculated with parental 4T-1luc2 neg Images of selected treatment groups of cell lines at day 24 post-treatment. ( Figure 1D ) The parental 4T-1luc2 was determined by caliper measurements every two weeks for 35 days. neg Tumor growth of cell lines. For each experiment shown, N = 5 mice per group. Five representative experiments are shown. ( Figure 1E ) At day 47 after treatment, as shown by Kaplan-Meir survival analysis, only in treatment group #13 (red circles) were all animals still alive. N = 5 mice per group. Three representative experiments are shown. Error bars = + / - SEM. *p<0.05, **p<0.01 by Tukey post hoc one-way ANOVA.

[0058] FIG. 2A to FIG. 2F: After removal of 4-1BBL, TGF-β receptor antagonism, but not repeated dosing, restored the efficacy of optimized treatment regimen #13. Treatment group #15 (#13 without 4-1BBL) and treatment group #16 (#15 + TGF-βR antagonist plus ruxolitinib) were introduced as rational modifications. ( Figure 2A ) Female Balb / c mouse cohorts were implanted subcutaneously with 500,000 4T1-luc2 cells and intratumoral injections of treatment #15 were administered on day 7 only, on day 7 and day 10, or on day 7, day 10, and day 14. Mice were imaged by IVIS on day 16 post-inoculation. (Figure 2B) Tumors and tumor growth were also measured and plotted biweekly by caliper. Administration of increasing numbers promoted growth delays, but ultimately did not confer durable tumor control. ( Figure 2C )A single intratumoral injection of treatment #15 was compared to a single intratumoral injection of treatment #16 (#15 + TGF-βR antagonism), and tumors were measured biweekly by caliper. For each experiment, N = 5 animals per group. One of three representative experiments is shown. (Figure 2D) Repeated dosing of treatment #16 was able to confer sustained anti-tumor efficacy in a dose-responsive manner. Cohorts of female Balb / c mice were implanted subcutaneously with 500,000 4-T1 TNBC cells and injected intratumorally with treatment #16 on days 9, 12, and 15. Each cohort received a designated portion of 1.00 times (100%) the treatment #16 dose. ( Figure 2E ) Kaplan-Meier survival analysis of D. For each experiment D / E, N = 5 animals per group. One of three representative experiments is shown. ( Figure 2F ) 500,000 4T-1luc-2 + Cells were implanted orthotopically in the mammary fat pad beneath the fourth nipple. Animals were treated intratumorally with vehicle control, galusertib at a concentration equal to that used in the optimization regimen, galusertib at a concentration three times that used in the optimization regimen, or the full optimization regimen 16 itself. Error bars = + / - SEM. For BD: *p<0.05, **p<0.01, corrected for multiple comparisons by Student's two-tailed unpaired t-test. For F: *p<0.05, **p<0.01, one-way ANOVA with Tukey post hoc.

[0059] FIG. 3A to FIG. 3J : Tumor treatment with regimen #16 resulted in elevated levels of circulating CD8 + IFN-γ + T cells and memory T cells, while significantly reducing exhausted and regulatory CD4 +T cells. Cohorts of female Balb / c mice were implanted subcutaneously with 500,000 4T-1 cells and each group was treated intratumorally with treatments 12 to 16 (all at dose level 1.00) on days 9 and 12, and circulating PBMCs were analyzed by retro-orbital bleeding and flow cytometry on day 15. ( Figure 3A ) Every 10 6 Absolute circulating CD8 + T cells. ( Figure 3B ) Compared with untreated controls, IFN-γ + CD8 + T cell fold increase. ( Figure 3C ) Circulating depleted CD4 + T cells (defined as CTLA-4 + 、PD-1 + ) percentage. Figure 3D ) compared with untreated controls, circulating regulatory CD4 + T cells (defined as CTLA-4 + 、Foxp3 + ). For each experiment AD, red line = group mean, N = 5 mice per group. One of three representative experiments is shown. Error bars = + / - SEM. *p<0.05, ***p<0.005 by Tukey post hoc one-way ANOVA. To characterize memory, circulating PBMCs were collected on days 20 and 35 and analyzed by flow cytometry. (Fig. 3E) Circulating CD8 + CD44 + CD127 + Comparison of cells. (Fig. 3F / Fig. 3G) Visualization of individual cohort members in vehicle control and treatment groups. Figure 3H ) Circulating CD4 between vehicle control and treatment #16 on days 20 and 35 after treatment + CD44 + CD127 + Comparison of cells. (Figure 3I / Figure 3J ) 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 of three representative experiments is shown. For E to J: error bars = + / - SEM. *p<0.05, **p<0.01 by Student's two-tailed unpaired t-test.

[0060] Figure 4A to Figure 4E: TNBC intratumoral injection protocol #16 produces transferable memory and propagates abscopal effects. (Figure 4A) The adoptive transfer experiment was performed as shown in the schematic. Briefly, mice inoculated with palpable TNBC tumors were treated three times with protocol #16. Thirty days after the last treatment, total splenocytes were harvested and 100,000 total splenocytes were adoptively transferred into naive recipients. The control cohort received 100,000 splenocytes from unvaccinated / untreated animals. Three days after the adoptive transfer, recipient mice were injected intraperitoneally with 500,000 4T-1-luc2 cells and imaged by IVIS another seven days later ( Figure 4B To characterize any apparent abscopal effects, cohorts of female Balb / c mice were implanted subcutaneously with 500,000 4T-1 parental (luc2 neg ) cells and injected intratumorally with regimen #16 or vehicle control on days 13, 17, and 20. On day 11, 250,000 4T1-luc2 cells were implanted subcutaneously at a distal site and imaged by IVIS on days 13 and 23, but not directly treated. (Figure 4C) Primary tumor growth was assessed by caliper measurement. ( Figure 4D ) Growth of untreated satellite lesions was assessed by IVIS luminescence. Figure 4E ) IVIS images of satellite tumor burden after primary tumors were treated with vehicle control or regimen #16 two days and 12 days after implantation. Representative of three experiments shown, N=5 mice per group. Error bars=+ / -SEM. *p<0.05 by Student's two-tailed unpaired t-test.

[0061] Figure 5A to Figure 5K : Optimized treatment regimen 16 reshapes the tumor microenvironment and improves invasive MOC-2 HNSCC. (Figure 5A) C57BL / 6 mouse cohorts 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 volume was determined by caliper measurements every two weeks. In subsequent experiments, animals were treated twice with vehicle or regimen 16, and tumors were subsequently resected, digested, and analyzed by flow cytometry to determine the effects of regimen 16 on the tumor microenvironment. ( Figure 5B ) CD8 was observed in MOC-2 MHSCC tumors treated with regimen 16 + A massive influx of T cells (Figure 5C). Representative CD4 / CD8 flow cytometry plots of vehicle-treated MOC-2 tumor microenvironment. Figure 5D ) Representative CD4 / CD8 flow cytometry plots of the microenvironment of MOC-2 tumors treated with regimen 16. All major populations of MDSCs were significantly reduced in regimen 16 treated tumors (Figure 5E). +CD11b + (Figure 5F) Ly6c + CD11b + and( Figure 5G )Ly6c + Gr-1 + (Figure 5H) In tumors treated with regimen 16, tumor-associated F4 / 80 + CD11b + Macrophages were also nearly eliminated. (Figure 5I) Representative F4 / 80CD11b flow cytometry of vehicle-treated MOC-2 tumor microenvironment. Figure 5J ) Representative F4 / 80CD11b flow cytometry of the MOC-2 tumor microenvironment treated with regimen 16. Figure 5K ) The optimized anti-tumor regimen also induced all CD45 + The number of cells was reduced three-fold. Representative of three experiments shown. N = 5 mice per group. Error bars = + / - SEM (A) and + / - SD (bar graph). *p < 0.05, **p < 0.01, ***p < 0.005 by Student's two-tailed unpaired t-test.

[0062] FIG. 6A to FIG. 6K : Optimized treatment regimen 16 eliminates MYCN-amplified neuroblastoma and induces dedifferentiation of resistant myeloid cells. Fig. 6A Cohorts of 129X1 / SvJ mice were subcutaneously implanted with single cell suspensions from Th-MYNC tumors and administered intratumoral injections of regimen 16 or vehicle control on days 7, 10, and 14. Tumor volumes were determined by caliper measurement. Circulating CD8 + Cell content. (Fig. 6B) Representative image of vehicle-treated PBMCs. (Fig. 6C) Representative image of protocol 16-treated PBMCs. In subsequent experiments, animals were treated twice with either vehicle or protocol 16, and tumors were subsequently resected, digested, and analyzed by flow cytometry to determine the effects of protocol 16 on the tumor microenvironment. Analysis showed that myeloid cell populations were in the process of transitioning from a tolerogenic phenotype to an inflammatory phenotype following treatment with protocol 16. (Fig. 6D) Representative image of the microenvironment of a vehicle-treated Th-MYCN tumor F4 / 80 + Analysis of intracellular arginase in macrophages. (Figure 6E) Representative F4 / 80 in the microenvironment of Th-MYCN tumors treated with regimen 16 + Analysis of intracellular arginase in macrophages. (Figure 6F) Representative CD11b in the microenvironment of vehicle-treated Th-MYCN tumors + Analysis of intracellular arginase in macrophages. Figure 6G ) Representative CD11b of the Th-MYCN tumor microenvironment treated with regimen 16+ Analysis of intracellular arginase in macrophages. (Figure 6H) Representative CD11c in the microenvironment of vehicle-treated Th-MYCN tumors + / PD-L1 + Cellular analysis. (Figure 6I) Representative CD11c in the microenvironment of Th-MYCN tumors treated with regimen 16 + / PD-L1 + Cellular analysis. (Figure 6J) Representative CD11c in the microenvironment of vehicle-treated Th-MYCN tumors + / CTLA-4 + Cell analysis. Figure 6K ) Representative CD11c in the Th-MYCN tumor microenvironment treated with regimen 16 + / CTLA-4 + Cell analysis. Representative of three experiments shown. N = 3 mice per group. Error bars = + / - SEM. ****p < 0.001 by Student's two-tailed unpaired t-test.

[0063] Figure 7A to Figure 7C : Antagonizing tumor immunosuppressive mechanisms alone does not confer antitumor effects. (Figure 7A) Combinations of agents injected intratumorally on day 12. (Figure 7B) 250,000 4T-1TNBC cells were implanted subcutaneously in the hindquarters of female Balb / c mice and injected intratumorally on day 12 with one of the four combinations described in (A). Tumor volumes were measured with calipers every 2 to 3 days. ( Figure 7C ) Quantification of day 21 results. For both B and C, n=5. One of three representative experiments is shown. Error bars=+ / -SEM. *p<0.05 by Student's two-tailed unpaired t-test with correction for multiple comparisons.

[0064] Figure 8A to Figure 8C : Combined agonism of three major intracellular PRR pathways (TLR, RIG-I, STING) confers a temporary anti-tumor effect. (Figure 8A) Combinations of agents injected intratumorally on day 12. (Figure 8B) 250,000 4T-1TNBC cells were implanted subcutaneously in the hindquarters of female Balb / c mice and injected intratumorally on day 12 with one of the four combinations described in (A). Tumor volumes were measured with calipers every 2 to 3 days. ( Figure 8C ) Quantification of day 21 results. For both B and C, n=5. One of three representative experiments is shown. Error bars=+ / -SEM. *p<0.05 by Student's two-tailed unpaired t-test with correction for multiple comparisons.

[0065] Fig. 9: Optimized Regimen 16 regressed orthotopic TNBC. 500,000 4T-1luc-2+ cells were implanted orthotopically in the mammary fat pad below the fourth nipple. Animals were treated intratumorally with vehicle control, galusertib at a concentration equal to that used in the optimized regimen, galusertib at a concentration three times that used in the optimized regimen, or the full optimized regimen 16 24 itself. Shown: IVIS imaging of individual animals, including Figure 2F Data shown in . N = 6 animals per group.

[0066] Figure 10A to Fig. 10C :Tumor treatment with formulation #16 produces elevated levels of tumor-resident CD8+IFN-g+T cells, while significantly reducing exhausted and regulatory CD4+T cells. Female Balb / c mouse cohorts were subcutaneously implanted with 500,000 4T-1 cells, and each group was treated intratumorally with treatments 9 to 16 (all dose levels 1.00) on days 9 and 12, and circulating PBMCs were analyzed by retro-orbital bleeding and flow cytometry on day 15. On day 15, tumors were excised and tumor microenvironment was analyzed. (Figure 10A) Absolute tumor-infiltrating CD8+T cells (left axis) and tumor-infiltrating CD8+T cells per 106 total cells ((right axis) (Figure 10B). Absolute tumor-infiltrating regulatory CD4+T cells (defined as CTLA-4+, Foxp3+) per 10,000 CD45+ cells. ( Fig. 10C ) Percentage of tumor-infiltrating exhausted CD4+ T cells (defined as CTLA-4+PD-1+ of total CD4+). For each experiment shown, red line = group mean, n = 5 mice per group. One of four representative experiments is shown. Error bars = + / - SEM. *p<0.05 by Tukey post hoc one-way ANOVA.

[0067] Figure 11A to Fig. 11C : Optimized intratumoral injection of regimen 16 skews the tumor microenvironment in TNBC toward a pro-inflammatory phenotype. 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. On day 15, tumors were resected and the microenvironment analyzed as described. (Figure 11A) Tumor infiltrating dendritic cells. (Figure 11B) M1 macrophages. ( Fig. 11C ) Myeloid-derived suppressor cells (MDSC). For each experiment shown, n = 5 mice per group. One of four representative experiments is shown 47. Error bars = + / - SEM. *p < 0.05 by Student's two-tailed unpaired t-test.

[0068] Fig.12: Optimized Regimen 16 dissipates invasive MOC-2 HNSC. 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. Shown: Individual animals containing data shown in Figure 5A. DETAILED DESCRIPTION

[0069] To account for biological redundancy and unpredictability, this study attempted to use rational and empirical strategies to optimize combinatorial approaches to cancer immunomodulation. The results showed that optimized combinations of intracellular pattern recognition receptor (PRR) agonists, immunosuppressive antagonists, and cytostatic / damage inducers can produce effective and durable antitumor responses in TNBC model systems that are generally considered non-immunogenic. (Brockstedt, Diagana et al., 2002, Feola, Capasso et al., 2018, Liu, Wang et al., 2018)

[0070] Tumors often lack the classic danger signals required to activate adaptive immunity and also frequently employ multiple immunomodulatory mechanisms to downregulate adaptive responses and facilitate evasion of immune surveillance. Given the diversity of different mechanisms that protect tumors from immune recognition, it is not surprising that single-agent immunomodulatory approaches have been largely unsuccessful in generating durable antitumor responses.

[0071] Therefore, in certain embodiments, methods and compositions of unique combinations of immunomodulators and cytostatics are provided herein, which repair 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 amplification model of neuroblastoma. The course of treatment optimized for TNBC produced a 50% complete response rate and eliminated metastatic spread in all animals tested at the highest dose. The immune response can be transferred between treatment recipients and naive donors by adoptive transfer, and a considerable remote effect on distant untreated lesions can be demonstrated experimentally. Similar results were observed in HNSCC and neuroblastoma models, and substantial remodeling of the tumor microenvironment was recorded in all model systems. The data show that targeting multiple pathways and mechanisms of action can produce significant synergistic effects. These results are also shown in other cancers including PDAC and melanoma, so the present method can be used to treat other non-immunogenic cancers.

[0072] I. Definitions

[0073] As used herein, "substantially free" with respect to a specified component is used herein to mean that the specified component is not intentionally formulated into the composition and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the specified component caused by any accidental contamination of the composition is much less than 0.05%, preferably less than 0.01%. Most preferred are compositions in which the amount of the specified component cannot be detected using standard analytical methods.

[0074] As used herein in the specification, "a" or "an" may mean one or more. As used herein in the claims, when used with the word "comprising", the word "a" or "an" may mean one or more than one.

[0075] Unless it is clear that only alternatives are referred to or the alternatives are mutually exclusive, the term "or" is used in the claims to mean "and / or", but the present disclosure supports the definition referring only to alternatives and "and / or". As used herein, "another" can mean at least a second or more.

[0076] The term "substantially" should be understood to mean that the methods or compositions include only the specified steps or materials and those steps or materials that do not materially affect the basic and novel characteristics of those methods and compositions.

[0077] As used herein, a composition that is "substantially free" of a particular substance or material contains ≤ 30%, ≤ 20%, ≤ 15%, more preferably ≤ 10%, even more preferably ≤ 5%, or most preferably ≤ 1% of that substance or material.

[0078] As used herein, the terms "substantially" or "approximately" may be applied to modify any quantitative comparison, value, measurement, or other representation that could be permitted to vary without resulting in a change in the basic function to which it is related.

[0079] The term "about" generally refers to within the standard deviation of the stated value as determined using standard analytical techniques for measuring the stated value. These terms can also refer to plus or minus 5% of the stated value.

[0080] As used herein, "substantially free" with respect to a specified component is used herein to mean that the specified component is not intentionally formulated into the composition and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the specified component caused by any accidental contamination of the composition is much less than 0.05%, preferably less than 0.01%. Most preferred are compositions in which the amount of the specified component cannot be detected using standard analytical methods.

[0081] The term "substantially free" is used to mean 98% of the listed component and the composition or particle is substantially free of less than 2% of the component.

[0082] "Treatment" or "treating" includes (1) inhibiting the disease in a subject or patient experiencing or displaying pathology or symptoms of the disease (e.g., arresting further development of the pathology and / or symptomology), (2) ameliorating the disease in a subject or patient experiencing or displaying pathology or symptoms of the disease (e.g., reversing the pathology and / or symptomology), and / or (3) achieving any measurable reduction in the disease in a subject or patient experiencing or displaying pathology or symptoms of the disease.

[0083] "Prevention" or "preventing" includes: (1) inhibiting the onset of a disease in a subject or patient who may be at risk and / or susceptible to the disease but does not yet experience or display any or all of the pathology or symptoms of the disease, and / or (2) slowing the onset of the pathology or symptoms of a disease in a subject or patient who may be at risk and / or susceptible to the disease but does not yet experience or display any or all of the pathology or symptoms of the disease.

[0084] In some embodiments, all compounds of the present disclosure can be used to prevent and treat one or more diseases or conditions discussed herein or otherwise. In some embodiments, one or more compounds characterized or exemplified herein as intermediates, metabolites and / or prodrugs can also be used to prevent and treat one or more diseases or conditions. Therefore, unless explicitly stated to the contrary, all compounds of the present invention are considered to be "active compounds" and "therapeutic compounds" intended to be used as active pharmaceutical ingredients (APIs). The actual suitability for human or veterinary use is usually determined using a combination of clinical trial protocols and regulatory procedures (such as those managed by the Food and Drug Administration (FDA)). In the United States, FDA is responsible for protecting public health by ensuring the safety, effectiveness, quality and assurance of human and veterinary drugs, vaccines and other biological products, and medical devices.

[0085] "Prophylactic treatment" includes: (1) reducing or alleviating the risk of developing a disease in a subject or patient who may be at risk and / or susceptible to the disease but does not yet experience or display any or all of the pathology or symptoms of the disease, and / or (2) slowing the onset of the pathology or symptomology of the disease in a subject or patient who may be at risk and / or susceptible to the disease but does not yet experience or display any or all of the pathology or symptoms of the disease.

[0086] 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 a transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human patients are adults, adolescents, infants, and fetuses.

[0087] The term "effective" as used in the specification and / or claims refers to an amount sufficient to achieve a desired, expected or desired result. When used in the context of treating a patient or subject with a compound, an "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount" means that the amount of the compound, when administered to a subject or patient to treat or prevent a disease, is an amount sufficient to achieve such treatment or prevention of the disease.

[0088] As used herein, the term “IC 50 ” refers to the inhibitory dose that is 50% of the maximal response obtained. This quantitative measurement indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological, biochemical, or chemical process (or component of a process, i.e., an enzyme, cell, cell receptor, or microorganism) by half.

[0089] "Anti-cancer" agents are capable of negatively affecting cancer cells / tumors in a subject, for example by promoting the killing of cancer cells, inducing apoptosis of cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to tumors or cancer cells, promoting an immune response to cancer cells or tumors, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject suffering from cancer.

[0090] "Aggressive cancers" as referred to herein are cancers that grow and spread more aggressively and have challenges that make them more difficult to treat than common tumor types. Cancer cells often become resistant to standard treatment options, and as a result, patients may quickly exhaust these options.

[0091] As generally used herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs and / or body fluids of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0092] "Pharmaceutically acceptable salt" refers to a salt of a compound of the invention that is pharmaceutically acceptable as defined above and that 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 acid addition salts formed with organic acids such as 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 monocarboxylic acids and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, Pharmaceutically acceptable salts include benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, enanthic acid, caproic 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, tert-butylacetic acid and trimethylacetic acid. Pharmaceutically acceptable salts also include base addition salts, which can be formed when the acidic protons present can react with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. The limiting examples of acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine and N-methylglucamine. It should be recognized that the specific anion or cation forming part of any salt of the present invention is not important, so long as the salt as a whole is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and methods of making and using them are provided in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth, ed., Verlag Helvetica Chimica Acta, 2002).

[0093] As used herein, a "durable" anti-tumor response refers to a persistent response that is capable of preventing the establishment 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 longer) after initiation or that can be adoptively transferred between subjects at least 3 months (e.g., at least 6 months, 12 months, 2 years, 3 years, 4 years, or 5 years or longer) after initiation.

[0094] II. Immunomodulators

[0095] Certain aspects of the embodiments relate to immunomodulators, including inhibitors and agonists of various pathways such as TGFβ pathways and PRR pathways. In specific aspects, provided herein are therapeutic mixtures comprising a combination of intracellular agonists, immunosuppressive antagonists, and cell inhibitors / damage inducers. PRR agonists may include STING agonists, TLR7 / 8 agonists, and RIG-I agonists. In specific aspects, the mixture comprises a STING agonist, a TLR7 / 8 agonist, a RIG-I agonist, a proteasome inhibitor, and an adenosine A3 receptor agonist.

[0096] A. PRR agonists

[0097] a. STING agonists

[0098] In some aspects, the methods and compositions of the present invention comprise an activator of the stimulator of interferon genes (STING) pathway, 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 to DNA in the cytoplasm, STING activates signal transduction through the TANK-binding kinase 1 (TBK-1) / IRF-3 axis, leading to IFN-β production. This pathway has been shown to play an important role in sensing DNA viruses and some autoimmune diseases. Recent data suggest that the STING pathway is absolutely necessary for inducing spontaneous T cell activation against tumor antigens in vivo. Tumor DNA is detected in tumor-infiltrating DCs, which leads to IFN-β production and T cell activation.

[0099] The STING agonist of the present invention can be selected from chemical activators of the STING pathway, which are selected from cyclic dinucleotides and xanthone 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. Xanthone analogs include but are not limited to tumor vascular disruptors 5,6-dimethylxanthone-4-acetic acid (DMXAA; Vadimezan or ASA404). In some aspects, the STING agonist is 2'3'-c-di-AM (PS) 2 (Rp, Rp), a dithioate phosphorothioate analog of the c-di-AMP, Rp isomer (also known as ADU-S100 or MIW815) (CAS No.: 1638750-95-4) (sodium salt).

[0100] b. TLR7 / 8 agonists

[0101] In some aspects, the methods and compositions of the present invention include an agonist of an immunostimulatory 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). A TLR agonist can be BCG, a TLR7 agonist (e.g., polyOICLC and imiquimod), a TLR8 agonist (e.g., resiquimod (R848)) or a TLR9 agonist (e.g., CPG 7909). In some aspects, a TLR7 agonist is CL075, CL097, CL264, CL307, GS-9620, poly (dT), imiquimod, gadiquimod, resiquimod (R848), loxoribine or ssRNA oligonucleotides. Exemplary TLR-9 agonists include CpG oligodeoxynucleotides (CpG ODN). Other TLR agonists are described, for example, in U.S. Patent Publication No. 2014 / 0005255; which is incorporated herein by reference.

[0102] c. RIG-I agonists

[0103] In certain aspects, the methods and compositions of the invention comprise 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.

[0104] RIG-I sample receptor (RLR) ligand known in the art refers to the activator of RIG-I, Mda5 and LGP2 signal transduction. These ligands include but are not limited to single-stranded RNA, double-stranded RNA and 5'-triphosphate RNA. RIG-I sample receptor ligand also refers to any modification introduced in RNA molecule, which can cause the combination and activation of RIG-I, Mda5 and LGP2, thereby producing RLR sample biological activity. In some aspects, RLR ligand can be a regulator of common adapter protein such as IPS-1 (also referred to as MAVS, VISA or CARDIF). For example, RIG-1 sample receptor ligand is selected from the group consisting of the following: MDA5 ligand, LGP2 ligand, ssRNA, dsRNA, 5'ppp-dsRNA, poly (dA: dT) and poly (I: C).

[0105] B. TGFβ antagonists

[0106] Transforming growth factor β (TGFβ) is a secreted protein that controls proliferation, cell differentiation and other functions of most cells. It is a cytokine that plays a role in immunity, cancer, bronchial asthma, pulmonary fibrosis, heart disease, diabetes and multiple sclerosis. There are at least three isoforms, called TGF-β1, TGF-β2, and TGF-β3. The TGF-β family is part of a superfamily of proteins called the transforming growth factor β superfamily, which includes inhibins, activins, anti-Mullerian hormone, bone morphogenetic protein, Decapentaplegic, and Vg-1.

[0107] In some aspects, the TGFβ inhibitor is galusarti (LY2157299), trabecen, risorumab, LY2382770, Lucanix, or PF-03446962.

[0108] C. Proteasome inhibitors

[0109] The proteasome is a protease complex that maintains optimal levels of intracellular proteins required for cell cycle progression, apoptosis, and normal cellular processes through ubiquitin-dependent or ubiquitin-independent protein degradation, thereby mediating many cellular mechanisms. The inhibition of the proteasome induces cell cycle arrest and apoptosis by regulating multiple pathways, including the stabilization of p53, the activation of C-Jun NH2-terminal kinase (JNK), and the inactivation of nuclear factor kappa-B (NFκB), which leads to the activation of intrinsic and extrinsic caspase cascades. In addition, inhibition of the proteasome can lead to the accumulation of unfolded proteins in the endoplasmic reticulum (ER), followed by the activation of the unfolded protein response (UPR) pathway, leading to apoptosis. The proteasome inhibitor of the present invention can 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.

[0110] Proteasome inhibitors that can be used in the present methods and compositions include, but are not limited to, copper(II) diethyldithiocarbamate, (a) peptide boronates such as bortezomib (also known as Velcade TMand PS341), dilanzomib (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, filutamide 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) epoxyketones, such as epoxomicin, oprozomib (also known as PR-047 or ONX 0912), PR-957 (also known as ONX 0914) and carfilzomib (also known as PR-171); and (e) β-lactones such as lactacystin, omuralide, salinosporamide A (also known as NPI-0052 and marizomib), salinosporamide B, belactosine, cinnabaramide, polyphenols, TMC-95, and PS-519.

[0111] D. Adenosine A3 receptor agonists

[0112] Adenosine receptors (ARs) are G protein-coupled receptors (GPCRs) that sense imbalances in energy / oxygen / nutrient demand and supply. Extracellular adenosine concentrations rise in response to hypoxia and other stresses, acting on four AR subtypes (A1AR, A 2A AR, A 2B AR and A3AR). A1AR,A 2A AR and A3AR agonists have been the subject of preclinical and clinical evaluation (Jacobson et al., Front. Cell. Neurosci., 2019). Adenosine A(3) receptors (A3AR) are coupled to G proteins involved in a variety of intracellular signaling pathways and physiological functions. Exemplary A3AR agonists include, but are not limited to, 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylformamide (2-Cl-IB-MECA; CF102, Namodenoson), IB-MECA (CF101), CP-608,039, CP-532,903, MRS5698, MRS5980, LJ-529 33 (4'-thio-Cl-IB-MECA) and MRS4322.

[0113] E.4-1BB agonists

[0114] In some aspects, the immunogenic composition of the present invention includes a co-stimulatory receptor 4-1BB agonist.4-1BB belongs to the TNF receptor family, which includes a variety of T cell co-stimulatory receptors, which have been targeted by agonist antibodies, including GITR, CD40, CD27, HVEM, LIGHT, APRIL and TWEAK.Exemplary 4-1BB agonists include but are not limited to 4-1BB agonist antibodies (e.g., tomilimumab (PF-05082566) or ureliumab (BMS-663513)), recombinant 4-1BBL (including but not limited to soluble form, matrix binding form, scaffold binding form) and 4-1BB aptamers.

[0115] III. How to use

[0116] Further provided herein are methods for treating or delaying cancer progression in an individual, such as preventing metastasis, comprising administering to the individual an immunogenic composition of an embodiment of the invention. The composition can be administered as an intratumoral injection.

[0117] Examples of cancers contemplated for treatment 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 in the lung, colon cancer, melanoma, and bladder cancer. In a specific aspect, the cancer is triple negative breast cancer.

[0118] Cancer can specifically be of the following histological types, but is not limited to these: tumor, malignancy; carcinoma; non-small cell lung cancer; renal cancer; renal cell carcinoma; clear cell renal cell carcinoma; lymphoma; blastoma; sarcoma; undifferentiated carcinoma; meningioma; brain cancer; oropharyngeal cancer; nasopharyngeal cancer; biliary tract cancer; 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; stomach cancer; pancreatic cancer; ovarian cancer; uterine cancer; cervical cancer; testicular cancer; colon cancer; rectal cancer; skin cancer; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; small cell lung cancer; breast cancer Carcinoma capitis; oral cancer; oropharyngeal cancer; nasopharyngeal cancer; respiratory tract cancer; genitourinary cancer; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatricoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrointestinal cancer; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma in familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; bronchoalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; oncocytic carcinoma; oncocytic adenocarcinoma; basophilic cell carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencircling sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; carcinoma of skin appendages; apocrine gland carcinoma; sebaceous gland carcinoma ; cerumen gland carcinoma; 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; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; glomus sarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant nevus; lentigo maligna melanoma; acral lentigo melanoma; nodular melanoma; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrohistiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; Muller mixed tumor; Wilms tumor; hepatoblastoma; carcinosarcoma; mesenchymal tumor, malignant; Brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; ovarian goiter, malignant; choriocarcinoma; mesonephroma, malignant; angiosarcoma; hemangioendothelioma, malignant; Kaposi sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma;Chondroblastoma, malignant; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Odontogenic tumor, malignant; Ameloblastic odontogenic sarcoma; Ameloblastoma, malignant; Ameloblastic fibrosarcoma; Endocrine or neuroendocrine or hematopoietic carcinoma; Pineal tumor, malignant; Chordoma; Carcinoma of tissue of central or peripheral nervous system; Glioma, malignant; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibrillary astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma; Primitive neuroectodermal; Cerebellar sarcoma; Ganglioneuroblastoma; Neuroblastoma; Retinoblastoma; Olfactory neurogenic tumor; Meningioma, malignant; Neurofibrosarcoma; Schwannoma, malignant; Granulocytoma, malignant; B-cell lymphoma; Malignant lymphoma; Hodgkin's disease; Hodgkin's; low-grade / follicular non-Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; mantle cell lymphoma; Waldenstrom's macroglobulinemia; other specified non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; granulocytic leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocyte leukemia; myeloid sarcoma; chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia; and / or hairy cell leukemia. ;

[0119] In some embodiments, the subject is a mammal, such as a primate, preferably a higher primate, such as a human (e.g., a patient suffering from a disorder described herein or at risk of suffering from a disorder described herein). In one embodiment, the subject needs to enhance the immune response. In certain embodiments, the subject is immunocompromised or at risk of immunocompromise. For example, the subject is undergoing or has undergone chemotherapy and / or radiation therapy. Alternatively or in combination, the subject is immunocompromised due to infection, or at risk of immunocompromise. In some aspects, the subject is a mammal. Mammals may include, but are not limited to, goats, cattle, pigs, dogs, cats, donkeys, monkeys, apes, rodents such as mice, hamsters and rabbits.

[0120] A. Formulation and Administration

[0121] The present disclosure provides pharmaceutical compositions comprising immunomodulators. When considering clinical applications, it is necessary to prepare pharmaceutical compositions in a form suitable for the intended application. In general, this requires the preparation of compositions substantially free of pyrogens and other impurities that may be harmful to humans or animals. People generally wish to use appropriate salts, buffers and lipids to deliver oligonucleotides to allow target cells to take up. Such methods and compositions are well known in the art, for example, as disclosed in U.S. Patents 6,747,014 and 6,753,423, which are incorporated herein by reference. The compositions of the present disclosure include oligonucleotides to a cell effective amount, which are dissolved or dispersed in a pharmaceutically acceptable carrier or medium.

[0122] Active compositions of the present disclosure may include classical pharmaceutical preparations. Administration according to these compositions of the present disclosure will be carried out by any common route, as long as the target tissue can be obtained by the route. Administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or introduced into the CNS, such as introduced into the cerebrospinal fluid. Such compositions are usually administered as pharmaceutically acceptable compositions, as described above.

[0123] In particular aspects, administration is by injection, such as intratumoral injection. Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injection solutions or dispersions. In all cases, the form should be sterile and should be fluid to the extent that there is easy injection. It must be stable under the conditions of preparation and storage, and must be preserved to resist the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium, which contains, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Suitable fluidity can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using a surfactant. Prevention of the effects of microorganisms can be brought by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents, such as 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.

[0124] Sterile injectable solutions are prepared by incorporating the desired amount of the active compound into an appropriate solvent, optionally along with a variety of other ingredients listed above, followed by filtration sterilization. Typically, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze drying techniques, which produce a powder of the active ingredient plus any additional desired ingredients from its previous sterile filtered solution.

[0125] 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 pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active ingredient, their use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the composition.

[0126] For oral administration, the compositions of the present disclosure may be incorporated with excipients. The compositions of the present disclosure may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins), and they are formed with inorganic acids (such as, for example, hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed by free carboxyl groups may also be derived from inorganic bases (such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide) and organic bases (such as isopropylamine, trimethylamine, histidine, procaine, etc.).

[0127] After preparation, the solution will be used in a manner compatible with the dosage formulation and in a therapeutically effective amount. The preparation is easy to use in a variety of dosage forms, such as injection solutions, drug release capsules, etc. For example, for parenteral administration in an aqueous solution, if necessary, the solution should be appropriately buffered, and the liquid diluent should first be made isotonic with enough saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, in view of the present disclosure, those skilled in the art will know the sterile aqueous media that can be used. For example, a dose can be dissolved in 1ml isotonic NaCl solution, then added to 1000ml subcutaneous injection or injected into the intended infusion site (see, for example, "Remington's Pharmaceutical Sciences" the 15th edition, pages 1035-1038 and 1570-1580). Some changes in dosage will inevitably occur according to the condition of the subject being treated. The person responsible for administration will determine the appropriate dose for individual subjects in any case. In addition, for human administration, the preparation should meet the sterility, pyrogenicity, general safety and purity standards required by the FDA Office of Biologics Standards. In some aspects, the dosage of each component in the immunogenic composition can be between 1 and 100 times the dosage shown in Table 2. For example, the dose of a proteasome inhibitor (e.g., copper (II) diethyldithiocarbamate) can be 120 μg to 12 mg, an adenosine A3 receptor agonist (e.g., 2-Cl-IB-MECA) can be 28 μg to 2.8 mg, a STING agonist (e.g., 2'3'-c-di-AM(PS))2(Rp,Rp)) can be 0.50 μg to 0.05 mg, a RIG-I agonist (e.g., Iyovec) can be 0.53 μg to 0.053 mg, a TLR7 / 8 agonist (e.g., resiquimod, R848) can be 21 μg to 2.1 mg, and a TGFβ inhibitor (e.g., galusarti) can be 42 μg to 4.2 mg, or any range derivable therein.

[0128] B. Combination therapy

[0129] In order to increase the effectiveness of the immunogenic compositions of the present invention, it may be necessary to combine these compositions with other agents that effectively treat the target disease. In some aspects, immune checkpoint inhibitors or vaccines may be administered to the subject. In some aspects, radiotherapy or chemotherapy is administered to the subject. In some aspects, targeted kinase inhibitors, such as BRAF inhibitors, are administered to the subject.

[0130] As a non-limiting example, the treatment of cancer can be implemented with the immunogenic compositions of the embodiments of the present invention and other anticancer agents. "Anticancerous" agents can have a negative impact on the cancer in the subject, such as by killing cancer cells, inducing apoptosis of cancer cells, reducing the growth rate of cancer cells, reducing the incidence or quantity of metastasis, reducing tumor size, inhibiting tumor growth, reducing the blood supply to tumors or cancer cells, promoting the immune response to cancer cells or tumors, preventing or inhibiting the progress of cancer, or increasing the life span of the subject suffering from cancer. More generally, these other compositions will be provided with a combined amount that effectively kills or inhibits cell proliferation. The process can involve contacting cells with anticancer peptides or nanoparticle complexes and one or more medicaments or one or more factors simultaneously. This can be achieved by contacting cells with a single composition or pharmaceutical preparation comprising two reagents, or by contacting cells with two different compositions or preparations (wherein one composition comprises the immunogenic composition, and the other comprises the second medicament) simultaneously.

[0131] Treatment using immunogenic composition can be before or after other medicaments treatment, and interval range is several minutes to several weeks.In the embodiment that other medicaments and immunogenic composition are respectively applied to experimenter, usually ensure that each delivery will not be separated by a long period of time, so that medicament and immunogenic composition can still play a favorable combination effect to cell.In this case, it can be considered that within about 12 to 24 hours each other, more preferably within about 6 to 12 hours each other, contact cell in two ways.In some cases, it may be necessary to significantly extend the treatment time period, wherein between corresponding application, through several days (for example, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days) to several weeks (for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks).

[0132] Various combinations may be employed where the immunogenic composition is "A" and a secondary agent (such as radiation therapy, chemotherapy, or an anti-inflammatory agent) is "B":

[0133] A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B

[0134] B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A

[0135] B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0136] In certain embodiments, administration of the immunogenic compositions of the embodiments of the present invention to a patient will follow the general protocol for administering chemotherapeutic agents, taking into account the toxicity of the carrier (if any). It is expected that treatment cycles will be repeated as needed. It is also contemplated that various standard therapies and surgical interventions may be combined with the hyperproliferative cell therapy.

[0137] a. Chemotherapy

[0138] Cancer therapy also includes various combination therapies. In some aspects, the immunogenic composition of the embodiment is administered (or formulated) with a 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, fosmatinib, gefitinib, imatinib, lapatinib, lenvatinib, muritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab, ruxolitinib, sacatinib, sorafenib, sunitinib, trastuzumab, vandetanib, AP23451, vemurafenib, MK-2206, GSK 690693, A-443654, VQD-002, miltefosine, perifosine, CAL101, PX-866, LY294002, rapamycin, temsirolimus, everolimus, rifolimus, avosidide, genistein, selumetinib, AZD-6244, vatalanib, P1446A-05, AG-024322, ZD1839, P276-00, GW572016 or a mixture thereof.

[0139] Still further combination chemotherapy includes, for example, alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, isoprosulfan and piposulfan; aziridines such as benzotepa, carboquinone, metodepa and uredepa; ethyleneimines and methylmelamines including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; caproic acid glycosides (especially bratacin and bratacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; potatostatin; CC-1065 (including its synthetic analogues adolesine, carzelesine and biszelesine); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycins (including the synthetic analogue KW-2 189 and CB1-TM1); eleutherobin; cynaroline; stolonol; sponge inhibitor; nitrogen mustards such as chlorambucil, chlornaphthamide, estramustine, ifosfamide, mechlorethamine, methoxychlor hydrochloride, melphalan, nembixin, phenambucil, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma 1I and calicheamicin omega 1I; enediyne anthracyclines, including enediyne anthracycline antibiotic A; bisphosphonates such as clodronate; esperamicins; and neocarcinogens Chromophores and related chromophores of enediyne antibiotics, aclarubicin, actinomycin, anthramycin, azaserine, bleomycin, actinomycin C, carabinin, carminomycin, carmomycin, chromomycin, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrroline-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, mexilomycin, mitomycins such as mitomycin C, mycophenolic acid, noramycin, olivetomycin, peplomycin, porphyromycin, puromycin, triferon-doxorubicin, rhodorubicin, streptozocin, streptozocin, tuberculocidin, ubenimex, zinotubicin, zoro bicins; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as norpterin, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiopurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calosterone, drostanolone propionate, cyclothiodine, melastane, testolactone; antiadrenal drugs such as mitotane, trilostane; folic acid supplements such as folinic acid; aceglucuronolide; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; betribucil; bisantrene; edatrexate; diphosphamide; colcemid; diacronil;Eflornithine; elliptinium acetate; epothilone; etoglucagon; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansines, such as maytansine and ansamitocin; mitoguanidine; mitoxantrone; mopidarol; nitriloquinone; pentostatin; belemet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; lisoxantrone; sizoran; spirogermanamine; tenuisporin acid; triazoline quinone; 2,2',2"-trichloro triethylamine; trichothecenes (particularly T-2 toxin, viracoulin A, baculosporin A, and serpentin); urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulanol; pipobroman; garcitosine; cytarabine ("Ara-C"); cyclophosphamide; taxanes, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; edoxaban; Toposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; noanto; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicamycin, gemcitabine, vinorelbine, farnesyl protein transferase inhibitors, trans-platinum, and pharmaceutically acceptable salts, acids or derivatives of any of the above substances. In certain embodiments, the compositions provided herein can be used in combination with gefitinib. In other embodiments, the embodiments of the present invention can be implemented in combination with Gleevac (e.g., about 400 to about 800 mg / day of Gleevac can be administered to a patient). In certain embodiments, one or more chemotherapeutic agents can be used in combination with the compositions provided herein. ;

[0140] b.Radiotherapy

[0141] Other factors that cause DNA damage and have been widely used include so-called gamma rays, X-rays and / or directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors, such as microwaves and UV irradiation, are also considered. All of these factors are likely to cause extensive damage to DNA, DNA precursors, replication and repair of DNA, and assembly and maintenance of chromosomes. The dosage range of X-rays ranges from 50 to 200 roentgen doses per day for a long period of time (3 to 4 weeks) to a single 2000 to 6000 roentgen dose. The dosage range of radioisotopes varies greatly and depends on the half-life of the isotope, the intensity and type of the emitted radiation, and the absorption of tumor cells.

[0142] When applied to cells, the terms "contacting" and "exposing" are used herein to describe the process of delivering a therapeutic composition and a chemotherapeutic or radiotherapeutic agent to or in direct juxtaposition with a target cell. For example, to achieve cell killing or stasis, the two agents are delivered to the cell in a combined amount effective to kill the cell or prevent it from dividing.

[0143] c.Gene therapy

[0144] In another embodiment, the secondary treatment is gene therapy, in which a therapeutic polynucleotide is administered before, after, or simultaneously with the administration of the therapeutic composition. Viral vectors for expressing gene products are well known in the art and include eukaryotic expression systems such as adenovirus, adeno-associated virus, retrovirus, herpes virus, lentivirus, poxvirus (including vaccinia virus) and papillomavirus (including SV40). Alternatively, administration of the expression construct can be accomplished by lipid-based carriers 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).

[0145] d.Surgery

[0146] Approximately 60% of cancer patients will undergo some type of surgery, including preventive, diagnostic or staging, curative, and palliative surgery. Curative surgery is a cancer treatment that can be used in combination with other therapies, such as the treatment provided herein, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapy.

[0147] Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised and / or destroyed. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatment also includes laser surgery, cryosurgery, electrosurgery and microscopically controlled surgery (Mohr's surgery). It is also expected that the present embodiment can be used in combination with the removal of normal tissue of superficial cancer, precancerous lesions or incidental amounts. In some aspects, tumor resection is performed after the immunogenic composition of the embodiment is used. In some aspects, tumor resection is performed 2 weeks, 3 weeks, 4 weeks or 5 weeks after treating tumors with the immunogenic composition of the present invention.

[0148] IV. Examples

[0149] The following examples are included to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to work well in the practice of the present invention, and therefore can be considered to constitute preferred modes for its practice. However, it will be appreciated by those skilled in the art in light of this disclosure that many changes may be made to the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present invention.

[0150] Example 1 - Multifactorial immune regulation of anti-tumor immunity

[0151] Optimizing Experience Generation for Antitumor Multimodal Therapy. The literature describes an exhaustive list of signaling pathways and mechanisms whose agonism or antagonism may modulate the immune system and influence the growth of various tumors; however, administration of many compounds that have been reported to abrogate or inhibit important elements of tumor-associated immunosuppression, including clodronate (M2 macrophage depletion), epacadostat (indoleamine 2,3-dioxygenase inhibition), Nω-hydroxy-nor-L-arginine (selective arginase inhibition), and LN G -monomethylarginine acetate (L-NMMA, nitric oxide synthase inhibitor) did not show a beneficial effect on tumor growth when injected intratumorally in the 4T-1 model of TNBC (in Figure 7A to Figure 7C In addition, nine days after administration to established tumors, the combination containing clodronate resulted in an observable acceleration of tumor growth (also Figure 7A to Figure 7C ). These results are somewhat surprising given that the importance of 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) A different approach using a combination of immune agonist agents produced slightly better results (in Figure 8A to Figure 8C Although monotherapy with any individual PRR agonist did not significantly slow tumor growth, a combination of agonists that stimulate 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% (also 95% CI, 2.3-1.1) nine days after intratumoral administration to established tumors. Figure 8A to Figure 8C). However, although these results were encouraging, 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.

[0152] Tumors are thought to avoid immune recognition through a variety of different mechanisms related to the absence of important danger signals, as well as active immunosuppression through recruitment of MDSC, macrophage polarization, and expression of immunosuppressive ligands and cytokines. (Beatty & Gladney, 2015, Vinay, Ryan et al., 2015) Therefore, it is hypothesized that a viable tumor therapy should address most of these mechanisms and should also employ some type of cytotoxic agent to induce cell death, thereby generating the desired antigen that may elicit a subsequent adaptive immune response. (Galluzzi, Buque et al., 2017, Yatim, Cullen et al., 2017) To this end, a cocktail of three different agents was created, each targeting the three major categories of MoA (providing danger, suppressing immunosuppression, and cytostatic / cytotoxic), and these cocktails were combined in various ways (Table 1) (designated as regimens 8 to 14 in Table 1) prior to treatment of established 4T-1 tumors. This rational and empirical process identified four different combinations of agents (Regimens 10, 12, 13, and 14) that significantly reduced luciferase expression in 4T-1 tumor cells two weeks after administration ( Figure 1A to Figure 1B ), but only two treatment combinations (regimens 12 and 13) were performed using the parental 4T-1luc2 neg The tumor size of the cell line remained significantly reduced four weeks after administration ( Figure 1C to Figure 1D To verify that the immune response might be directed against the native 4T-1 antigen rather than just the heterologous luciferase antigen, luc2 neg The parent is important. Of the two formulations, only one (Scheme 13) had 100% of the cohort members still alive at six weeks after treatment ( Figure 1E ). Consistent with earlier data, this combination of agents has no inhibitors of immunosuppression, consisting only 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 (Mitchell, 2003, Skrott & Cvek, 2012).

[0153] Considering the data in Figure 8, it is unexpected that the most effective scheme includes 4-1BBL, which indicates that the presence of 4-1BBL antagonizes the anti-tumor effects of TLR, STRING and RLH agents. In addition, in preclinical and early human clinical trials, 4-1BB agonists have been shown to be toxic at therapeutic doses (Lee, Salek-Ardakani et al., 2009, Segal, Logan et al., 2017). Therefore, 4-1BB agonists are removed from scheme 13 (subsequently designated as scheme 15, i.e., 15=13 without the addition of 4-1BBL), and attempt to reproduce equivalent efficacy by alternative reasonable modifications. Repeated dosing schemes were attempted, demonstrating that three doses confer better efficacy than two doses, and that two doses are better than one dose ( Figure 2A to Figure 2B); however, even the three-dose regimen 15 could not recapitulate the efficacy of the original regimen 13 formulation with 4-1BBL. TGF-β is considered to be one of the most important immunosuppressive cytokines in the tumor microenvironment, which can mediate tumor escape from immune surveillance (Batlle & Massague, 2019), and inhibition of TGF-β signaling has previously shown activity against TNBC in mouse xenograft models (Bhola, Balko et al., 2013) and has also shown clinical benefits in early human trials (Rodon, Carducci et al., 2015). Given these observations, a single-dose formulation lacking 4-1BB agonism (Regimen 15) was next compared to a single-dose regimen containing the TGF-β kinase signaling inhibitor galactosidase (Regimen 16). This modification resulted in a significant increase in efficacy ( Figure 2C ).

[0154] Finally, the boosting regimen was combined with regimen 16 and dose-response analysis was performed at 1-fold, 0.5-fold, 0.125-fold, 0.0625-fold, and 0.03125-fold concentrations. Figure 2D to Figure 2E As shown, both the 1x and 0.5x doses of regimen 16 significantly inhibited tumor growth, resulting in durable 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 conferred intermediate levels of tumor control, while the 0.03125x dose produced an effect that was indistinguishable from the vehicle control. Figure 1C to Figure 1D The experiments outlined in this study used 4T-1luc2 neg The parental cell line was used to verify that the protective immune response was independent of the presence of the xenobiotic luciferase. Next, the therapeutic model was validated in an in situ setting (Zhang et al., 2019). In this series of experiments, 500,000 4T-1luc-2 +The cells were implanted in the mammary fat pad beneath the fourth nipple. Animals were randomized on day 7 post-implantation and treated intratumorally with vehicle control, galusartin at a concentration equal to that used in the optimized regimen, galusartin at a concentration three times that used in the optimized regimen, or the full optimized regimen 16 itself. Fig. 9 and Figure 2F As shown, treatment regimen 16 gave results in the orthotopic setting that were identical to those observed ex situ. In addition, galusinib alone showed no effect on tumor growth. Treatment regimen 16 ( Figure 2C The components listed in ) were henceforth considered as the final optimized solution for subsequent characterization in multiple model systems.

[0155] Effect of the optimized treatment regimen on the 4T-1TNBC tumor microenvironment. We next sought to characterize the effects of the optimized treatment regimen on the immune system and tumor microenvironment. Established 4T-1 tumors were treated with optimized regimen 16, re-treated three days later, and analyzed an additional three days after the second treatment. Animals were retro-orbitally bled, and optimized regimen 16 was compared not only to vehicle control but also to suboptimal regimens 12 to 15. The optimized regimen induced circulating CD8 + T cells increased sevenfold, while the next best regimens (13 and 15) induced only a 3- to 4-fold increase ( Figure 3A ), while other regimens had almost no effect at all. Regimen 16 induced circulating CD8 + The increase in CD8 + IFN-γ + The cells increased significantly by 2.5-fold, while the other treatment groups showed no statistical difference from the vehicle control ( Figure 3B ). In addition, both regimens 13 and 16 virtually eliminated circulating depleted CD4 + and regulatory T cells ( FIG. 3C to FIG. 3D ). The results were similar in the tumor microenvironment, with regimen 16 inducing the highest number of tumor-infiltrating CD8 + cells, while tumor-infiltrating CD4 + The number of cells was significantly reduced and Tregs were almost eliminated ( Figure 10A to Fig. 10C ). Circulating PBMCs in mice given the optimized regimen 16 also expressed CD8 + ( Figure 3E to Figure 3G) and CD4 + ( Figures 3H to 3J ) CD44 in both compartments + CD127 +The significant and prolonged expansion of T cells indicates the development of a durable T cell memory response (Boettler, Panther et al., 2006, Huster, Busch et al., 2004). The development of this memory phenotype is through the CD4 + CD25 + Foxp3 + Regulatory T cells and PD-1 + CD4 + The results are consistent with those predicted by the reduction of exhausted T cells (Boettler et al., 2006, Liu, Putnam et al., 2006). Comparison of the bone marrow cell compartment between 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 + M1 macrophages increased significantly, while the MDSC population (defined as CD11b + Gr-1 + Arginase + ) significantly reduced ( Figure 11A to Fig. 11C ).

[0156] Effect of optimized treatment regimen on memory development. To verify the existence of persistent memory, mice were implanted with 4T-1 tumors and treated with optimized regimen 16 on days 12, 16, and 20 after implantation. Thirty days later (day 50 after implantation), spleen cells were harvested from the treated mice and adoptively transferred to the naive cohort. The control group was adoptively transferred with spleen cells harvested from tumor-inoculated mice, which were injected intratumorally with only solvent control. Three days later, mice were injected intraperitoneally with 500,000 4T-1-luc2 cells, and seven days later, both cohorts were imaged. As Figure 4A to Figure 4B As shown, mice adoptively transferred with cells from treated animals were able to clear large intraperitoneal tumor boluses, whereas mice adoptively transferred with naive splenocytes developed larger abdominal masses. The ability of antitumor immunity to be adoptively transferred from a T cell splenocyte population suggests that there may be abscopal effects in treated animals. To determine whether treatment with the optimized tumor formulation could confer abscopal effects, mice were implanted with “primary” luc2 neg 4T-1 tumors and "secondary" luc2 +This experimental design is important in that it demonstrates that any immune response controlling the growth of untreated metastases is derived from native 4T-1 antigens and not from foreign luciferase antigens. neg The tumors were treated as metastatic lesions, allowing the experimental kinetics in all mice to be performed with uniform temporal kinetics, independent of the stochastic nature of spontaneous metastatic spread. The “primary” luc2 neg Tumor.luc2 + “Metastasis” was imaged at baseline on day 13 and again on day 23. Figure 4C to Figure 4E As shown, in the treatment of primary luc2 neg luc2 in mice with primary tumors treated with vehicle control + Compared with untreated luc2 + The lesions showed a six-fold reduction in size, indicating a demonstrable abscopal effect.

[0157] Effect of the optimized regimen on other refractory model systems. To determine whether the optimized treatment regimen is applicable to refractory or non-immunogenic model systems other than TNBC, a series of experiments were repeated in a cohort of animals implanted subcutaneously with the invasive HNSCC MOC-2 cell line. As shown in Figures 5A and 11, treatment of MOC-2 HNSCC with the optimized regimen 16 resulted in a significant reduction in the size or complete resolution of these tumors. In subsequent experiments, animals were treated twice with either vehicle or regimen 16, and tumors were subsequently resected, digested, and analyzed by flow cytometry to determine the effect of regimen 16 on the tumor microenvironment. In these experiments, a large number of CD8 + T cell influx ( FIG. 5B to FIG. 5D In addition, all major MDSC populations were significantly reduced in tumors treated with regimen 16 ( Figure 5E to Figure 5G ), and tumor-associated macrophages were almost eliminated ( Figure 5H to Figure 5J ). The optimized anti-tumor regimen also induced all CD45 + A three-fold reduction in the number of cells ( Figure 5K ).

[0158] Next, the study moved into a second difficult-to-treat model system, high-risk, MYCN-amplified neuroblastoma. Single-cell suspensions were generated from seven-week-old Th-MYCN homozygous tumors and transplanted ectopically into the flanks of isogenic wild-type mice. Tumors were then treated with three consecutive injections of regimen 16 or vehicle control, and growth was monitored by caliper measurements. Fig. 6A As shown, treatment with regimen 16 was sufficient to completely eliminate MYCN-amplified neuroblastoma, with total CD8 + Extensive systemic proliferation of peripheral blood spleen cells (i.e. Figure 6B to Figure 6C). As with the HNSCC MOC-2 model, subsequent experiments were performed in which tumors were treated with either two regimen 16 administrations or vehicle and then the microenvironment of the resolved tumors was analyzed. Compared to the MOC-2 model, where the myeloid cell compartment was substantially resolved after two treatments, the resolution of the myeloid 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 regimen 16 treatment. Significantly, characterization of this intermediate step showed that myeloid cells first dedifferentiate from a pro-tumorigenic and immunosuppressive phenotype before resolution. Although no CD11c was observed between treated and mock-treated tumor cell populations, + 、CD11b + 、F4 / 80 + 、Gr-1 + Ly6C + There was a significant difference in cell number (not shown), but F4 / 80 + (Fig. 6D to Fig. 6E) and CD11b + (Figure 6F to Figure 6G ) macrophage population ceased to express arginase, indicating a shift from immunosuppressive T H 2 phenotype changes. In addition, CD11c + The APC group stopped PD-L1( Figure 6H to Figure 6I) and CTLA-4 (Figure 6J to Figure 6K ) expression of both, again indicating a shift from a tolerogenic to an inflammatory phenotype.

[0159] The current work shows that a formulation consisting of an innate intracellular signaling ligand, a cytotoxic agent / cytostatic agent, and a TGF-β signaling inhibitor produced a highly effective antitumor response 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 the aggressive 4T-1 TNBC model, treatment effectively prevented metastasis, cured the primary tumor in 50% of animals receiving the highest dose, and treatment apparently involved the generation of cellular memory as determined by adoptive transfer experiments. The composition of the formulation is derived from a simultaneous rational and empirical development process designed to optimize the synergy between a variety of different agents that act through three broad mechanisms thought to be important for generating antitumor immunity. These three mechanisms are 1) providing danger signals by connecting intracellular pattern recognition receptors that upregulate interferon signaling and propagate T H 1 immune response (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) inhibition of what may be the most important mediator of tumor-associated immunosuppression (i.e., TGF-β) (Beatty & Gladney, 2015, Sinha et al., 2005a, Sinha et al., 2005b, Vinay et al., 2015), and 3) introduction of cytotoxic agents to generate endogenous DAMP signals (Galluzzi et al., 2017, Yatim et al., 2017) and provide antigenic material through cell damage, against which downstream adaptive immune responses can be elicited. While the literature suggests that a very wide variety of different agents can be used to achieve these broad goals, the ways in which any group of individual agents might interact could not be predicted prior to the important empirical experiments documented here. Indeed, some promising combinations of agents did not confer any antitumor effects. This was particularly true for formulations of combination agents designed to inhibit multiple mechanisms of tumor-associated immunosuppression, at least in the non-immunogenic 4T-1 TNBC model system in which optimization was performed. These observations may not be entirely unpredictable given the recent failure of a randomized phase III study of the IDO inhibitor epalrestat in combination with pembrolizumab (Garber, 2018). The broad applicability of the TNBC optimization regimen was subsequently validated in the Th-MYCN and MOC-2 HNSCC model systems.

[0160] It is well known that successful clinical responses to anti-PD-1 checkpoint inhibition are significantly associated with tumor mutational burden (Cristecu et al., 2018; Mandal et al., 2019). It is thought that a greater number of coding mutations increases the likelihood of generating sufficiently high-affinity T cell epitopes against which productive T cell responses may spontaneously arise; however, in cancer patients, this process occurs under steady-state homeostatic conditions or in the context of cellular damage and DAMP release following cytotoxic chemotherapy and radiotherapy. In contrast, multiple PRR signaling ligands combined with inhibition of immunosuppressive TGF-β signaling alter homeostasis during treatment with optimized regimens by initiating interferon signaling, which subsequently mediates increased MHC expression, upregulation of the immunoproteasome, maturation of myeloid DCs, activation of plasmacytoid DCs, and a range of other immune-enhancing effects that are not typically induced by standard-of-care chemotherapy and radiotherapy (Hervas-Stubbs et al., 2011). These effects shift the balance between tolerance and immunity and may allow not only the generation of T cell responses against high-affinity neoepitopes but also the persistence of responses against intermediate and low-affinity T cell clones. These may respond not only to neoepitopes but also to upregulated developmental or differentiation antigens and to post-translational modifications that are relatively unique to the tumor (Ilyas et al., 2015).

[0161] Characterization of the tumor-resolving microenvironment in all three model systems provides an intriguing window into at least one of the mechanisms by which the optimized treatment regimens exert their significant antitumor effects. In all model systems, interim analyses of treatments for MDSCs demonstrated either a significant reduction in myeloid cell burden or captured a snapshot in time of MDSC dedifferentiation, as they downregulated immunosuppressive markers such as PD-L1. This was also true for M2 macrophages, which had reduced or lost expression of arginase, indicating a shift toward an M1 inflammatory phenotype. CD11c + Cells were also affected, downregulating the expression levels of CDLA-4 and PD-L1; the changes observed in various bone marrow compartments were accompanied by CD8 + Cell influx into the tumor bed as well as circulating CD8 + Systemic upregulation of cells.

[0162] In summary, the current study shows that a unique combination of immunomodulators and cytostatics designed and optimized by both rational and empirical methods can reshape the tumor microenvironment and improve tumor types with poor prognosis, including 4T-1TNBC models and high-risk neuroblastoma models of HNSCC, which are generally considered non-immunogenic (Brockstedt et al., 2002, Feola et al., 2018, Liu et al., 2018) and difficult to treat. The course of treatment optimized for 4T-1TNBC produced a 50% complete response rate and eliminated metastatic spread in all animals tested at the highest dose. The results in the HNSCC model were similar, but more robust in high-risk neuroblastoma, where all tumors were resolved after treatment. The immune response can be transferred between treatment recipients and naive donors through adoptive transfer, and considerable afar effects have also been demonstrated. The results show that targeting multiple immune stimulatory and immunosuppressive pathways can produce significant synergistic effects.

[0163] Example 2 - Materials and Methods

[0164] Mice. Female Balb / c mice or C57BL / 6 animals of both sexes, 6 to 10 weeks old, were purchased from Baylor College of Medicine or Jackson Laboratory (Bar Harbor, ME). Th-MYCN mice on the 129X1 / SvJ background were a kind gift from Dr. William Weiss (UCSF, San Francisco, CA) and were maintained in house. All mice were maintained in accordance with the specific IACUC requirements of Baylor College of Medicine and according to animal protocol AN-8375.

[0165] Tumor implantation and measurements. 500,000 4T-1TNBC cells (+ / -luc2 transduced) were injected subcutaneously into the hindquarters / back of each mouse and allowed time to reach the posterior margin to palpation (approximately 50 to 100 mm) prior to treatment. 3 ). For the in situ 4T-1 experiments, according to Zhang et al. 34 500,000 4T-1 cells were implanted into the mammary fat pad using the method described in

[14] . After tumors were established, experimental cohorts were assembled by stratified randomization. Simple randomization was attempted to minimize confounding variables such as cohort treatment order, tumor measurement order, and cage location. Tumors were treated with the designated antitumor regimen or vehicle control and measured by IVIS imaging (if applicable) or calculated by caliper measurements. 100,000 MOC-2 HNSCC cells were injected subcutaneously into the hindquarters / back of each mouse and allowed to become palpable (approximately 50 to 100 mm) prior to treatment. 3 Tumor size and progression were determined by caliper measurements. According to Kroesen et al. 12Single cell suspensions were generated from Th-MYCN tumors using the same method. The suspension was injected subcutaneously into the hindquarters / back of each mouse and allowed time to reach a point of apposition to palpation (approximately 50 to 100 mm) prior to treatment. 3 Tumor size and progression were determined by caliper measurement. The formula (longest length 2 ) x shortest length, and the tumor volume was calculated by two-dimensional measurement.

[0166] 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 minutes, and bioluminescence was measured after 60 seconds of exposure using an IVIS imaging system (Caliper Life Sciences, Waltham, MA). Imaging was performed every 2 to 4 days.

[0167] Tumor microenvironment analysis. According to the manufacturer's instructions, the mouse tumor dissociation kit and dissociator of Miltenyi Biotec (Gaithersburg, MD) were used to analyze tumor microenvironment leukocytes. In brief, the tumor was excised, cut into smaller 2 to 4 mm pieces, and then placed in a separate gentleMACS C tube containing 2.5 ml enzyme mixture (2.35 ml RPMI-1640, 100 μl enzyme D, 50 μl enzyme R and 12.5 μl enzyme A). The tube was then inverted and locked into the gentleMACS Octodissociater with a heater, and the appropriate program was run to break the tumor into a single cell suspension. After the program was completed, the suspension was filtered through a 70 nm cell strainer to remove any remaining debris, and the cells were precipitated, washed 2 times with PBS, and subsequently stained to be analyzed 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.

[0168] Analysis of circulating PBMC. Mice were bled retro-orbitally at specified time intervals, and samples were appropriately mixed with EDTA to prevent coagulation. Red blood cells were lysed by treatment with ammonium chloride (Sigma-Aldrich) according to the manufacturer's instructions. The leukocyte pellet was washed once with PBS and resuspended in 1% PBS, 0.01% normal goat serum + sodium azide and stored 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.

[0169] Antitumor agents. To optimize the TNBC model, all agents were dissolved in DMSO or sterile water based on solubility requirements and included 85 μg / 50 μl of copper (II) diethyldithiocarbamate (VWR, Radnor, PA), 20 μg / 50 μl of 2-C1-IB-MECA (Millipore, Burlington, MA), 75 μg / 50 μl of DMXAA (R&D Systems, Minneapolis, MN), 375 ng / 50 μl of 5'ppp-dsRNA / Lyovec (Invivogen, San Diego, CA), 15 μg / 50 μl of R848 (Invivogen), 30 μg / 50 μl of galusarti (Selleckchem, Houston, TX), 5 μg / 50 μl of epcadostat (Selleckchem), 6 μg / 50 μl of N w -Hydroxy-nor-L-arginine (Sigma-Aldrich, St. Louis, MO), 100 ng / 50 μl of L-NMMA (Abcam, Boston, MA), 80 μg / 50 μl of clodronate (Abcam), and 1 μg / 50 μl of 4-1BB ligand (NovusBio). The combination treatment was reconstituted as a 10x stock solution, combined as needed, and diluted to 1x concentration with appropriate diluent. Intratumoral injection treatment was performed using a 28-gauge needle as a 50 μl push. In HNSCC and neuroblastoma models, the mouse-specific STING agonist DMXAA was replaced by the broader species-responsive STING agonist 2'3'-c-di-AM(PS)2(Rp,Rp) (715 ng / 50 μl). Other reagent concentrations in the HNSCC and neuroblastoma models included 172 μg / 50 μl of copper(II) diethyldithiocarbamate, 40 μg / 50 μl of 2-C1-IB-MECA, 755 ng / 50 μl of 5'ppp-dsRNA / Lyovec, 30 μg / 50 μl of R848, and 60 μg / 50 μl of galusartin.

[0170] Adoptive transfer. Spleens were harvested, physically separated, and reduced to a single cell suspension through a 70 μM cell strainer (Corning, Glendale, AZ). After lysis of red blood cells with ammonium chloride (ThermoFisher Scientific, Waltham, MA), leukocytes were washed, precipitated, and resuspended in PBS before intraperitoneal injection into naive recipient mice. Forty-eight hours later, adoptively transplanted animals were injected intraperitoneally with 500,000 4T1-luc2 tumor cells and then imaged by IVIS.

[0171] Flow Cytometric Analysis. All flow cytometric analyses were performed using an LSR II flow cytometer (BD Biosciences) and analyzed with FlowJo version 10.0.00003 for MacIntosh (Tree Star, Inc., Ashland, OR).

[0172] Statistical analysis. Unless otherwise stated, the significance of differences was determined by one-way analysis of variance (ANOVA) with Tukey's honestly significant difference (HSD) post hoc test. Paired 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 shown as mean ± SEM or SD as specified. All analyses were performed using Prism software version 8.3 for iOS (GraphPad Software) 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 in a sample size of n = 5, assuming σ = 20% of μ, α = 0.05, and a power of 0.8. Therefore, five mice were typically used per cohort unless otherwise stated. The normality and variance of the data were assessed graphically.

[0173]

[0174]

[0175]

[0176]

[0177] ***

[0178] All methods disclosed and claimed herein can be made and performed in accordance with the present disclosure without undue experimentation. Although the compositions and methods of the present invention have been described in conjunction with preferred embodiments, it is apparent to those skilled in the art that the methods and steps or the order of steps in the methods described herein may be changed without departing from the concept, spirit and scope of the present invention. More specifically, it is apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein while achieving the same or similar results. It is apparent to those skilled in the art that all such similar substitutions and modifications are deemed to be within the spirit, scope and concept of the present invention as defined by the appended claims.

[0179] References

[0180] The following references, to the extent they provide exemplary procedural or other details supplementary to those set forth herein, are expressly incorporated herein by reference.

[0181] Batlle E,Massague J(2019)Transforming Growth Factor-beta Signaling inImmunity and Cancer.Immunity 50:924-940

[0182] Beatty GL,Gladney WL(2015)Immune escape mechanisms as a guide for cancer immunotherapy. Clin Cancer Res 21:687-92

[0183] Bhola NE, Balko JM, Dugger TC, Kuba MG, Sanchez V, Sanders M, Stanford J, Cook RS, Arteaga CL (2013) TGF-beta inhibition enhances chemotherapy action against triple-negative breast cancer. J Clin Invest 123:1348-58

[0184] Boettler T, Panther E, Bengsch B, Nazarova N, Spangenberg HC, Blum HE, Thimme R (2006) Expression of the interleukin-7 receptor alpha chain (CD127) on virus-specific CD8+ T cells identifies functionally and phenotypically defined memory T cells during acute resolving hepatitis B virus infection. J Virol 80:3532-40

[0185] Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, Drake CG, Camacho LH, Kauh J, Odunsi K, Pitot HC, Hamid O, Bhatia S, Martins R, Eaton K, Chen S, Salay TM, Alaparthy S, Grosso JF, Korman AJ et al. (2012) Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med 366:2455-65

[0186] Brockstedt DG, Diagana M, Zhang Y, Tran K, Belmar N, Meier M, Yang A, Boissiere F, Lin A, Chiang Y (2002) Development of anti-tumor immunity against a non-immunogenic mammary carcinoma through in vivo somatic GM-CSF, IL-2, and HSVtk combination gene therapy. Mol Ther 6:627-36

[0187] Brunet JF,Denizot F,Luciani MF,Roux-Dosseto M,Suzan M,Mattei MG,Golstein P(1987)

[0188] A new member of the immunoglobulin superfamily--CTLA-4.Nature 328:267-70Carroll EC,Jin L,Mori A,Munoz-Wolf N,Oleszycka E,Moran HBT,Mansouri S,McEntee CP,Lambe E,Agger EM,Andersen P,Cunningham C,Hertzog P,Fitzgerald KA,Bowie AG,Lavelle EC(2016)The Vaccine Adjuvant Chitosan Promotes CellularImmunity via DNA Sensor cGAS-STING-Dependent Induction of Type IInterferons.Immunity 44:597-608

[0189] Chester C,Sanmamed MF,Wang J,Melero I(2018)Immunotherapy targeting 4-1BB:mechanistic rationale,clinical results,and future strategies.Blood 131:49-57

[0190] Clowes GHA,Baeslack FW(1905)Further Evidence of Immunity AgainstCancer in Mice after Sontaneous Recovery.Med News 87:968-71

[0191] Cristescu R, Mogg R, Ayers M, Albright A, Murphy E, Yearley J, Sher X, Liu XQ, Lu H, Nebozhyn M, Zhang C, Lunceford JK, Joe A, Cheng J, Webber AL, Ibrahim N, Plimack ER, Ott PA, Seiwert TY, Ribas A et al. (2018) Pan-tumor genomic biomarkers for PD-1 checkpoint blockade-based immunotherapy. Science 362

[0192] Decker WK, Safdar A (2009) Bioimmunoadjuvants for the treatment of neoplastic and infectious disease: Coley's legacy revisited. Cytokine Growth Factor Rev 20:271-81

[0193] Decker WK, Safdar A (2011) Cytokine adjuvants for vaccine therapy of neoplastic and infectious disease. Cytokine Growth Factor Rev 22:177-87

[0194] Feola S, Capasso C, Fusciello M, Martins B, Tahtinen S, Medeot M, Carpi S, Frascaro F, Ylosmaki E, Peltonen K, Pastore L, Cerullo V (2018) Oncolytic vaccines increase the response to PD-L1 blockade in immunogenic and poorly immunogenic tumors. Oncoimmunology 7:e1457596

[0195] Galluzzi L,Buque A,Kepp O,Zitvogel L,Kroemer G(2017)Immunogenic celldeath in cancer and infectious disease.Nat Rev Immunol 17:97-111

[0196] Garber K(2018)A new cancer immunotherapy suffers a setback.Science360:588

[0197] Graham JB,Graham RM(1959)The effect of vaccine on cancerpatients.Surg Gynecol Obstet 109:131-8

[0198] Gross L(1943)Intradermal Immunization of C3H Mice against a SarcomaThat Originated in an Animal of the Same Line.Cancer Res 3:326-33

[0199] Hamilton MJ,Bosiljcic M,Lepard NE,Halvorsen EC,Ho VW,Banath JP,Krystal G,Bennewith KL(2014)Macrophages are more potent immune suppressors exvivo than immature myeloid-derived suppressor cells induced by metastaticmurine mammary carcinomas.J Immunol 192:512-22

[0200] Hellmann MD, Ciuleanu TE, Pluzanski A, Lee JS, Otterson GA, Audigier-Valette C, Minenza E, Linardou H, Burgers S, Salman P, Borghaei H, Ramalingam SS, Brahmer J, Reck M, O'Byrne KJ, Geese WJ, Green G, Chang H, Szustakowski J, Bhagavatheeswaran P et al. (2018) Nivolumab plus Ipilimumab in Lung Cancer with a High Tumor Mutational Burden. N Engl J Med 378:2093-2104

[0201] Hervas-Stubbs S, Perez-Gracia JL, Rouzaut A, Sanmamed MF, Le Bon A, Melero I (2011) Direct effects of type I interferons on cells of the immune system. Clin Cancer Res 17:2619-27

[0202] Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, Gonzalez R, Robert C, Schadendorf D, Hassel JC, Akerley W, van den Eertwegh AJ, Lutzky J, Lorigan P, Vaubel JM, Linette GP, Hogg D, Ottensmeier CH, Lebbe C, Peschel C et al. (2010) Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 363:711-23

[0203] Hubalek M, Czech T, Muller H (2017) Biological Subtypes of Triple-Negative Breast Cancer. Breast Care (Basel) 12:8-14

[0204] Huster KM, Busch V, Schiemann M, Linkemann K, Kerksiek KM, Wagner H, Busch DH (2004) Selective expression of IL-7 receptor on memory T cells identifies early CD40L-dependent generation of distinct CD8+ memory T cell subsets. Proc Natl Acad Sci U S A 101:5610-5

[0205] Ilyas S, Yang JC (2015) Landscape of Tumor Antigens in T Cell Immunotherapy. J Immunol 195:5117-22

[0206] Jacobson et al., Front. Cell. Neurosci., 2019

[0207] Jafari SM, Panjehpour M, Aghaei M, Joshaghani HR, Enderami SE (2017) A3 Adenosine Receptor Agonist Inhibited Survival of Breast Cancer Stem Cells via GLI-1 and ERK1 / 2 Pathway. J Cell Biochem 118:2909-2920

[0208] Kawai T, Akira S (2009) The roles of TLRs, RLRs and NLRs in pathogen recognition. Int Immunol 21:317-37

[0209] Krieg AM(2007)Development of TLR9 agonists for cancer therapy.J ClinInvest 117:1184-94

[0210] Leach DR,Krummel MF,Allison JP(1996)Enhancement of antitumor immunityby CTLA-4blockade.Science 271:1734-6

[0211] Lee SW,Salek-Ardakani S,Mittler RS,Croft M(2009)Hypercostimulationthrough 4-1BB distorts homeostasis of immune cells.J Immunol 182:6753-62

[0212] Liu L,Wang Y,Miao L,Liu Q,Musetti S,Li J,Huang L(2018)CombinationImmunotherapy of MUC1 mRNA Nano-vaccine and CTLA-4Blockade EffectivelyInhibits Growth of Triple Negative Breast Cancer.Mol Ther 26:45-55

[0213] Liu W,Putnam AL,Xu-Yu Z,Szot GL,Lee MR,Zhu S,Gottlieb PA,Kapranov P,Gingeras TR,Fazekas de St Groth B,Clayberger C,Soper DM,Ziegler SF,BluestoneJA(2006)

[0214] CD127 expression inversely correlates with FoxP3 and suppressivefunction of human CD4+T reg cells.J Exp Med 203:1701-11

[0215] Mandal R, Samstein RM, Lee KW, Havel JJ, Wang H, Krishna C, Sabio EY, Makarov V, Kuo F, Blecua P, Ramaswamy AT, Durham JN, Bartlett B, Ma X, Srivastava R, Middha S, Zehir A, Hechtman JF, Morris LG, Weinhold N et al. (2019) Genetic diversity of tumors with mismatch repair deficiency influences anti-PD-1 immunotherapy response. Science 364:485-491

[0216] Marcus L, Lemery SJ, Keegan P, Pazdur R (2019) FDA Approval Summary: Pembrolizumab for the Treatment of Microsatellite Instability-High Solid Tumors. Clin Cancer Res 25:3753-3758

[0217] Marra A, Viale G, Curigliano G (2019) Recent advances in triple negative breast cancer: the immunotherapy era. BMC Med 17:90

[0218] Miller JF, Mitchell GF, Weiss NS (1967) Cellular basis of the immunological defects in thymectomized mice. Nature 214:992-7

[0219] Mitchell BS (2003) The proteasome--an emerging therapeutic target in cancer. N Engl J Med 348:2597-8

[0220] Overman MJ, Lonardi S, Wong KYM, Lenz HJ, Gelsomino F, Aglietta M, Morse MA, Van Cutsem E, McDermott R, Hill A, Sawyer MB, Hendlisz A, Neyns B, Svrcek M, Moss RA, Ledeine JM, Cao ZA, Kamble S, Kopetz S, Andre T (2018) Durable Clinical Benefit With Nivolumab Plus Ipilimumab in DNA Mismatch Repair-Deficient / Microsatellite Instability-High Metastatic Colorectal Cancer. J Clin Oncol 36:773-779

[0221] Park BH, Biggar WD, Good RA (1974) Minnesota experience in bone-marrow transplantation in man, 1968 to June 1973. Transplant Proc 6:379-83

[0222] Rizvi NA, Hellmann MD, Snyder A, Kvistborg P, Makarov V, Havel JJ, Lee W, Yuan J, Wong P, Ho TS, Miller ML, Rekhtman N, Moreira AL, Ibrahim F, Bruggeman C, Gasmi B, Zappasodi R, Maeda Y, Sander C, Garon EB et al. (2015) Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer. Science 348:124-8

[0223] Rodon J,Carducci MA,Sepulveda-Sanchez JM,Azaro A,Calvo E,Seoane J,Brana I,Sicart E,Gueorguieva I,Cleverly AL,Pillay NS,Desaiah D,Estrem ST,Paz-Ares L,Holdhoff M,Blakeley J,Lahn MM,Baselga J(2015)First-in-human dose studyof the novel transforming growth factor-beta receptor I kinase inhibitorLY2157299monohydrate in patients with advanced cancer and glioma.Clin CancerRes 21:553-60

[0224] Segal NH,Logan TF,Hodi FS,McDermott D,Melero I,Hamid O,Schmidt H,Robert C,Chiarion-Sileni V,Ascierto PA,Maio M,Urba WJ,Gangadhar TC,Suryawanshi S,Neely J,Jure-Kunkel M,Krishnan S,Kohrt H,Sznol M,Levy R(2017)Results from an Integrated Safety Analysis of Urelumab,an Agonist Anti-CD137Monoclonal Antibody.Clin Cancer Res 23:1929-1936

[0225] Shi G,Vistica BP,Nugent LF,Tan C,Wawrousek EF,Klinman DM,Gery I(2013)Differential involvement of Th1 and Th17 in pathogenic autoimmune processestriggered by different TLR ligands.J Immunol 191:415-23

[0226] Simpson KD,Templeton DJ,Cross JV(2012)Macrophage migration inhibitoryfactor promotes tumor growth and metastasis by inducing myeloid-derivedsuppressor cells in the tumor microenvironment.J Immunol 189:5533-40

[0227] Sinha P,Clements VK,Bunt SK,Albelda SM,Ostrand-Rosenberg S(2007)Cross-talk between myeloid-derived suppressor cells and macrophages subvertstumor immunity toward a type 2 response.J Immunol 179:977-83

[0228] Sinha P,Clements VK,Ostrand-Rosenberg S(2005a)Interleukin-13-regulated M2macrophages in combination with myeloid suppressor cells blockimmune surveillance against metastasis.Cancer Res 65:11743-51

[0229] Sinha P,Clements VK,Ostrand-Rosenberg S(2005b)Reduction of myeloid-derived suppressor cells and induction of M1 macrophages facilitate therejection of established metastatic disease.J Immunol 174:636-45

[0230] Skrott Z,Cvek B(2012)Diethyldithiocarbamate complex with copper:themechanism of action in cancer cells.Mini Rev Med Chem 12:1184-92

[0231] Smith HO,Wilcox KW(1970)A restriction enzyme from Hemophilusinfluenzae.I.Purification and general properties.J Mol Biol 51:379-91

[0232] Spranger S,Javorovic M,Burdek M,Wilde S,Mosetter B,Tippmer S,BigalkeI,Geiger C,Schendel DJ,Frankenberger B(2010)Generation of Th1-polarizingdendritic cells using the TLR7 / 8 agonist CL075.J Immunol 185:738-47

[0233] Steinman RM,Cohn ZA(1973)Identification of a novel cell type inperipheral lymphoid organs of mice.I.Morphology,quantitation,tissuedistribution.J Exp Med 137:1142-62Stetson DB,Medzhitov R(2006)Recognition ofcytosolic DNA activates an IRF3-dependent innate immune response.Immunity 24:93-103

[0234] Sun L, Wu J, Du F, Chen X, Chen ZJ (2013) Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339:786-91

[0235] Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, Powderly JD, Carvajal RD, Sosman JA, Atkins MB, Leming PD, Spigel DR, Antonia SJ, Horn L, Drake CG, Pardoll DM, Chen L, Sharfman WH, Anders RA, Taube JM et al. (2012) Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 366:2443-54

[0236] Vinay DS, Ryan EP, Pawelec G, Talib WH, Stagg J, Elkord E, Lichtor T, Decker WK, Whelan RL, Kumara H, Signori E, Honoki K, Georgakilas AG, Amin A, Helferich WG, Boosani CS, Guha G, Ciriolo MR, Chen S, Mohammed SI et al. (2015) Immune evasion in cancer: Mechanistic basis and therapeutic strategies. Semin Cancer Biol 35 Suppl: S185-S198

[0237] Waks AG, Winer EP (2019) Breast Cancer Treatment: A Review. JAMA 321:288-300

[0238] Wolchok JD, Chiarion-Sileni V, Gonzalez R, Rutkowski P, Grob JJ, Cowey CL, Lao CD, Wagstaff J, Schadendorf D, Ferrucci PF, Smylie M, Dummer R, Hill A, Hogg D, Haanen J, Carlino MS, Bechter O, Maio M, Marquez-Rodas I, Guidoboni M et al. (2017) Overall Survival with Combined Nivolumab and Ipilimumab in Advanced Melanoma. N Engl J Med 377:1345-1356

[0239] Xagorari A, Chlichlia K (2008) Toll-like receptors and viruses: induction of innate antiviral immune responses. Open Microbiol J 2:49-59

[0240] Yatim N, Cullen S, Albert ML (2017) Dying cells actively regulate adaptive immune responses. Nat Rev Immunol 17:262-275

[0241] Zhang W, Wang G, Xu ZG, Tu H, Hu F, Dai J, Chang Y, Chen Y, Lu Y, Zeng H, Cai Z, Han F, Xu C, Jin G, Sun L, Pan BS, Lai SW, Hsu CC, Xu J, Chen ZZ et al. (2019) Lactate Is a Natural Suppressor of RLR Signaling by Targeting MAVS. Cell 178:176-189e15

[0242] Zhao B,Du F,Xu P,Shu C,Sankaran B,Bell SL,Liu M,Lei Y,Gao X,Fu X,ZhuF,Liu Y,Laganowsky A,Zheng

[0243] Zinkernagel RM, Doherty PC (1974) Restriction of in vitro T cell-mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic orsemiallogeneic system. Nature 248:701-2

[0244] U.S. Patent No. 6,747,014

[0245] U.S. Patent No. 6,753,423

[0246] U.S. Patent Publication No. 2014 / 0005255.

Claims

1. An immunogenic composition comprising at least 4 of the following: a transforming growth factor β (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. The composition according to claim 1, wherein the composition comprises a transforming growth factor β (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. The composition according to claim 1, wherein the composition comprises a transforming growth factor β (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. The composition of claim 1, 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.

5. The composition of claim 1, 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.

6. The composition of claim 1, wherein the composition comprises at least 5 of the following: a transforming growth factor β (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. The composition of claim 1, wherein the composition comprises at least 5 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.

8. The composition of claim 1, wherein the composition comprises a transforming growth factor β (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. The composition of claim 1, 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.

10. The composition of any one of claims 1 to 9, wherein the TGFβ antagonist is galusenti (LY2157299), trabedocan, risorumab, 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 xanthone 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 xanthone analog.

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

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, gadiquimod, 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. The composition of any one of claims 1 to 17, wherein the proteasome inhibitor is copper (II) diethyldithiocarbamate, Bortezomib, Dilanzomib, Ixazomib, MG132, MG115, IPSI 001, Filuramide B, ALLN, Leupeptin, Epoxomicin, Oprozomib, PR-957, Carfilzomib, Lactacystin, Omuralide, Salinosporamide A, Salinosporamide B, Belactosine, Cinnabaramide, Polyphenols, TMC-95 or PS-519.

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-methylformamide (2-Cl-IB-MECA), IB-MECA (CF101), CP-608,039, CP-532,903, MRS5698, MRS5980, LJ-529 33 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 aptamer.

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

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

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

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

25. A method for stimulating an anti-tumor immune response in a subject, the method comprising administering to the subject an effective amount of at least four of the following: a transforming growth factor β (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. The method of claim 25, wherein 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 are administered to the subject.

27. The method of claim 25, wherein a transforming growth factor β (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 are administered to the subject.

28. The method of claim 25, wherein 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 are administered to the subject.

29. The method of claim 25, wherein a transforming growth factor β (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 are administered to the subject.

30. The method of claim 25, wherein the subject is administered at least 5 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. The method of claim 25, wherein the subject is administered at least 5 of the following: a transforming growth factor β (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. The method of claim 25, wherein 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 to the subject.

33. The method of claim 25, wherein the subject is administered a transforming growth factor β (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. The method of any one of claims 25 to 33, wherein the TGFβ antagonist is galusenti (LY2157299), trabedocan, risorumab, 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 xanthone 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. The method of any one of claims 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. The method of claim 35, wherein the STING agonist is a xanthone analog.

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

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, gadiquimod, 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. The method of any one of claims 25 to 41, wherein the proteasome inhibitor is copper (II) diethyldithiocarbamate, Bortezomib, Dilanzomib, Ixazomib, MG132, MG115, IPSI 001, Filuramide B, ALLN, Leupeptin, Epoxomicin, Oprozomib, PR-957, Carfilzomib, Lactacystin, Omuralide, Salinosporamide A, Salinosporamide B, Belactosine, Cinnabaramide, Polyphenols, TMC-95, or PS-519.

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-methylformamide (2-Cl-IB-MECA), IB-MECA (CF101), CP-608,039, CP-532,903, MRS5698, MRS5980, LJ-529 33 or MRS4322.

44. according to the method described in 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 aptamer.

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

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. 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 the administering is performed prior to surgical intervention.

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

51. The method of any one of claims 50, wherein the intratumoral injection does not comprise 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 administration of the immunogenic composition results in an increase in circulating CD8+ T cells, M1 macrophages, and / or tumor infiltrating dendritic cells.

55. The method of claim 54, wherein the CD8+ T cells are CD8 + IFN-γ + cell.

56. The method of any one of claims 25 to 55, wherein administration of the immunogenic composition results in a persistent T cell memory response, such as by binding to a CD44 + CD127 + T cells compared to CD44 + CD127 + Measured by the increase in T cells.

57. The method of any one of claims 25 to 56, wherein the method does not comprise administering cell therapy to the subject at the time of administering the immunogenic composition.

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

59. A method for treating a subject having cancer, the method comprising administering to the subject an effective amount of at least four of the following: a transforming growth factor β (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. The method of claim 59, wherein 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 are administered to the subject.

61. The method of claim 59, wherein a transforming growth factor β (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 are administered to the subject.

62. The method of claim 59, wherein 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 are administered to the subject.

63. The method of claim 59, wherein a transforming growth factor β (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 are administered to the subject.

64. The method of claim 59, wherein the subject is administered at least 5 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. The method of claim 59, wherein the subject is administered at least 5 of the following: a transforming growth factor β (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. The method of claim 59, wherein 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 to the subject.

67. The method of claim 59, wherein the subject is administered a transforming growth factor β (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. The method of any one of claims 59 to 67, wherein the TGFβ antagonist is galusenti (LY2157299), trabedocan, risorumab, 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 xanthone 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. The method of any one of claims 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. The method of claim 69, wherein the STING agonist is a xanthone analog.

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

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, gadiquimod, loxoribine, or a ssRNA oligonucleotide.

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. The method of any one of claims 59 to 75, wherein the proteasome inhibitor is copper (II) diethyldithiocarbamate, Bortezomib, Dilanzomib, Ixazomib, MG132, MG115, IPSI 001, Filuramide B, ALLN, Leupeptin, Epoxomicin, Oprozomib, PR-957, Carfilzomib, Lactacystin, Omuralide, Salinosporamide A, Salinosporamide B, Belactosine, Cinnabaramide, Polyphenols, TMC-95, or PS-519.

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-methylformamide (2-Cl-IB-MECA), IB-MECA (CF101), CP-608,039, CP-532,903, MRS5698, MRS5980, LJ-529 33 or MRS4322.

78. according to the method described in 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 aptamer.

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

80. The method of any one of claims 59 to 77, wherein the administering is performed prior to 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 injecting the immunogenic composition.

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. 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 more than one injection.

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

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

87. The method of any one of claims 85 to 86, wherein the intratumoral injection does not comprise a hydrogel.

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

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

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 administration results in an increase in circulating CD8+ T cells, M1 macrophages, and / or tumor infiltrating dendritic cells.

92. The method of claim 91, wherein the CD8+ T cells are CD8 + IFN-γ + cell.

93. The method of any one of claims 59 to 92, wherein administration results in a persistent T cell memory response, such as by binding to CD44 + CD127 + T cells compared to CD44 + CD127 + Measured by the increase in T cells.

94. The method of any one of claims 59 to 94, wherein the method does not include administering cell therapy to the subject while administering the transforming growth factor β (TGFβ) antagonist, 4-1BB agonist, stimulator of interferon genes (STING) agonist, Toll-like receptor 7 / 8 (TLR7 / 8) agonist, RIG-I agonist, proteasome inhibitor, and adenosine A3 receptor agonist.

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

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

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

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

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

100. A method for preventing tumor metastasis in a subject having cancer, the method comprising administering to the subject an effective amount of at least four of the following: a transforming growth factor β (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. The method of claim 100, wherein 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 are administered to the subject.

102. The method of claim 100, wherein a transforming growth factor β (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 are administered to the subject.

103. The method of claim 100, wherein 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 are administered to the subject.

104. The method of claim 100, wherein a transforming growth factor β (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 are administered to the subject.

105. The method of claim 100, wherein the subject is administered at least 5 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. The method of claim 100, wherein the subject is administered at least 5 of the following: a transforming growth factor β (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. The method of claim 100, wherein 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 to the subject.

108. The method of claim 100, wherein a transforming growth factor β (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 to the subject.

109. The method of any one of claims 100 to 108, wherein the TGFβ antagonist is galusarti (LY2157299), trabedocan, risorumab, 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 xanthone 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 any one of claims 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 xanthone analog.

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

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, gadiquimod, loxoribine, or a ssRNA oligonucleotide.

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. The method of any one of claims 100 to 116, wherein the proteasome inhibitor is copper (II) diethyldithiocarbamate, Bortezomib, Dilanzomib, Ixazomib, MG132, MG115, IPSI 001, Filuramide B, ALLN, Leupeptin, Epoxomicin, Oprozomib, PR-957, Carfilzomib, Lactacystin, Omuralide, Salinosporamide A, Salinosporamide B, Belactosine, Cinnabaramide, Polyphenols, 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-methylformamide (2-Cl-IB-MECA), IB-MECA (CF101), CP-608,039, CP-532,903, MRS5698, MRS5980, LJ-529 33 or MRS4322.

119. according to the method described in 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 aptamer.

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

121. The method of any one of claims 100 to 118, wherein the administering is performed prior to 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 administering comprises injecting the immunogenic composition.

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

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

128. The method of any one of claims 126 to 127, wherein the intratumoral injection does not comprise a hydrogel.

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

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 administration results in an increase in circulating CD8+ T cells, M1 macrophages, and / or tumor infiltrating dendritic cells.

132. The method of claim 131, wherein the CD8+ T cells are CD8 + IFN-γ + cell.

133. The method of any one of claims 100 to 132, wherein administration results in a persistent T cell memory response, such as by binding to CD44 + CD127 + T cells compared to CD44 + CD127 + Measured by the increase in T cells.

134. The method of any one of claims 100 to 134, wherein the method does not include administering cell therapy to the subject while administering the transforming growth factor β (TGFβ) antagonist, stimulator of interferon genes (STING) agonist, Toll-like receptor 7 / 8 (TLR7 / 8) agonist, RIG-I agonist, proteasome inhibitor, and adenosine A3 receptor agonist.

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

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

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

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

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

140. An immunogenic composition comprising at least 4 of the following: a transforming growth factor β (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, the composition being for use in treating cancer by administering the composition to a subject.

141. A 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 of claim 140, wherein the composition comprises a transforming growth factor β (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. A 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 of claim 140, wherein the composition comprises a transforming growth factor β (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. A composition for use according to claim 140, wherein the composition comprises at least 5 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.

146. A composition for use according to claim 140, wherein the composition comprises at least 5 of the following: a transforming growth factor β (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. A 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 of claim 140, wherein the composition comprises a transforming growth factor β (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. The composition for use of any one of claims 140 to 148, wherein the TGFβ antagonist is galusenti (LY2157299), trabedecantan, risorimab, LY2382770, Lucanix, or PF-03446962.

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

151. The composition for use of 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 xanthone analog.

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

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

156. The composition for use of 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. A composition for use according to any one of claims 140 to 156, wherein the proteasome inhibitor is copper (II) diethyldithiocarbamate, Bortezomib, Dilanzomib, Ixazomib, MG132, MG115, IPSI001, Filuramide B, ALLN, Leupeptin, Epoxomicin, Oprozomib, PR-957, Carfilzomib, Lactacystin, Omuralide, Salinosporamide A, Salinosporamide B, Belactosine, Cinnabaramide, Polyphenols, TMC-95 or PS-519.

158. A 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-methylformamide (2-Cl-IB-MECA), IB-MECA (CF101), CP-608,039, CP-532,903, MRS5698, MRS5980, LJ-529 33 or MRS4322.

159. A 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 aptamer.

160. The composition for use of claim 159, wherein the 4-1BB agonist antibody is tomilizumab or usrelizumab.

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

162. The composition for use of 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 of any one of claims 140 to 162, wherein the composition is administered prior to surgical intervention.

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

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

166. A composition for use according to 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 two or more times.

168. The composition for use of claim 165, wherein administration of the immunogenic composition results in an increase in circulating CD8+ T cells, M1 macrophages, and / or tumor infiltrating dendritic cells.

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

170. A composition for use according to any one of claims 140 to 169, wherein the composition results in a persistent T cell memory response, such as by binding to a CD44 + CD127 + T cells compared to CD44 + CD127 + Measured by the increase in 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. The composition for use of any one of claims 140 to 172, wherein the intratumoral injection does not comprise a hydrogel.

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

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

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

177. A 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. The composition for use of 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, hormone therapy, anti-angiogenic therapy, or immunotherapy.

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