Compositions and methods for enhancing the therapeutic efficacy of cancer treatments

Abstract

The present disclosure relates to compositions and methods for enhancing the therapeutic effect of cancer treatment.Disclosed herein are methods and combination therapies for treating cancer, which significantly reduce tumor growth in treated individuals, increase survival rate, and increase complete response rate.The present disclosure demonstrates that adjuvants (such as vaccine adjuvants) can enhance the effectiveness of in situ tumor vaccine approaches combined with radiation therapy and checkpoint inhibitor treatment, and promote the immune system's response to tumor cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 408,610, filed September 21, 2022, the entirety of which is incorporated herein by reference.

[0002] INCORPORATION-BY-REFERENCE OF SEQUENCE LISTINGS PROVIDED ELECTRONICALLY This application contains a Sequence Listing that has been submitted as an electronic text file entitled "22-0925-WO_ST26.xml," having a byte size of 6,159 bytes and created on September 20, 2023. The information contained in this electronic file is incorporated herein by reference in its entirety. [Background technology]

[0003] Summary of disclosure FIELD OF THE INVENTION The present disclosure relates to compositions and methods for enhancing the therapeutic efficacy of cancer treatments.

[0004] Technical background Radiation therapy (RT) has been demonstrated in mouse models and cancer patients to produce an in situ vaccination (ISV) effect by inducing tumor cell death so that tumor cells present tumor antigens to immune cells. Unfortunately, RT also adversely affects the tumor microenvironment, which can inhibit immune cell activity against tumors. This is why RT generally does not induce systemic immune activation, and the occurrence of subsequent antitumor responses outside the radiation field (i.e., abscopal responses) remains extremely rare when RT is used. Checkpoint inhibitors are an emerging clinical class of molecules that block immune cell checkpoint pathways that lead to immune stimulation. In addition to inhibiting checkpoint pathways to stimulate immune cells, the release of antigens from tumors upon radiation exposure should result in an enhanced tumor-killing response. However, radiation has not routinely translated into enhanced clinical responses to immune checkpoint inhibition (ICI). Therefore, additional treatments to further stimulate the immune system are needed to enhance the anticancer activity of radiation in combination with checkpoint inhibitors. Summary of the Invention [Means for solving the problem]

[0005] Summary of disclosure The present disclosure provides compositions and methods for enhancing the therapeutic efficacy of cancer treatments.

[0006] In a first aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of an adjuvant to a tumor in the subject, wherein the adjuvant is administered intratumorally, administering a therapeutically effective amount of radiation therapy and / or local ablative therapy to the tumor, administering a therapeutically effective amount of an immune checkpoint inhibitor to the subject, and inducing an immune response against the cancer.

[0007] In one embodiment of the first aspect, the adjuvant comprises a TLR4 agonist. In one embodiment of the first aspect, the TLR4 agonist comprises one or more of monophosphoryl lipid A, monophosphoryl lipid A-504, monophosphoryl triacyl lipid A, monophosphoryl 3-deacyl lipid A, monophosphoryl tetraacyl lipid A, monophosphoryl hexaacyl lipid A, 3-deacyl, D-(+)-trehalose 6,6'-dibehenate, and dimethyldioctadecylammonium (bromide salt). In one embodiment of the first aspect, the therapeutically effective amount of the adjuvant comprises about 20 μg to about 70 mg or about 0.5 to about 5 mg / kg. In one embodiment of the first aspect, the radiation therapy comprises external beam radiation therapy (EBRT) and / or internal radiation therapy. In one embodiment of the first aspect, the radiation therapy is EBRT. In one embodiment of the first aspect, the internal radiation therapy comprises brachytherapy and / or a radiopharmaceutical. In one embodiment of the first aspect, the therapeutically effective amount of radiation therapy is about 2 to about 20 Gy. In one embodiment of the first aspect, the therapeutically effective amount of radiation therapy is administered at a gradient dose of about 2 Gy / min. In one embodiment of the first aspect, the local ablative treatment comprises radiofrequency ablation, microwave ablation, and / or cryoablation. In one embodiment of the first aspect, the immune checkpoint inhibitor comprises one or more therapeutic agents that inhibit CTLA-4, PD-1, and / or PD-L1. In one embodiment of the first aspect, the immune checkpoint inhibitor comprises an anti-CTLA-4 antibody.

[0008] In a second aspect, the present disclosure provides a composition for treating cancer, the composition comprising a therapeutically effective amount of an adjuvant, a therapeutically effective amount of an immune checkpoint inhibitor, and a pharmaceutically acceptable carrier or diluent.

[0009] In one embodiment of the second aspect, the adjuvant comprises a TLR4 agonist. In one embodiment of the second aspect, the TLR4 agonist comprises one or more of monophosphoryl lipid A, monophosphoryl lipid A-504, monophosphoryl triacyl lipid A, monophosphoryl 3-deacyl lipid A, monophosphoryl tetraacyl lipid A, monophosphoryl hexaacyl lipid A, 3-deacyl, D-(+)-trehalose 6,6'-dibehenate, and dimethyldioctadecylammonium (bromide salt). In one embodiment of the second aspect, the immune checkpoint inhibitor comprises one or more therapeutic agents that inhibit CTLA-4, PD-1, and / or PD-L1. In one embodiment of the second aspect, the immune checkpoint inhibitor comprises an anti-CTLA-4 antibody.

[0010] In a second aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of a TLR4 agonist to a tumor in the subject, wherein the TLR4 agonist is administered intratumorally, administering a therapeutically effective amount of EBRT to the tumor, administering a therapeutically effective amount of an anti-CTLA-4 antibody to the subject, and at least one of reducing tumor volume, increasing overall survival of the subject, and increasing complete response rate in the subject.

[0011] In one embodiment of the third aspect, the TLR4 agonist comprises one or more of monophosphoryl lipid A, monophosphoryl lipid A-504, monophosphoryl triacyl lipid A, monophosphoryl 3-deacyl lipid A, monophosphoryl tetraacyl lipid A, monophosphoryl hexaacyl lipid A, 3-deacyl, D-(+)-trehalose 6,6'-dibehenate, and dimethyldioctadecylammonium (bromide salt). In one embodiment of the third aspect, the therapeutically effective amount of the TLR4 agonist comprises about 0.5 to about 5 mg / kg. In one embodiment of the third aspect, the therapeutically effective amount of EBRT is about 2 to about 20 Gy administered at a gradient dose of about 2 Gy / min. In one embodiment of the third aspect, the therapeutically effective amount of the anti-CTLA-4 antibody is about 10 mg / kg.

[0012] In one embodiment according to any of the first or third aspects or embodiments thereof, the cancer comprises one or more solid tumors. In one embodiment, the cancer is melanoma or prostate cancer.

[0013] In one embodiment according to any of the first or third aspects or embodiments thereof, the method further comprises increasing the production of tumor-associated Th1-associated IgG2c anti-tumor antibodies.

[0014] In one embodiment according to any of the first or third aspects or embodiments thereof, the method further comprises inducing a systemic anti-tumor immune response.

[0015] These and other features and advantages of the present invention will be more fully understood from the following detailed description taken in conjunction with the appended claims, which should be noted that the claims are defined by the recitations therein, not the specific recitations of the features and advantages described herein.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings are included to provide a further understanding of the methods and compositions of the present disclosure, and are incorporated herein and constitute a part of this specification. The drawings illustrate one or more embodiments of the present disclosure and, together with the description, serve to explain the principles and operation of the present disclosure. Unless otherwise specified, all data presented are reported as mean ± SEM. For all graphs, * , p<0.05; ** , p<0.01; *** , p<0.001; and **** , p<0.0001. [Brief explanation of the drawings]

[0017] [Figure 1-1]MPL enhances the efficacy of RT+C4 in B78 melanoma and Myc-CaP prostate cancer models. (1A) Mice bearing single B78 or Myc-CaP flank tumors were treated with PBS (p<0.05 vs. RT+C4 and RT+C4+MPL), external beam radiation (RT, 12 Gy; p<0.05 vs. RT+C4+MPL), RT+anti-CTLA-4 (C4, 200 μg; p<0.05 vs. PBS, RT, and RT+C4+MPL), RT+C4+MPL (MPL, 20 μg; p<0.05 vs. PBS, RT, RT+C4, and MPL), or MPL alone (p<0.05 vs. RT+C4 and RT+C4+MPL). Tumor response and animal survival by individual animal per group are shown for B78 (in 1B, 1C, and 1D (p<0.05 vs. PBS-RT+C4 and RT+C4+MPL; p<0.05 vs. RT-RT+C4+MPL; p<0.05 vs. RT+C4-PBS, RT, and RT+C4+MPL; p<0.05 vs. RT+C4+MPL-PBS, RT, RT+C4, and MPL; p<0.05 vs. MPL-RT+C4 and RT+C4+MPL)). Mice that showed a complete response to treatment with either RT+C4 or RT+C4+MPL (1E) were rechallenged with the same tumor that had initially rejected them (1F). For Myc-CaP, tumor response and animal survival by individual animal per group are shown (1G (p<0.05 vs. PBS-RT+C4+MPL; p<0.05 vs. RT-RT+C4+MPL, p<0.05 vs. RT+C4-RT+C4+MPL; p<0.05 vs. RT+C4+MPL-PBS, RT, RT+C4, and MPL, p<0.05 vs. MPL-RT+C4+MPL), 1H, and and 1I (p<0.05 for PBS-RT+C4 and RT+C4+MPL; p<0.05 for RT-RT+C4 and RT+C4+MPL; p<0.05 for RT+C4-PBS, RT, RT+C4+MPL and MPL; p<0.05 for RT+C4+MPL-PBS, RT, RT+C4 and MPL; p<0.05 for MPL-RT+C4 and RT+C4+MPL).Mice that showed a complete response to treatment with either RT + C4 or RT + C4 + MPL (1J) were rechallenged with the same tumor that initially rejected them (1K). N = 10–16 mice per group. Significance was determined by linear mixed-effects regression analysis with Tukey's multiple comparison test for tumor growth (significant differences were defined by *, with asterisks indicating which group the sample was significantly different from, p<0.05). Survival analysis was performed using log-rank tests and Kaplan-Meier estimates with Cox regression (significant differences were defined by *, with asterisks indicating which group the sample was significantly different from, p<0.05). Significance was determined by chi-square tests for complete response rates. MPL, monophosphoryl lipid; RT, radiation therapy; EBRT, external beam radiation therapy; PBS, phosphate-buffered saline. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 1-5] Same as above. [Figure 1-6] Same as above. [Figure 1-7] Same as above.

[0018] [Figure 2-1]MPL promotes Th1 antibody class switching and correlates with the depth of tumor response. To determine the presence of antitumor antibodies, serum was isolated from B78 tumor-bearing mice on days 15 and 30 after treatment initiation. The serum was incubated with B78 cells, and antibody classes were determined using secondary antibodies against IgG, IgG1, and IgG2c (2A). The IgG2c:IgG1 ratio at day 15 was increased in RT+C4+MPL compared with the other groups, where total IgG remained unchanged (2B, 2C). A similar trend in the IgG2c:IgG1 ratio was observed at day 30, but did not reach statistical significance (2D, 2E). In a treatment-independent manner, mice were reclassified based on the depth of response using RECIST 1.1 criteria [PD: progression N=33, SD: stable disease N=6, PR: partial response N=7, CR: complete response N=9], and the IgG2c:IgG1 ratio was quantified (2F). Mice experiencing any response (SD, PR, or CR, N = 22) were also pooled, and the IgG2c:IgG1 ratio was compared with that of non-responder mice (PD, N = 33) (2G). Within treatment groups that included mice experiencing complete responses (RT + C4 and RT + C4 + MPL), IgG2c:IgG1 was quantified in mice experiencing complete responses (RT + C4, N = 2 and RT + C4 + MPL, N = 7) and compared with non-responder mice (RT + C4, N = 14 and RT + C4 + MPL, N = 9). The IgG2c:IgG1 ratio was elevated in complete responder mice treated with RT + C4 + MPL (2H), but not in RT + C4 (2I). One-way ANOVA with Tukey's truly significant difference (HSD) test to adjust for multiple comparisons was used to assess the statistical significance of mean differences observed in the IgG2c:IgG1 ratio. For comparison between two groups, Student's t test was performed. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above. [Figure 2-5] Same as above.

[0019] [Figure 3-1]MPL reprograms the immune microenvironment to promote M1 and Th1 polarization. Flow cytometry analysis of tumor immune cell infiltrates as a percentage of total viable cells [total macrophages (CD11b+F4 / 80+), M1 macrophages (CD80+), M2 macrophages (CD206+), M1:M2 macrophage ratio, and CD8+ T cells (CD8+)] (Figures 3A-3E) and lymph node immune populations [classical dendritic cells (CD11c+CD103+MHC-II+CD80+), Th1 cells (TBET+CD4+), resident memory CD4 T cells (CD4+CD103+), Ifnγ-producing CD8 T cells (CD8+Ifnγ+), and resident memory CD8 T cells (CD8+CD103+)] (Figures 3F-3J) is shown 15 days after treatment initiation in B78 melanoma. N = 7 mice per group. Tumors from a separate cohort of mice (3K) were subjected to cytokine profiling. Cytokine and chemokine concentrations in tumor lysates were measured by multiplex immunoassay. Hierarchical clustering analysis was performed, and assay results were expressed as Z-scores for each cytokine or chemokine. Addition of MPL to RT+C4 increased the expression of TLR4-associated cytokines (3L), increased Th1 cytokines (3M), decreased Th2 cytokines (3N), and increased several pro-inflammatory M1 cytokines compared with RT+C4 (3O). N = 6 mice per group. CR, complete response; MFI, median fluorescence intensity; MPL, monophosphoryl lipid; RT, radiation therapy; TBET, T-box transcription factor 21. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 3-4] Same as above. [Figure 3-5] Same as above. [Figure 3-6] Same as above. [Figure 3-7] Same as above.

[0020] [Figure 4-1]MPL directly activates macrophages and influences Th cell polarization. Bone marrow-derived macrophages were cultured in the presence of increasing amounts of MPL (0, 5, 20, 100, and 500 ng / mL) and harvested for qPCR analysis 24 hours later to quantify polarization (4A), pro-inflammatory cytokines (4B), and anti-inflammatory markers (4C). This was repeated using freshly isolated CD4 and CD8 cells from mouse spleens to determine CD4 polarization (4D), CD4 activation (4E), and CD8 activation (4F) after MPL treatment. (For 4A–4F, data corresponding to MPL concentrations of 5, 20, 100, and 500 ng / mL are presented from left to right for each gene measured.) The potential of MPL-stimulated macrophages to activate CD8 T cells and polarize CD4 T cells was determined using a coculture system (4G). MPL-stimulated macrophages increased activation of CD8 T cells (CD69+) when cocultured (4H), but not in the presence of CD4 T cells (4I). MPL-stimulated macrophages increased Th1 polarization (CXCR3+) (4J), decreased Th2 polarization (CXCR4+) (4K), and increased regulatory T cell polarization (CD25+FOXP3+) (4L) when cocultured. The same trends were observed with the addition of CD8 T cells (4M-4O). One-way ANOVA with Tukey's truly significant difference (HSD) test to adjust for multiple comparisons was used to assess the statistical significance of the mean differences observed in gene expression (significant difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). N=5 replicates per group. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 4-4] Same as above. [Figure 4-5] Same as above.

[0021] [Figure 5-1]Serum antibodies bound to tumor cells enhance MPL-induced activation of macrophages. Bone marrow-derived macrophages were irradiated in culture (12 Gy), and the medium was immediately replaced with fresh medium containing 100 ng / mL MPL. After 24 h, cells were harvested for qPCR analysis of polarization and activation markers (5A). The ability of serum-derived antitumor antibodies to activate macrophages was tested using a coculture system (5B). Macrophages were cultured with PBS, MPL (100 ng / mL), serum from mice that had been rendered disease-free from B78 tumors by treatment with RT, C4, and MPL, or both MPL and serum. After 24 h, cells were harvested for analysis of polarization (5C) and activation markers (5D). To test whether macrophages could be activated in the presence of tumor cells, macrophages were cultured with or without 100 ng / mL MPL, and after 24 h, B78 cells were added with or without serum from disease-free mice. After 24 hours of co-culture, macrophages were harvested for analysis of polarization (5E) and activation markers (5F). To confirm the tumor specificity of the serum antibodies, this was repeated with the unrelated cell line Myc-CaP (5G, 5H). One-way ANOVA with Tukey's truly significant difference (HSD) test to adjust for multiple comparisons was used to assess the statistical significance of the observed mean differences in gene expression (significant difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). N=3 replicates per group. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 5-4] Same as above. [Figure 5-5] Same as above. [Figure 5-6] Same as above. [Figure 5-7] Same as above.

[0022] [Figure 6-1]Radiation downregulates TLR4 expression on macrophages and MHC-II expression on B16 tumor cells. Cytotoxic CD4+ cells are not generated by the combination treatment, but B cells are moderately activated by radiation but not by MPL or serum. In vitro cultured macrophages were treated with PBS, anti-CTLA-4 (C4, 5 μg), radiation (RT, 12 Gy), or RT + C4, and TLR4 expression was quantified using flow cytometry 24 hours after treatment (6A). In vitro cultured B16 melanoma cells were treated with PBS or radiation (RT, 12 Gy), and MHC-II expression was quantified using flow cytometry 3 days after treatment (6B). B16 tumors were treated with PBS or RT + C4 + MPL, and MHC-II expression was quantified using flow cytometry 15 days after treatment (6C). CD4+ cells were also harvested and co-cultured with B16 cells that received either 0 or 12 Gy of RT 7 days prior to co-culture, and tumor cell killing was quantified using Annexin V staining (6D). In vitro cultured B cells were treated with either PBS, MPL (100 μg), serum from disease-free mice, or MPL + serum, and activation markers were quantified using qPCR (6E). In vitro cultured B cells were treated with either PBS, C4 (5 μg), RT (12 Gy), or RT + C4, and activation markers were quantified using qPCR (6F). [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above. [Figure 6-5] Same as above.

[0023] [Figure 7-1]Serum antibody-induced macrophage activation is dependent on Fcγ receptors and is important for antitumor responses. The significance of Fcγ receptors was tested in vivo. Wild-type and Fcγ receptor-deficient mice were treated with either PBS or RT+C4+MPL (three times) and followed for tumor growth and survival (7A, 7B (p<0.05 vs. PBS WT-3×WT and p<0.05 vs. 3×WT-PBS WT)). N=5 mice per group. Bone marrow-derived macrophages were irradiated in culture (12 Gy), and the medium was immediately replaced with fresh medium containing 100 ng / mL MPL. After 24 h, cells were harvested for qPCR analysis of Fcγ receptor expression (7C). To determine the importance of Fcγ receptors for macrophage activation, macrophages were cultured with PBS, MPL (100 ng / mL), serum from mice that had been rendered disease-free from B78 tumors by RT+C4+MPL treatment, or both MPL and serum. After 24 hours, cells were harvested for analysis of polarization (7D) and activation markers (7E). To test whether macrophages could be activated in the presence of tumor cells, macrophages were cultured with or without 100 ng / mL MPL. After 24 hours, B78 cells were added with or without serum from disease-free mice. After 24 hours of coculture, macrophages were harvested for analysis of polarization (7F) and activation markers (7G). N = 3 replicates per group. To test whether serum-derived antitumor antibodies could initiate ADCC (antibody-dependent cellular cytotoxicity), unstimulated and MPL-stimulated macrophages were cocultured with B78 cells with or without serum. After 24 hours, cells were harvested and analyzed by flow cytometry. CD45-Annexin V+ cells are plotted (7H). N = 5 replicates per group. One-way ANOVA with Tukey's truly significant difference (HSD) test to adjust for multiple comparisons was used to assess the statistical significance of observed mean differences in gene expression. Linear mixed-effects regression analysis with Tukey's multiple comparison test was used to compare tumor growth. Kaplan-Meier estimation with log-rank tests and Cox regression was performed for survival analysis (significant differences defined by *, p<0.05, with asterisks indicating which samples were significantly different from which groups). [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 7-4] Same as above. [Figure 7-5] Same as above. [Figure 7-6] Same as above.

[0024] [Figure 8-1]MPL-induced immune responses depend on macrophages and Th1 CD4 T cells. The importance of different immune cell populations for the antitumor response generated by RT+C4+MPL was determined using antibody-based depletion. B78 tumor-bearing mice were treated with either PBS or RT+C4+MPL (3x) and depleted of macrophages (αCD115), NK cells (αNK1.1), CD4 T cells (αCD4), CD8 T cells (αCD8), or CD4 T cells and CD8 T cells were depleted for either both (αCD115 / CD8) and tumor growth and survival were followed (8A, 8B (p<0.05 for PBS-3x+IgG, 3x+αNK1.1, and 3x+αCD8; p<0.05 for 3x+IgG-PBS, 3x+αCD115, 3x+αNK1.1, and 3x+αCD4 / CD8; p<0.05 for 3x+αCD115-3x+IgG, 3x+αNK1.1, and 3x+αCD8; p<0.05 for 3x+αNK1.1) p<0.05 for -PBS, 3x+αCD115, 3x+αCD4, and 3x+αCD4 / CD8; p<0.05 for 3x+αCD4-3x+IgG, 3x+αNK1.1, and 3x+αCD8; p<0.05 for 3x+αCD8-PBS, 3x+αCD115, 3x+αCD4, and 3x+αCD4 / CD8; and p<0.05 for 3x+αCD4 / CD8-3x+IgG, 3x+αNK1.1, and 3x+αCD8. N = 5–10 mice per group.Serum was collected 15 days after RT for antitumor antibody quantification (8C (PBS-p<0.0001 for CD8, NK, and 3x; 3x-CD115, p<0.0001 for CD4+CD8, CD4, and PBS; CD115-p<0.05 for CD8 and NK and p<0.0001 for CD4+CD8, CD4, and 3x; NK-p<0.05 for CD115 and p<0.0001 for CD4+CD8, CD4, and PBS; CD4-CD115, p<0.0001 for CD8, NK, and 3x; CD8-CD1 p<0.05 for 15 and p<0.0001 for CD4+CD8, CD4 and PBS; and p<0.0001 for CD4+CD8-CD115, CD8, NK and 3x), 8D (p<0.05 for NK-CD4+CD8 and p<0.01 for CD4; p<0.01 for CD4-CD8 and NK; p<0.01 for CD8-CD4+CD8 and CD4; and p<0.05 for CD4+CD8-NK, p<0.01 for CD8). The importance of CD4 T cells was determined using TBET-deficient mice. Wild-type and TBET-deficient mice (TBET KO) were treated with either PBS or RT + C4 + MPL (three times) and tumor growth was followed (8E (p<0.05 vs. PBS WT-3xWT; and p<0.05 vs. 3xWT-PBS WT)). N=5 mice per group. On day 15 after RT, serum was collected for anti-tumor antibody quantification (8F (p<0.05 vs. 3xWT-3xPBSWT; p<0.01 vs. PBS TBET KO and 3xTBETKO)). Tumors and draining lymph nodes were collected for invasion analysis (8G, 8H). Tumor growth was compared using linear mixed-effects regression analysis with Tukey's multiple comparison test. Kaplan-Meier estimation using the log-rank test and Cox regression was performed for survival analysis (significant differences, p<0.05, are defined by *, with asterisks indicating which group the sample is significantly different from).One-way ANOVA with Tukey's truly significant difference (HSD) test adjusting for multiple comparisons was used to assess the statistical significance of observed mean differences in lung metastases (significant difference, *, p<0.05; **, p<0.01; ***, p<0.001; and ****, p<0.0001; the color of the asterisk indicates which group the sample is significantly different from). [Figure 8-2] Same as above. [Figure 8-3] Same as above. [Figure 8-4] Same as above.

[0025] [Figure 9-1]MPL enhances systemic immune responses independently of CD8 T cells. To determine whether CD8 T cells are required for the generation of systemic immune responses, mice bearing B78 primary tumors and B16 lung metastases (250,000 B16 cells injected via the tail vein immediately after RT) were treated with either PBS, RT, RT + C4, RT + C4 + MPL, or MPL. In addition, a separate cohort of mice treated with RT + C4 + MPL was also treated with a CD8-depleting antibody. Tumor volume and survival were tracked (9A (p<0.05 for PBS-RT, RT+C4, RT+C4+MPL, and RT+C4+MPL+αCD8; p<0.05 for RT-PBS and RT+C4+MPL; p<0.05 for RT+C4-PBS and RT+C4+MPL; p<0.05 for RT+C4+MPL-PBS, RT, RT+C4, and MPL; p<0.05 for RT+C4+MPL+αCD8-PBS and MPL; and p<0.05 for MPL-RT+C4, RT+C4+MPL, and RT+C4+MPL+αCD8), 9B (p<0.05 for PBS-RT+C4+MPL and RT+C4+MPL+αCD8; p<0.05 for RT-PBS, RT+C4+MPL, and RT+C4+MPL+αCD8). p<0.05; p<0.05 vs. RT+C4-PBS and MPL; p<0.05 vs. RT+C4+MPL-PBS, RT, RT+C4, and MPL; p<0.05 vs. RT+C4+MPL+αCD8-PBS, RT, RT-C4, and MPL; and p<0.05 vs. MPL-RT+C4+MPL and RT+C4+MPL+αCD8. At the time of death or 60 days after treatment initiation, lungs were excised and the number of metastases was calculated (9C). N=5-6 mice per group. Tumor growth was compared using linear mixed-effects regression analysis with Tukey's multiple comparison test. Kaplan-Meier estimation using log-rank tests and Cox regression was performed for survival analysis (significant differences, p<0.05, with asterisks indicating which group the sample was significantly different from).One-way ANOVA with Tukey's truly significant difference (HSD) test adjusting for multiple comparisons was used to assess the statistical significance of observed mean differences in lung metastases (significant difference, *, p<0.05; **, p<0.01; ***, p<0.001; and ****, p<0.0001; the color of the asterisk indicates which group the sample is significantly different from). [Figure 9-2] Same as above.

[0026] [Figure 10-1] Depletion confirmation. Multiple depletion studies were performed to deplete T cells (10A), NK cells (10B), and macrophages (10C). In systemic disease studies, CD8+ T cells were depleted (10D). [Figure 10-2] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0027] Detailed Description It is to be understood that certain aspects of the specification described herein are not limited to the specific embodiments presented and may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting unless specifically defined herein. Furthermore, certain embodiments disclosed herein can be combined with other embodiments disclosed herein, as recognized by those skilled in the art, without limitation.

[0028] All publications, patents, and patent applications cited herein are expressly incorporated by reference in their entirety for all purposes.

[0029] Throughout this specification, unless the context specifically dictates otherwise, the terms "comprise" and "include" and variations thereof (e.g., "comprises," "comprising," "includes," and "including") will be understood to indicate the inclusion of a stated component, feature, element, or step, or group of components, features, elements, or steps, but not the exclusion of any other component, feature, element, or step, or group of components, features, elements, or steps. Any of the terms "comprising," "consisting essentially of," and "consisting of" may be substituted for either of the other two terms while retaining their ordinary meaning.

[0030] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0031] In some embodiments, the amounts or percentages disclosed herein can vary by ±10, 20, or 30% amounts from the disclosed values ​​and still be within the contemplated disclosed range.

[0032] Unless otherwise indicated or apparent from the context and the understanding of one of ordinary skill in the art, values ​​herein expressed as ranges can assume any specific value or subrange within the range set forth in different embodiments of this disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0033] As used herein, ranges and amounts may be expressed as "about" a particular value or range. About includes the exact amount. For example, "about 5%" means "about 5%" and also "5%." The term "about" may also refer to ±10% of a given value or range of values. Thus, about 5% also means, for example, 4.5% to 5.5%.

[0034] As used herein, the terms "or" and "and / or" are utilized to describe multiple components in combination with or exclusive of each other. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z."

[0035] "Pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio, or that have been approved by the U.S. Food and Drug Administration as otherwise acceptable for use in humans or domestic animals.

[0036] As used herein, the terms "therapeutic amount," "therapeutically effective amount," or "effective amount" may be used interchangeably and refer to the amount of a compound or material (i.e., a "therapeutic agent") made available through an appropriate route of administration to provide a therapeutic benefit to a patient for a disorder, condition, or disease. The amount of a compound that constitutes a "therapeutic amount," "therapeutically effective amount," or "effective amount" will vary depending on the compound, the disorder and its severity, and the age of the subject being treated, but can be routinely determined by one of ordinary skill in the art.

[0037] As used herein, "treating" or "treatment" includes treatment of a disorder, condition, or disease described herein in a subject, preferably a human; i. inhibiting a disease or disorder, i.e., arresting its development; ii. alleviating the disease or disorder, i.e. causing regression of the disorder; iii. slowing the progression of the disorder; and / or iv. Inhibiting, alleviating, ameliorating, or slowing the progression of one or more symptoms of a disease or disorder For example, the terms "treating," "treat," or "treatment" refer to either preventing the onset or worsening of, providing symptomatic relief from, or curing a patient's disorder, condition, or disease.

[0038] In view of the present disclosure, the methods and compositions described herein can be configured by one of skill in the art to meet desired needs.

[0039] As used herein, the terms "patient," "subject," and "individual" may be used interchangeably and refer to an animal. For example, a patient, subject, or individual may be a mammal, such as a human, being treated for a disorder, condition, or disease.

[0040] As used herein, the terms "disorder," "condition," and "disease" refer to, for example, cancer and its associated comorbidities.

[0041] It should be noted that terms such as "preferably," "generally," and "typically" are not used herein to limit the scope of the methods and compositions described herein or to imply that a particular feature is critical, essential, or even essential to the structure or function of the claimed subject matter.

[0042] In view of the present disclosure, the methods and compositions described herein can be configured by one of skill in the art to meet desired needs.

[0043] overview

[0044] Disclosed herein are methods and combination therapies for treating cancer that significantly reduce tumor growth, increase survival rates, and increase complete response rates in treated individuals. This disclosure demonstrates that adjuvants (e.g., vaccine adjuvants) can enhance the effectiveness of in situ tumor vaccine approaches combined with radiation therapy and checkpoint inhibitor treatment to promote the immune system's response to tumor cells.

[0045] method

[0046] In one embodiment, the present disclosure provides a method for treating cancer in a subject in need thereof, which may include administering a therapeutically effective amount of an adjuvant to a tumor in the subject, wherein the adjuvant is administered intratumorally. The method may further include administering a therapeutically effective amount of radiation therapy to the tumor and administering a therapeutically effective amount of an immune checkpoint inhibitor to the subject. The method may result in the induction of an immune response against the cancer, which treats the disease in the subject.

[0047] In some embodiments, the adjuvant is a vaccine adjuvant. In some embodiments, the adjuvant is lipopolysaccharide or a derivative thereof. In some embodiments, the adjuvant is the lipopolysaccharide (LPS) component of the cell wall of Salmonella entericais. In some embodiments, the adjuvant is a TLR4 agonist. Examples of TLR4 agonists contemplated for use herein include those having the structure shown below: Structure 1 (monophosphoryl lipid A): [ka] ; Structure 2 (Monophosphoryl lipid A-504): [ka] ; Structure 3 (monophosphoryl triacyl lipid A): [ka] ; Structure 4 (Monophosphoryl 3-deacyl lipid A): [ka] ; Structure 5 (monophosphoryl tetraacyl lipid A): [ka] ; Structure 6 (Monophosphorylhexaacyl lipid A): [ka] ; Structure 7 (3-Deacyl, D-(+)-Trehalose 6,6'-Dibehenate): [ka] ; and Structure 8 (Dimethyldioctadecylammonium (bromide salt)): [ka] .

[0048] Further examples of adjuvants and compositions thereof contemplated herein include those described in U.S. Patent No. 9,241,988, which is incorporated by reference. Additionally, bacterially derived monophosphoryl lipid A, such as that provided by Avanti Polar Lipids, Inc. (Avanti No. 699200), may also be used.

[0049] In addition to naturally derived adjuvants, further examples of adjuvants contemplated for use herein include synthetically derived adjuvants, such as structural analogs of monophosphoryl lipid A, including those available from Croda International Plc, including 3D(6-acyl)-PHAD™, 3D-PHAD™ and PHAD™.

[0050] The therapeutically effective amount of adjuvant contemplated for use herein can be about 1 μg to about 100 μg, about 50 μg to about 90 μg, about 10 μg to about 80 μg, about 15 μg to about 70 μg, or about 20 μg to about 50 μg per dose, or any other amount that treats or supports the treatment of the subject receiving the adjuvant. For example, a therapeutically effective amount of adjuvant can be about 20 μg. For example, a therapeutically effective amount of TLR4 agonist can be about 0.01 to about 20 mg / kg, or about 0.1 to about 10 mg / kg, or about 0.5 to about 5 mg / kg.

[0051] In some embodiments, radiation therapy contemplated for use herein may be external beam radiation therapy (EBRT) and / or internal radiation therapy such as brachytherapy and radiopharmaceuticals. In some preferred embodiments, the radiation therapy is EBRT.

[0052] In some embodiments, a therapeutically effective amount of radiation treatment can range from about 2 to about 20 Gy.

[0053] In some embodiments, the therapeutically effective amount of radiation therapy is administered in gradient doses of about 1 Gy / min, about 2 Gy / min, or any other amount that treats or assists in the treatment of the subject receiving the adjuvant.

[0054] In some embodiments, a therapeutically effective amount of radiation therapy is administered by continuous decay of the radiopharmaceutical.

[0055] In some embodiments, radiation therapy may be combined with or substituted for other locally ablative treatments, such as radiofrequency ablation, microwave ablation, and / or cryoablation techniques.

[0056] In some embodiments, the immune checkpoint inhibitor comprises one or more therapeutic agents that inhibit CTLA-4, PD-1, and / or PD-L1. Examples of therapeutic agents contemplated herein include antibodies and antigen-binding fragments thereof, and small molecule inhibitors. For example, the immune checkpoint inhibitor contemplated for use herein may be an anti-CTLA-4 antibody. Specific anti-CTLA-4 antibodies contemplated for use herein are known in the art.

[0057] Therapeutically effective amounts of immune checkpoint inhibitors contemplated for use herein can be about 1 μg to about 400 μg, about 20 μg to about 300 μg, about 40 μg to about 200 μg, about 15 μg to about 70 μg, or about 20 μg to about 50 μg per dose, or any other amount that treats or supports the treatment of a subject receiving the adjuvant. For example, a therapeutically effective amount of an immune checkpoint inhibitor can be about 200 μg. For example, a therapeutically effective amount of an immune checkpoint inhibitor can be about 1.0 to about 50 mg / kg, or about 5 to about 25 mg / kg, or about 10 to about 20 mg / kg, or about 5 mg / kg, or about 10 mg / kg, or about 15 mg / kg, or about 20 mg / kg.

[0058] In another embodiment, the present disclosure provides a method for treating cancer in a subject in need of cancer treatment. The method includes administering a therapeutically effective amount of a TLR4 agonist to a tumor of the subject, wherein the TLR4 agonist is administered intratumorally. The method further includes administering a therapeutically effective amount of EBRT to the tumor, administering a therapeutically effective amount of an anti-CTLA-4 antibody to the subject, and at least one of reducing tumor volume, increasing the overall survival time of the subject, and increasing the complete response rate of the subject. Additional outcomes contemplated herein include an increase in local control rate, progression-free survival, and a decrease in the rate of new distant metastasis.

[0059] Any type of cancer that has a solid tumor can be treated according to the methods described herein. For example, cancers that can be treated include melanoma and prostate cancer, among others.

[0060] In another embodiment, the present disclosure provides a method of treating cancer in a subject in need thereof, which increases the production of Th1-associated, IgG2c anti-tumor antibodies associated with solid tumors. In another embodiment, the present disclosure provides a method of treating cancer in a subject in need thereof, which induces a systemic anti-tumor immune response.

[0061] In some embodiments, the method of the present disclosure may administer one or more adjuvants before, simultaneously with, or after administration of one or more immune checkpoint inhibitors. The components of the administered composition may be administered via the same administration route (e.g., intravenously) or different administration routes (e.g., one component is administered intravenously and another component is administered intratumorally). Any variation in the timing and / or route of administration is contemplated herein.

[0062] composition

[0063] In other embodiments, the present disclosure provides therapeutic and pharmaceutical compositions (which may be generally referred to as "compositions") for treating cancer. For example, compositions contemplated herein may include a therapeutically effective amount of an adjuvant, and / or a therapeutically effective amount of an immune checkpoint inhibitor, and a pharmaceutically acceptable carrier or diluent.

[0064] For example, compositions contemplated herein may include a therapeutically effective amount of a TLR4 agonist, such as one or more of monophosphoryl lipid A, monophosphoryl lipid A-504, monophosphoryl triacyl lipid A, monophosphoryl 3-deacyl lipid A, monophosphoryl tetraacyl lipid A, monophosphoryl hexaacyl lipid A, 3-deacyl, D-(+)-trehalose 6,6'-dibehenate, and dimethyldioctadecylammonium (bromide salt).

[0065] Additionally, compositions contemplated herein may include a therapeutically effective amount of one or more immune checkpoint inhibitors, e.g., antibodies and antigen-binding fragments thereof or small molecules that inhibit CTLA-4, PD-1, and / or PD-L1.

[0066] Certain compositions contemplated herein include monophosphoryl lipid A and a pharmaceutically acceptable carrier or diluent. The composition may also include an anti-CTLA-4 antibody.

[0067] The compositions described herein can be formulated as separate compositions that are given to a subject simultaneously or sequentially, or can be combined into a single composition immediately before administration. Alternatively, the compositions contemplated herein can refer to a single composition that contains all components. In certain embodiments, the compositions of the present disclosure can contain one or more secondary therapeutic agents. Examples of suitable secondary therapeutic agents include intratumoral injection nanoparticles, microparticles, oncolytic viruses, immunotherapy, such as cytokines, monoclonal antibodies and immunocytokines, and chemotherapy, such as cyclophosphamide.

[0068] Compositions of the present disclosure may be formulated into a form suitable for virtually any mode of administration, including, for example, injection, intratumoral administration, transdermal, oral, topical, ocular, buccal, systemic, nasal, rectal, vaginal, etc., or administration by inhalation or insufflation. Compositions that can be delivered (e.g., formulated to be administered) intravenously, intratumorally, intraperitoneally, and / or intratracheally are also contemplated herein.

[0069] In some embodiments, the compositions of the present disclosure are included in pharmaceutical compositions having at least one pharmaceutically acceptable carrier, solvent, adjuvant, or diluent.

[0070] The term "pharmaceutical composition" may be used in its broadest sense and encompasses all pharmaceutically applicable compositions containing at least one active substance and, optionally, carriers, adjuvants, components, etc. The term "pharmaceutical composition" also encompasses compositions containing an active substance in the form of a derivative or prodrug, such as a pharmaceutically acceptable salt and / or ester. The preparation of pharmaceutical compositions for different routes of administration is within the capabilities of those skilled in the art of pharmaceutical chemistry. The exact nature of the carrier, excipient, or diluent used in a pharmaceutical composition depends on the desired use of the pharmaceutical composition. A pharmaceutical composition may optionally contain one or more additional compounds, such as a therapeutic agent or other compounds, as described elsewhere herein.

[0071] The compositions described herein can be administered orally, topically, parenterally, by inhalation or spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intratumoral, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques, etc.

[0072] Preparations for parenteral administration can be in the form of aqueous or non-aqueous isotonic sterile injection or suspension.These solutions and suspensions can be prepared from sterile powder or granules with one or more carriers or diluents mentioned for use in oral preparations.The compound can be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and / or various buffer solutions.Other adjuvants and modes of administration are well known and widely known in the pharmaceutical field.

[0073] The therapeutic compositions or pharmaceutical compositions thereof described herein will generally be used in an amount effective to achieve the intended result, for example, an amount effective to treat or prevent the particular disease being treated (e.g., a therapeutically effective amount). Therapeutic benefit refers to the eradication or amelioration of the underlying disorder being treated and / or one or more symptoms associated with the underlying disorder, such that the patient reports an improvement in mood or symptoms, although the patient may still be suffering from the underlying disorder. Therapeutic benefit can also generally include halting or slowing the progression of the disease.

[0074] The amount of therapeutic composition administered can be based on a variety of factors, including, for example, the particular condition being treated, the mode of administration, whether the desired benefit is prophylactic and / or therapeutic, the severity of the condition being treated and the age and weight of the patient, the patient's genetic profile, and / or the bioavailability of the particular therapeutic composition.

[0075] Determining the effective dosage of a compound for a particular use and mode of administration is well within the capabilities of those skilled in the art. An effective dosage can be initially estimated, for example, from in vitro activity and metabolism assays. For example, the initial dosage of a therapeutic composition for use in animals may be determined based on the EC of a particular therapeutic composition as measured in an in vitro assay. 50 The therapeutic composition may be formulated to achieve a circulating blood concentration or serum concentration of 0.01 or higher. Taking into account the bioavailability of a particular therapeutic composition via a desired administration route, it is well within the ability of a person skilled in the art to calculate the dosage to achieve such a circulating blood concentration or serum concentration. The initial dosage of the therapeutic composition may also be estimated from in vivo data, such as animal models. Animal models useful for testing the effectiveness of therapeutic compositions for treating or preventing the various diseases mentioned above are well known in the art. Animal models suitable for testing the bioavailability of therapeutic compositions are also well known. A person of ordinary skill in the art can routinely adapt such information to determine the dosage of a particular therapeutic composition suitable for human administration.

[0076] In addition to the amounts of active agents (e.g., adjuvants and ICI agents or chemotherapy) described elsewhere herein, dosages may also range from about 0.0001 mg / kg / day, 0.001 mg / kg / day, or 0.01 mg / kg / day to about 100 mg / kg / day, but may be higher or lower depending on, among other factors, the activity of the therapeutic agent, the bioavailability of the therapeutic composition, other pharmacokinetic properties, the mode of administration, and various other factors, including the specific disease being treated, the location of the disease in the body, the severity of the disease, the genetic profile, age, health, sex, diet, and / or weight of the subject. Dosage amounts and intervals may be individually adjusted to provide levels of the therapeutic composition sufficient to maintain the desired therapeutic effect. For example, therapeutic compositions may be administered once a week, several times a week (e.g., every other day), once a day, or multiple times a day, depending, among other factors, on the mode of administration, the specific indication being treated, and the judgment of the prescribing physician. In cases of local administration or selective uptake, such as intratumoral injection, the effective local concentration of the therapeutic composition may not be related to plasma concentration. One skilled in the art will be able to optimize the effective dosage without undue experimentation.

[0077] In a further aspect, kits are contemplated herein that include one or more compositions administered according to the treatment methods described herein. Such contemplated kits may further include instructions on how to administer the applied one or more compositions. [Example]

[0078] Example The following examples illustrate certain embodiments of the present disclosure and various uses thereof. They are provided for illustrative purposes only and should not be construed as limiting the scope of the disclosure in any way.

[0079] Example 1: Local TLR4 stimulation enhances in situ vaccination through the induction of CD8 T cell-independent Th1 polarization overview Background: Radiation therapy (RT) has been demonstrated to produce an in situ vaccination (ISV) effect in mouse models and cancer patients, but this has not routinely translated into enhanced clinical responses to immune checkpoint blockade (ICI). We investigated whether monophosphoryl lipid A (MPL), a commonly used vaccine adjuvant, could enhance an ISV regimen consisting of a combination of RT and ICI.

[0080] Materials / Methods: We used syngeneic mouse models of melanoma (B78) and prostate cancer (Myc-CaP). Tumor-bearing mice were administered either RT (12 Gy, day 1), RT plus anti-CTLA4 (C4, days 3, 6, and 9), MPL (20 μg IT injection, days 5, 7, and 9), RT plus C4 plus MPL, or PBS control. To assess the effect of MPL on the irradiated tumor microenvironment, primary tumors with tumor-draining lymph nodes were harvested for immune cell infiltration analysis and cytokine profiling, and serum was collected for analysis of antitumor antibody populations.

[0081] Results: The combination of RT, C4, and MPL significantly reduced tumor growth and increased survival and complete response rates compared with RT and C4 in both B78 and Myc-CaP models. MPL favorably reprogrammed the irradiated tumor immune microenvironment toward M1 macrophages and Th1 TBET+ CD4+ T cell polarization. Furthermore, MPL significantly increased intratumoral expression of several Th1- and M1-associated proinflammatory cytokines. In coculture models, MPL-stimulated macrophages directly activated CD8 T cells and polarized CD4+ cells toward a Th1 phenotype. MPL treatment significantly increased the production of Th1-associated IgG2c antitumor antibodies, which are necessary for and predictive of antitumor responses to RT, C4, and MPL, and enabled macrophage-mediated, antibody-dependent direct tumor cell killing by MPL-stimulated macrophages. Macrophage-mediated tumor cell killing was dependent on FcγR expression. In a metastatic model, RT and MPL generated a systemic antitumor immune response that enhanced the response to ICIs and was dependent on macrophages and CD4+, but not CD8+, T cells.

[0082] Conclusion: We report the potential of MPL to enhance the ISV effect of the RT+C4 combination through FcγR, macrophage, and TBET+CD4+ Th1 cell-dependent mechanisms. To our knowledge, this is the first report describing the generation of a CD8+ T cell-independent, Th1-polarized systemic antitumor immune response and subsequent generation of immunological memory. These findings support the potential for vaccine adjuvants to enhance the efficacy of in situ tumor vaccine approaches.

[0083] Introduction The majority of cancer patients will receive radiation therapy at some point during clinical care (1). While previously thought of as primarily a cytotoxic treatment, growing evidence suggests that radiation has a variety of immunomodulatory effects within the tumor microenvironment. Radiation (RT) can induce immunogenic tumor cell death and the release of tumor-specific antigens (2, 3), upregulation of immune susceptibility markers such as Fas and MHC-I (4, 5), and production of inflammatory cytokines such as type 1 interferon (6-8), as well as other inflammatory cytokines and damage-associated molecular patterns (9-11) that affect immune cell trafficking and activation. Through these mechanisms, RT can generate an in situ vaccination effect, transforming the patient's own tumor into a nest of enhanced antigen presentation, helping to generate diverse tumor-specific T cell responses (i.e., abscopal responses) that can propagate distal to disease, outside the RT field (12-16).

[0084] In contrast, radiation also induces changes within the tumor microenvironment that are potentially detrimental to the development of antitumor immunity. These may include blunting effector immune cell infiltration within the tumor by recruiting suppressive regulatory T cells, as well as increasing the infiltration and activation of inhibitory macrophage and myeloid-derived suppressor cell lineages (17-21). These inhibitory mechanisms likely underlie, in part, the clinical finding that abscopal responses after RT alone are extremely rare (22). Targeting such deleterious effects is one approach in which immunotherapy may be used to enhance the efficacy of radiation therapy.

[0085] Immune checkpoint inhibitors (ICIs; e.g., anti-PD-L1 and anti-CTLA-4 inhibitors) are a class of immunotherapies that modulate tumor immune tolerance by blocking specific inhibitory receptor-ligand interactions on the surface of immune cells, thereby overcoming T cell inhibition or exhaustion (23). In patients with highly immunogenic tumors, such as some melanomas, ICIs can restore the effectiveness of the antitumor immune response and can result in complete and durable tumor regression, even in the setting of advanced metastatic disease (24-28). However, ICIs have not shown clinical benefit in treating poorly immunogenic tumors, such as prostate cancer, which are characterized by low levels of T cell infiltrates and low mutational burden and rarely produce mutagenic neoantigens (29-32). Furthermore, even patients with highly immunogenic tumors who initially respond to ICIs often exhibit disease progression over time (33).

[0086] Several groups have exploited the immune-stimulating effects of RT to improve responses to ICI treatment, achieving remarkable success in preclinical models (4, 34-40). Furthermore, through mechanistic preclinical studies, it has become increasingly clear that RT likely requires combination with immunotherapy, such as ICI, to generate clinically meaningful abscopal responses (7, 40-42). While early clinical studies combining RT with ICIs have shown promise, clinical responses remain limited (16, 36, 43-45). Therefore, there is an immediate clinical need to increase both the rate and depth of responses to RT and ICI combination therapy.

[0087] Monophosphoryl lipid A (MPL) is a derivative of the lipopolysaccharide (LPS) component of the cell wall of Salmonella entericais. MPL promotes immune activation in mice and humans through activation of toll-like receptor 4 (TLR4) and has significantly reduced toxicity compared to LPS (46). Clinically, MPL has been used as an adjuvant in several infectious disease vaccines, including the HBV vaccine Fendrix (46) and the HPV vaccine Cervarix (47). Similar to conventional vaccines, in situ vaccination regimens rely on promoting antigen recognition, which can be enhanced by coadministration of adjuvants. Furthermore, MPL may overcome additional deleterious effects of RT that are not addressed by ICIs, such as preventing the activation of inhibitory macrophage and myeloid-derived suppressor cell lineages. By promoting the reprogramming of innate cell populations within the tumor microenvironment to a pro-inflammatory phenotype, MPL may enhance the antitumor response generated by the combination of RT and ICIs and function as an adjuvant for in situ vaccination.

[0088] Here, we investigated the potential of MPL to function as an adjuvant for an in situ vaccine regimen combining RT and anti-CTLA-4 in immunologically cold murine melanoma and prostate cancer models. We demonstrated that intratumorally injected MPL polarized CD4 T cells toward a Th1 phenotype, induced the production of functional antitumor antibodies, and directly activated and polarized macrophages within the tumor microenvironment toward an M1 phenotype, enabling macrophage-mediated direct tumor cell killing via a Th1 CD4 T cell-dependent mechanism and promoting the proliferation of a systemic antitumor immune response independent of CD8 T cells.

[0089] material and method research design

[0090] The objectives of this study were to determine whether the conventional vaccine adjuvant MPL could enhance the antitumor response of an in situ vaccination regimen consisting of a combination of RT and anti-CTLA-4, and to determine the mechanism by which MPL enhances the antitumor effect. In our study, tumors were established intradermally in mice, external beam radiation was delivered, PBS or anti-CTLA-4 was injected intraperitoneally, and PBS or MPL was injected intratumorally. Tumor growth and overall survival were recorded. Serum was collected and analyzed for the presence and characterization of antitumor antibodies, and tumors and tumor-draining lymph nodes were collected for immune infiltration analysis. The day before treatment initiation, mice were randomized into experimental groups / treatments. Generally, experimental groups consisted of at least 5–6 mice, although up to 10 mice were used in some experiments. To determine the effect of MPL on immune cell populations, we harvested macrophages from bone marrow for in vitro monoculture and coculture in the presence of MPL, and isolated CD4 and CD8 cells from the spleen. To test which immune cell populations are important for antitumor efficacy, we used antibody-mediated depletion of macrophages, NK cells, CD4+, and CD8+ T cells. To confirm the necessity of antitumor antibodies for generating sufficient antitumor immune responses, we used Fcγ receptor-deficient mice.

[0091] cell line

[0092] The mouse melanoma B78-D14 (B78) cell line, derived from the B16 melanoma as previously described, was obtained from Ralph Reisfeld (Scripps Research Institute) in 2002 (63). The mouse prostate cancer Myc-CaP cell line was obtained from the American Type Culture Collection (ATCC). B78 and B16 cells were grown in RPMI-1640 supplemented with 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. Myc-CaP cells were grown in DMEM supplemented with 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. Cell line authentication was performed within 6 months of use using morphology, growth curves, and mycoplasma testing according to ATCC guidelines.

[0093] Mouse tumor model

[0094] Mice were housed and treated under a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Wisconsin-Madison (Protocol No. M005670). Female C57BL / 6, FcγR- / - (FcγR-deficient C57BL / 6.129P2-Fcer1gtm1RavN12) and male FVBn mice were purchased from Taconic at 6–8 weeks of age. Female TBET- / - (B6.129S6-Tbx21tm1Glm / J strain #:004648) mice were purchased from The Jackson Laboratory. B78 and Myc-CaP tumors were engrafted by subcutaneous flank injection of 2 × 10 and 1 × 10 tumor cells, respectively. Tumor size was determined using calipers, and volume was approximated as (width × length) / 2. Mice were randomized immediately before treatment when tumors were fully established (100–150 mm3), approximately 4 weeks after tumor implantation for B78 and 3 weeks for Myc-CaP. The day of radiation was defined as "day 1" of treatment. For the metastatic model, 250,000 B16 cells were injected via tail vein immediately after radiation. Anti-CTLA-4 (IgG2c, clone 9D9, NeoClone) was administered intraperitoneally at 200 μg on days 3, 6, and 9. MPL (Sigma catalog no. SBR00012) was administered intratumorally at 20 μg on days 5, 7, and 9. T cell, NK cell, and macrophage depletion was performed as previously described (12, 94). Depletion was confirmed on day 15 of treatment (Figures 9A–9D). Mice were euthanized when tumor size exceeded 15 mm in the longest dimension or whenever recommended by an independent animal health monitor for morbidity or moribund behavior.

[0095] radiation

[0096] In vivo RT delivery was performed using an RS225 cell irradiator (Xstrahl). In vivo RT delivery was performed using an X-RAD 320 X-ray biological cabinet irradiator (Precision X-Ray, Inc.). Mice were immobilized using a custom-made lead jig that exposed the right flank while shielding the rest of the mouse. In all cases, EBRT was prescribed at 12 Gy. The dose rate of RT delivery in all experiments was approximately 2 Gy / min. Dosimetry calibration and monthly quality assurance checks were performed on these irradiators by University of Wisconsin Medical Physics staff.

[0097] Serum antibody analysis

[0098] To assess the presence of antitumor antibodies in treated mice, blood was collected on days 15 and 30 for analysis as previously described (55). Briefly, serum components were isolated and frozen at -80°C until ready for analysis, at which point the serum was thawed and co-incubated with B78 cells for antibody labeling. Labeled cells were washed, and tumor-bound antibodies were detected using secondary antibodies [anti-mouse IgG-FITC (405305; Biolegend), anti-mouse IgG1-PE (406607; Biolegend), anti-mouse IgG2b-PE (406708; Biolegend), anti-mouse IgG2c-FITC (NBP2-68518; Novus)] and a viability stain (DAPI).

[0099] cell culture

[0100] Macrophages were isolated from freshly harvested bone marrow as previously described (95-97). Briefly, isolated tibiae were flushed with RMPI medium, and the flow-through was collected and centrifuged. The cell pellet was resuspended in RBC lysis buffer (Biolegend catalog no. 420302) and filtered (70 μM). The filtered cells were seeded onto non-tissue culture-treated plates in minimal essential medium Eagle's Alpha Modification (Alpha MEM) with nucleosides supplemented with 10% FBS and 30 ng / mL M-CSF (Biolegend catalog no. 576408). After 24 h, the macrophage-containing culture supernatant was collected and seeded onto tissue culture-treated plates in Alpha MEM supplemented with 10% FBS and 120 ng / mL M-CSF.

[0101] CD4 and CD8 T cells were isolated from freshly harvested spleens of naive mice. Spleens were homogenized, filtered (70 μM), and centrifuged. The cell pellet was resuspended in RBC lysis buffer and filtered (70 μM). CD4 and CD8 cells were sorted from total splenocytes using MACS column sorting (Miltenyi Biotec CD8a Cat. No. 130-104-075, CD4 Cat. No. 130-104-454) according to the manufacturer's instructions.

[0102] To determine the direct effects of MPL on macrophages, CD4 and CD8 T cells, freshly isolated cells were cultured in 6-well plates containing Alpha MEM medium (supplemented with 120 ng / mL M-CSF for macrophages) in the presence of increasing amounts of MPL (5, 20, 100, and 500 ng / mL). After 24 hours, cells were harvested and RNA was isolated for analysis by qPCR.

[0103] In vitro co-culture

[0104] Bone marrow-derived macrophages were seeded in 12-well plates (200,000 cells / well) containing Alpha MEM supplemented with 120 ng / mL M-CSF and treated with either 100 ng / mL MPL or PBS control. After 24 hours, either CD4, CD8, or both (500,000 cells / well) were added to the cultures. CD4 and CD8 cells were harvested after 24 hours and analyzed for activation markers using flow cytometry.

[0105] For coculture with tumor cells, bone marrow-derived macrophages (harvested from wild-type or Fcγ receptor-deficient C57BL / 6 mice) were seeded into 6-well plates (500,000 cells / well) containing Alpha MEM supplemented with 120 ng / mL M-CSF and treated with either 100 ng / mL MPL or PBS control. After 24 h, either B78 melanoma or Myc-CaP cells were added (200,000 cells per well). To test whether serum-derived antitumor antibodies could activate macrophages, 5 μL of serum obtained from mice bearing disease-free B78 tumors was also added to the cocultures. After 24 h, cells were harvested and analyzed for polarization and activation markers using qPCR.

[0106] Cell killing assay

[0107] Tumor bone marrow-derived macrophages (harvested from wild-type or Fcγ receptor-deficient C57BL / 6 mice) were seeded into 48-well plates (400,000 cells / well) containing Alpha MEM supplemented with 120 ng / mL M-CSF and treated with either 100 ng / mL MPL or PBS control. After 24 h, B78 melanoma cells were added (20,000 cells per well, effector-to-target ratio 20:1) with or without 5 μL of serum obtained from disease-free B78 tumor-bearing mice. After 24 h, cells were harvested by gentle scraping and washed twice with PBS. For cytotoxic CD4 T cell assessment, CD4 T cells were isolated from the spleens of B16 tumor-bearing mice treated with either RT + C4 + MPL or PBS control on day 15 posttreatment and cocultured with B16 cells receiving 0 or 12 Gy of RT. After 24 hours, cells were harvested by gentle scraping and washed twice with PBS. In both cases, single-cell suspensions were labeled with a CD45 antibody (anti-CD45-PE-Cy7, BioLegend, 157206) for 30 minutes at 4°C and washed three times with flow buffer (PBS containing 2% FBS and 2 mM EDTA). The single-cell suspensions were then labeled with the apoptosis marker Annexin V using the FITC Annexin V / Dead Cell Apoptosis Kit (ThermoFisher Scientific, catalog number V13242) according to the manufacturer's instructions. Flow cytometry was performed using an Attune NxT flow cytometer (ThermoFisher). Data were analyzed using FlowJo software to quantify the percentage of CD45-Annexin V+ cells.

[0108] Gene expression analysis

[0109] Cells treated in vitro with MPL, RT, or a combination of these were washed with cold PBS, and TRIzol™ Reagent (ThermoFisher Scientific, Cat. No. 15596026) was added to the plate. Cells were harvested by scraping on ice. For tumor tissue analysis, tumors were harvested and samples were homogenized in TRIzol using a Bead Mill Homogenizer (Bead Ruptor Elite, Omni International, Cat. No. 19-040E). For in vitro and in vivo samples, total RNA was extracted using the RNeasy Mini Kit (QIAGEN, Germany, Cat. No. 74106) according to the manufacturer's instructions. The extracted RNA was subjected to complementary cDNA synthesis using the QuantiTect Reverse Transcription Kit (QIAGEN, Germany, Cat. No. 205314) according to the manufacturer's instructions. Quantitative polymerase chain reaction (qRT-PCR) was performed using Taqman Fast Advanced qPCR Master Mix. Thermal cycling conditions (QuantStudio 6, Applied Biosystems) included UDG activation at 50°C for 2 minutes, followed by a Dual-Lock™ DNA polymerase activation step at 95°C for 2 minutes, followed by 40 cycles of each PCR step (denaturation) at 95°C for 1 second and (annealing / extension) at 60°C for 20 seconds. Melting curve analysis was performed to ensure the specificity of the corresponding qRT-PCR reactions. For data analysis, Ct values ​​were exported to an Excel file, and fold changes normalized to untreated control samples were calculated using the ΔΔCt method. Hprt was used as an endogenous control. A complete list of Taqman probes is included in Table 1.

[0110] [Table 1]

[0111] Flow cytometry

[0112] Flow cytometry was performed as previously described (65), using fluorescent beads (UltraComp Beads eBeads, 176 Invitrogen, #01-2222-42) to determine compensation and the fluorescence minus one (FMO) method to determine gating. For in vivo analysis, tumors and tumor-draining lymph nodes were harvested and gently dissociated. For in vitro analysis, nonadherent CD4 and CD8 cells were collected from culture plates and washed twice with PBS. In both cases, whole cells were treated with CD16 / 32 antibody (BioLegend) to prevent nonspecific binding. Live cell staining was performed using Ghost Red Dye 780 (Tonbo Biosciences) according to the manufacturer's instructions. After live / dead staining, single-cell suspensions were labeled with surface antibodies for 60 min at 4°C and washed three times with flow buffer (PBS containing 2% FBS + 2 mM EDTA). For intracellular staining, cells were fixed and stained for internal markers with permeabilization solution according to the manufacturer's instructions (BD Cytofix / Cytoperm™). Flow cytometry was performed using an Attune NxT flow cytometer (ThermoFisher). Data were analyzed using FlowJo software. A complete list of antibody targets, clones, and fluorophores is shown in Table 2.

[0113] [Table 2]

[0114] Tumor cytokine multiplex immunoassay

[0115] On day 15, tumors were harvested and weighed. Tumor samples (5 μL / mg) were lysed in 20% cell lysis buffer (Cell Signaling Technology) containing PMSF and supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Scientific). Each tumor was homogenized in a bead-beater tube, and the lysates were stored at -80°C. The concentrations of 32 cytokines and chemokines in tumor lysates (MILLIPLEX MAP Mouse Cytokine / Chemokine Magnetic Bead Panel, Millipore) were determined by multiplex immunoassay according to the manufacturer's instructions. The MAGPIX System (Millipore) was used to read multiplex plates. Concentrations were determined using standard curves and their respective median fluorescence intensity (MFI) readings (Milliplex Analyst, Millipore). Data were subjected to logarithmic and Z646 (Z) transformation followed by unbiased hierarchical clustering.

[0116] statistical analysis

[0117] Prism 8 (GraphPad Software) and R version 4.0.2 (The R Foundation) were used for all statistical analyses. One-way ANOVA with Tukey's truly significant difference (HSD) test to adjust for multiple comparisons was used to assess the statistical significance of mean differences observed in gene expression and immune cell quantification. Student's t-tests were used for comparisons between two groups. For tumor growth analysis, linear mixed models after log-transformation of tumor volume were fitted for treatment and day. Day and the interaction between treatment and day were fixed effects. Tukey's adjustment for multiplicity was used when testing differences in the slope of log-transformed tumor volume. The Kaplan-Meier method was used to estimate survival distributions for overall survival. Cox regression models were fitted, and pairwise comparisons of overall survival were made using the log-rank test with Benjamini-Hochberg adjustment for p-values ​​between factor levels. The chi-square test was used to compare complete response rates.

[0118] result Intratumoral MPL enhances the antitumor response produced by the combination of RT and anti-CTLA-4

[0119] Clinically, melanoma is typified by a high tumor mutation burden, which can result in an abundance of mutationally generated neoantigens, often resulting in an endogenous antitumor immune response that is exhausted or otherwise ineffective in clinically detectable tumors (48). While some melanoma patients respond well to ICIs, the absence of inflammatory signals, the presence of suppressive immune lineages, limited tumor cell immune susceptibility (e.g., downregulation of MHC-I), and ineffective antigen presentation can result in functionally "cold" tumors that do not respond to ICIs alone (49-51). Therefore, to improve outcomes in patients resistant to ICIs, we utilized the syngeneic B78 mouse melanoma model. This model exhibits many hallmarks of resistance to ICIs, including low baseline MHC-I expression and low numbers of tumor-infiltrating lymphocytes (12, 52). In mice bearing B78 flank tumors, we tested the ability of MPL to function as an adjuvant to a combination treatment consisting of RT and anti-CTLA-4. We randomized mice to receive either RT (12 Gy delivered on day 1), RT + anti-CTLA-4 (C4; 200 μg intraperitoneal injection on days 3, 6, and 9), RT + C4 + MPL (20 μg intratumoral injection on days 5, 7, and 9), RT + MPL, MPL + C4, C4 alone, MPL alone, or PBS control (Figure 1A). In our model, we used type 1 interferon induction as a marker after RT and selected MPL dosing regimens to coincide with pre-peak, peak, and post-peak immune activation, with peak expression occurring 7 days after RT (21). We observed a significant decrease in tumor volume with the RT+C4+MPL combination compared with the single-agent treatment groups (PBS vs. RT+C4+MPL p<0.001; RT vs. RT+C4+MPL p<0.001; MPL vs. RT+C4+MPL p<0.001) (Figures 1B, 1C). This resulted in a significant increase in overall survival (PBS vs. RT+C4+MPL median survival 31 days vs. UND, p<0.001; RT vs. RT+C4+MPL median survival 31 days vs. UND, p<0.001; MPL vs. RT+C4+MPL median survival 31 days vs. UND, p<0.001) (Figure 1D).Compared to RT+C4, the RT+C4+MPL combination resulted in a significant reduction in tumor volume (p=0.003) and increased overall survival (RT+C4 vs. RT+MPL+C4 median survival 44 days vs. UND, p=0.002) (Figures 1B-1D). Furthermore, the addition of MPL significantly increased the complete response (CR) rate produced by the RT+C4 combination (RT+C4 vs. RT+MPL+C4 CR% 12.5% ​​vs. 44%) (Figure 1E). In both the RT+C4-treated and RT+C4+MPL-treated groups, disease-free mice rejected rechallenge with B78 cells, demonstrating the development of a specific antitumor immune response (Figure 1F).

[0120] We then sought to test our combination treatment strategy in a separate model of prostate cancer. In contrast to melanoma, prostate tumors generally arise from driver mutations or oncogenic translocations, and these tumors have low mutational burden, limited antitumor immune responses, and poor responses to ICIs (31, 53), even when delivered in combination with RT (32). We confirmed these findings using a syngeneic Myc-CaP prostate cancer model. The combination of RT+C4 failed to significantly reduce tumor growth compared with RT alone (p=0.094) or the PBS control (p=1). In contrast, we observed a significant reduction in tumor volume (p<0.001) and increased overall survival (RT+C4 vs. RT+MPL+C4 median survival 23.5 days vs. UND, p=0.017) with the addition of MPL to RT+C4 (Figures 1G-1I). Furthermore, we observed an increased CR rate (RT+C4 vs. RT+MPL+C4 CR% 16.7% vs. 50%), which approached significance (p=0.0833) (Figure 1J). At 90 days post-treatment, mice were disease-free and refused rechallenge, confirming immune memory (Figure 1K).

[0121] MPL polarizes towards a Th1 phenotype and predicts response to in situ vaccination

[0122] To test whether MPL polarizes CD4+ cells toward a Th1 phenotype in the irradiated tumor microenvironment, we treated B78 tumor-bearing mice with PBS, RT, RT + C4, RT + C4 + MPL, or MPL alone and collected serum on days 15 and 30 after RT. We selected days 15 and 30 as collection time points, which coincide with the peak of intratumoral immune activation and antitumor antibody production previously observed in our B78 tumor model (54, 55). We incubated B78 cells with serum isolated from these mice and detected antitumor antibodies by flow cytometry. We then quantified the levels of selected IgG subclasses using fluorescently labeled secondary antibodies (anti-IgG, anti-IgG1, and anti-IgG2c) by flow cytometry (Figure 2A). In C57BL / 6 mice, the antibody class IgG2c is associated with Th1 polarization, while IgG1 is associated with Th2 polarization (56). Using IgG2c:IgG1 as a marker of Th1 polarization, we observed a statistically significant increase in the IgG2c:IgG1 ratio in the RT+C4+MPL group compared to all others at day 15 after treatment (Figure 2B). We found that the total anti-B78 IgG antibody population, measured at day 15, was unchanged by treatment (Figure 2C). At day 30, the IgG2c / IgG1 ratio increased another 100-fold compared to day 15, with RT+C4+MPL showing the highest ratio compared to the other groups, which trended toward significance (Figure 2D). We also found that the total anti-B78 IgG antibody population, measured at day 30, was unchanged by treatment (Figure 2E).

[0123] To determine whether the antibody class ratio correlated with the depth of response, we classified serum antibody populations by Response Evaluation Criteria in Solid Tumors (RECIST) criteria in a treatment-independent manner. The IgG2c:IgG1 ratio was significantly increased in responding mice compared with mice with progressive disease, with the highest ratio in the CR group and the second highest in the partial response (PR) group (Figure 2F). In the RT 180 + MPL + C4 group, mice with CR had a significantly increased IgG2c / IgG1 ratio compared with non-responding mice (Figure 2G), which likely underlies the loss of statistical significance when comparing RT + MPL + C4 with the other groups (Figure 2D). This association between the IgG2c:IgG1 ratio and CR was not observed in the RT + C4 group, suggesting that the addition of MPL increases the complete response rate in a Th1-mediated manner (Figure 2H).

[0124] MPL polarizes macrophages toward the M1 phenotype and promotes T cell activation

[0125] Previous studies have shown that MPL mainly activates M1 macrophages (57), so the inventors next attempted to determine the effect of MPL on the irradiated tumor immune microenvironment. The inventors randomized B78 tumor-bearing mice to either PBS, RT, RT + C4, RT + C4 + MPL, or MPL alone, and harvested tumors and tumor-inflow lymph nodes on day 15 after RT. The inventors observed a significant difference in the number of macrophages in the RT + C4 + MPL group compared to RT alone (Figure 3A). The combination of RT + C4 significantly increased the proportion of anti-tumor M1 macrophages (F4 / 80+CD11b+CD80+) among all macrophages (CD11b+F480+) compared to the PBS, RT, and MPL groups. This increase was further enhanced by the addition of MPL (Figure 3B). Furthermore, the proportion of M2 macrophages (F4 / 80+CD11b+CD206+) among all macrophages (CD11b+F480+) was significantly decreased in the RT + C4 + MPL group compared to the PBS, RT + C4, and MPL groups (Figure 3C). This resulted in a significant increase in the M1:M2 ratio in the RT + C4 + MPL group compared to all others (Figure 3D). Additionally, the inventors observed a significant increase in the number of CD8 T cells present in the tumors in both the RT + C4 and RT + C4 + MPL groups compared to the PBS control (Figure 3E).

[0126] We next sought to determine whether MPL affected antigen presentation in the context of the irradiated tumor immune microenvironment and to characterize CD4 Th populations after MPL treatment to confirm our antitumor antibody characterization findings. Within tumor-draining lymph nodes, we observed comparable increases in type 1 dendritic cells between RT+C4 and RT+C4+MPL, both of which were significantly increased compared with PBS, RT, and MPL (Figure 3F). The proportion of Th1 cells (CD4+TBET+) was significantly increased in the RT+C4+MPL group compared with all others, consistent with the increased Th1-associated IgG2c antibody class switching observed after RT+C4+MPL treatment (Figure 3G). We observed a significant increase in CD103+CD4+ memory T cells in RT+C4 compared with PBS, RT, and MPL. This was further enhanced by the addition of MPL (Figure 3H). Furthermore, RT+C4+MPL resulted in a significant increase in IFNγ+CD8 T cells and CD103+CD8+ memory T cells compared to all other groups (Figures 3I, 3J).

[0127] Given our findings of tumor immune cell infiltration, we next profiled the cytokine repertoire within the irradiated tumor immune microenvironment after MPL treatment, focusing on TLR4 activation, Th polarization, and macrophage polarization. We observed a significant increase in keratinocyte-derived chemokine (KC, CXCL1) and macrophage inflammatory protein 2 (MIP2, CXCL2) in both the RT + C4 + MPL and MPL groups, consistent with TLR4 signaling activation (Figures 3K and 3L) (58). RT + C4 + MPL significantly increased the expression of the Th1-associated cytokine IL-12 compared to all other groups and IL-2 compared to all other groups, except for RT + C4, where the increase trended toward significance (Figure 3M). RT + C4 favored Th2 signaling, resulting in a significant increase in the Th2 cytokines IL-4 and IL-5 compared to all other groups (Figure 3N). Finally, RT+C4+MPL significantly increased several pro-inflammatory cytokines associated with M1 polarization, including I 224 L-1α, IL-1β, lipopolysaccharide-inducible CXC chemokines (LIX, CXCL5), TNFα, and GM-CSF, compared with all other groups (Figure 3O).

[0128] MPL induces CD8 T cell activation through direct stimulation and M1 polarization of macrophages

[0129] Given our observation that MPL significantly altered the infiltration and polarization of macrophages, CD4+, and CD8+ T cells within the irradiated tumor immune microenvironment, we sought to determine the direct effects of MPL on these populations. We cultured bone marrow-derived macrophages in the presence of increasing doses of MPL and used qPCR to quantify the expression of Arg1 and Nos2, markers of M2 and M1 macrophages, respectively (59). We observed a dose-dependent increase in both Arg1 and Nos2, with significantly greater expression of Nos2 than Arg1 at doses of 100 ng / ml and 200 ng / ml, strongly supporting M1 polarization (Figure 4A). We observed a similar trend of MPL promoting the expression of pro-inflammatory cytokines (Figure 4B) compared with anti-inflammatory cytokines (Figure 4C).

[0130] We next tested whether in vitro MPL treatment could directly polarize naive splenic CD4 T cells. Using Cxcr3 and Cxcr4 as markers of Th1 and Th2 cells, respectively (60, 61), we observed minimal changes in Cxcr3 and Cxcr4 expression, indicating that MPL treatment does not directly affect CD4 T cell polarization (Figure 4D). When MPL was added to CD4 and CD8 cells in culture, we again observed minimal increases in the expression of proinflammatory cytokines, suggesting that MPL does not directly affect T cell activation (Figures 4E and 4F), consistent with a previous report investigating the effects of LPS on T cell populations (54).

[0131] We hypothesized that MPL might favorably polarize and activate T cells through direct activation of macrophages. To test this, we cocultured CD8 cells with macrophages stimulated with MPL or vehicle control (PBS) and quantified CD69 expression as a marker of activation. We observed that macrophages stimulated with MPL significantly increased CD69 expression on CD8 T cells compared with CD8 T cells cultured alone or with unstimulated macrophages (Figure 4H). Interestingly, when CD4 cells were added to the culture, we no longer observed increased CD69 expression on CD8 cells (Figure 4I). To examine CD4 polarization, we cocultured CD4 cells with macrophages stimulated with MPL or PBS. We observed a significant increase in CXCR3 expression and a decrease in CXCR4 expression in CD4 cells cocultured with stimulated macrophages (Figure 4J, 4K). Interestingly, when CD4 T cells were co-cultured with activated macrophages, a significant increase in the proportion of regulatory T cells (CD4+CD25+FOXP3+) was also observed (Figure 4L). This may partially explain the loss of CD69 expression on CD8 T cells when CD4 T cells were added to the co-culture. We observed a similar trend in CXCR3 and CXCR4 expression on CD4+ cells, as well as an increase in the proportion of regulatory T cells when CD8 T cells were added to the co-culture (Figure 4M-4O).

[0132] Radiation and antitumor antibodies synergize with MPL treatment to activate macrophages

[0133] Macrophages are one of the immune cell populations least susceptible to radiation-induced cell death (62). Tumor-associated macrophage populations irradiated with this regimen are likely to survive and are subsequently exposed to intratumorally delivered MPL as part of our treatment strategy. To test whether RT could synergize with MPL to further increase macrophage activation, we delivered 12 Gy of RT to macrophages in culture, immediately replaced the growth medium with fresh medium containing 100 ng / mL MPL, and harvested cells for analysis 24 h later. We observed that the addition of RT to MPL further increased the expression of the pro-inflammatory marker Ifnβ1 and the M1 marker Nos2 compared to MPL treatment alone (Figure 5A). Interestingly, RT did not affect the expression of TLR4 on macrophages, suggesting that the enhanced activation extends beyond MPL-TLR4 receptor binding (Figure 6A).

[0134] Given our observation that MPL can activate and favorably polarize macrophages, CD8 and CD4 T cells, we next sought to determine whether the antitumor antibodies generated through the combination of RT, C4, and MPL contribute to the observed immune cell activation or function solely as a predictive biomarker for CR. We collected serum 30 days after RT from mice bearing B78 melanoma tumors that had been rendered disease-free after combined RT, C4, and MPL treatment. We cultured bone marrow-derived macrophages, either alone (mono) or cocultured with B78 cells, in the presence of MPL and / or serum from mice bearing B78 tumors that had been rendered disease-free after RT, MPL, and C4 (Figure 5B). In macrophages grown in monoculture, we again observed a significant increase in Arg1 and Nos2 expression with the addition of MPL, which strongly promoted Nos2 expression. The addition of serum did not significantly increase the expression of either gene, nor did the addition of serum to MPL-stimulated macrophages further increase expression compared to MPL stimulation alone (Figure 5C). A similar trend was observed for the activation markers Il-1a and Ifnβ1 (Figure 5D). When macrophages were cocultured with B78 cells, we observed a significant increase in the expression of Nos2, but not Arg1, after MPL treatment. Treatment with serum alone did not significantly increase the expression of either gene. However, when serum was added to MPL-stimulated macrophages, we observed a further significant increase in the expression of both Arg1 and Nos2, and the addition of MPL promoted Nos2 expression (Figure 5E). A similar trend to that seen with Nos2 was observed for the activation markers Il-1a and Ifnβ1 (Figure 5F). This suggests that antitumor antibodies can further increase the activation of MPL-stimulated macrophages only in the presence of tumor cells. We confirmed that the effect of serum in this coculture experiment was tumor cell-specific using a control study in which macrophages were cocultured with Myc-CaP cells, an unrelated B78 tumor eradicated by RT+MPL+C4 in serum-depleted mice.When macrophages were co-cultured with Myc-CaP cells and serum from mice rendered disease-free from B78 melanoma tumors by RT+MPL+C4, we did not observe an increase in the expression of macrophage polarization or activation markers with the addition of serum compared with MPL treatment alone (Figures 5G, 5H).

[0135] Given that serum only has an activating effect in the context of MPL treatment, we hypothesized that MPL enhances the binding and recognition of anti-tumor antibodies bound to tumor cells. To test this, we first compared the anti-tumor effects of the combination of RT, C4, and MPL in wild-type and FcγR- / - mice. FcγR deficiency abolished the anti-tumor response and survival benefit of RT, C4, and MPL in FcγR- / - mice compared with wild-type mice (Figures 7A and 7B). We then further investigated the effects of RT and MPL on bone marrow-derived macrophages cultured in vitro. We delivered 12 Gy of RT to macrophages in culture, immediately replaced the growth medium with fresh medium containing 100 ng / mL MPL, and harvested the cells for analysis 24 hours later. We observed that MPL treatment significantly increased the expression of activating FcγR1 and FcγR4 and inhibitory FcγR2. The addition of radiation to MPL further increased the expression of these Fcγ receptors with FcγR1 and FcγR4, a significant increase compared to that seen with FcγR2 (Figure 7C). To confirm that the observed synergistic activation of macrophages by MPL and serum was dependent on FcγR expression, we cultured bone marrow-derived Fcγ receptor-deficient macrophages monocultured and cocultured them with B78 cells in the presence of MPL and / or serum. In FcγR- / - macrophages grown in monoculture, we again observed a significant increase in Arg1 and Nos2 expression with the addition of MPL, which strongly promoted Nos2 expression. The addition of serum further increased the expression of Arg1 (Figures 7D and 7E), somewhat similar to that observed for wild-type macrophages, but not Nos2, Il-1a, or Ifnβ1 (Figures 7D and 7E). When FcγR− / − macrophages were cocultured with B78 cells, we observed a significant increase in the expression of Nos2, but not Arg1, after MPL treatment similar to that observed in wild-type macrophages.In contrast to wild-type macrophages, the addition of serum to cocultures with B78 cells did not significantly increase the expression of either gene, and the addition of serum to MPL-stimulated FcγR- / - macrophages did not further increase expression compared to MPL stimulation alone (Figure 7F). A similar trend was observed for the activation markers Il-1a and Ifnβ1, confirming that the enhanced macrophage activation following serum treatment of B78 tumors is dependent on Fcγ receptor expression (Figure 7G).

[0136] To test whether serum antibodies could induce macrophage-mediated antibody-dependent cellular cytotoxicity, we cocultured macrophages with B78 cells containing or without MPL and serum. Using Annexin V staining as a marker of apoptosis, we observed a five-fold increase in Annexin V staining in B78 cells cocultured with macrophages stimulated with MPL and serum compared with stimulation with each agent alone or unstimulated macrophages. The observed increase in Annexin V staining was lost when macrophages lacked Fcγ receptors (Figure 7H).

[0137] Given the functional importance of serum antibodies, we next sought to determine the effect of our treatment regimen on B cells. We cultured B cells and treated them with either MPL, serum, MPL plus serum, or a PBS control. In separate experiments, we also cultured B cells and treated them with RT, anti-CTLA-4, RT plus anti-CTLA-4, or a PBS control. We did not observe increased B cell activation after MPL or serum treatment, and only moderate activation after RT (Figures 6B and 6C), suggesting that B cells were not directly affected by our treatment regimen.

[0138] MPL-induced immunity is dependent on Th1 cells

[0139] Given our observation that MPL can not only promote the generation of antitumor antibodies but also directly activate macrophages, which in turn promote Th1 polarization of CD4 T cells and activation of CD8 T cells, we sought to determine which of these immune cells is important for the antitumor response of the RT+C4+MPL combination. We compared the efficacy of RT+MPL+C4 treatment in mice in which specific immune cell lineages had been depleted by intraperitoneal injection of lineage-specific depleting antibodies. This included mice depleted of macrophages (αCD115), NK cells (αNK1.1), CD4 T cells (αCD4), CD8 T cells (αCD8), or both CD4 and CD8 T cells (αCD4 / CD8). The loss of macrophages and CD4 cells significantly reduced the antitumor response compared to non-depleted mice. Interestingly, the loss of NK cells and CD8 cells did not affect the antitumor response (Figure 8A) or overall survival (Figure 8B), respectively.

[0140] As previously performed, we collected serum on day 15 after RT for anti-tumor antibody quantification. We again observed a significant increase in the IgG2c:IgG1 ratio with the RT + C4 + MPL combination compared to the PBS control. While depletion of NK cells or CD8 T cells did not affect the IgG2c:IgG1 ratio, macrophage depletion and CD4 T cell depletion significantly reduced the IgG2c:IgG1 ratio compared to the RT + C4 + MPL combination, suggesting that Th1-associated anti-tumor antibody class switching is dependent on both macrophages and CD4 T cells (Figure 8C). Interestingly, depletion of CD4 T cells significantly reduced total IgG production, suggesting that anti-tumor antibody production is at least partially dependent on CD4 T cells (Figure 8D).

[0141] We hypothesized that the loss of Th1 cells was responsible for the loss of treatment efficacy when CD4 cells were depleted. To test this, we compared the antitumor effects of the combination of RT + C4 + MPL in wild-type and TBET- / - mice. TBET deficiency abolished the antitumor response and the production of Th1-associated antitumor antibodies after RT + C4 + MPL treatment in TBET- / - mice compared with wild-type mice (Figures 8E and 8F). We observed a significant decrease in intratumoral CD4 cell infiltration and the M1:M2 macrophage ratio in TBET- / - mice compared with wild-type mice (Figure 8G). Furthermore, within the tumor-draining lymph nodes, we observed a significant decrease in Th1 C 360 D4 T cells and IFNγ+ CD4 T cells in TBET- / - mice compared with wild-type mice (Figure 8H). Taken together, these data suggest that Th1 CD4 T cells are central to the mechanisms underlying the generation of antitumor immunity by the combination of RT+C4+MPL.

[0142] MPL treatment promotes systemic immunity independent of CD8 T cells

[0143] Given that the loss of CD8 cells did not affect the antitumor response, we sought to determine whether CD8 cells are required for the generation of systemic immunity. We used a systemic disease model consisting of a B78 primary tumor and intravenously injected B16 melanoma cells to model heterogeneous metastatic disease. B16 cells are the parent of B78 and share common tumor neoantigens that can be recognized by T cells (12, 54, 63). Treatment with RT + MPL + C4 significantly reduced primary tumor proliferation and significantly increased survival compared to RT + C4 (Figures 9A and 9B). Immediately after death or at day 60, lungs were harvested to determine metastatic burden. The addition of MPL significantly reduced lung metastatic burden compared to RT + C4 (Figure 9C). Interestingly, the enhanced antitumor response, survival, and reduced lung metastatic burden observed with RT + C4 + MPL were independent of CD8 T cells (Figures 9A-9C).

[0144] Recent evidence suggests that CD4 cells can directly kill tumor cells through MHC-II-mediated recognition (73). We measured MHC-II expression on B16 tumor cells in vitro after RT and in vivo after the combination of RT, C4, and MPL, and observed downregulation of MHC-II expression in both cases compared with the PBS control (Figures 6D and 6E). Furthermore, we isolated CD4 cells from B16 tumor-bearing mice 15 days after RT, C4, and MPL treatment and cocultured them with B16 melanoma cells. We observed no change in tumor cell killing compared with CD4 cells isolated from tumor-bearing mice treated with the PBS control (Figure 6F). Together, these data suggest that macrophages are the primary cytotoxic cells activated by the combination treatment.

[0145] Consideration We demonstrated that intratumoral injection of the vaccine adjuvant MPL could enhance the antitumor immune response generated by RT, thereby enhancing the response to anti-CTLA-4 checkpoint blockade. This resulted from the favorable effect of MPL on the polarization of both M1 macrophages and Th1 CD4 T cells in the irradiated tumor microenvironment. Consistent with the polarization of Th1 CD4 T cells, we observed that MPL induced the production of IgG2c-dominant antitumor antibodies and upregulation of IgG2c high-affinity Fcγ receptors I and IV on macrophages. MPL-stimulated macrophages exposed to antitumor antibodies on tumor cells showed increased activation and direct killing of tumor cells in vitro. Depletion of macrophages, Th1 CD4 T cells, or loss of Fcγ receptor expression completely abolished the antitumor immune response in vivo. Finally, we demonstrate the ability of our combined treatment regimen to generate local and systemic immune responses through a CD8 T cell-independent mechanism.

[0146] CD8 T cells have classically been considered the "gold standard" in immunotherapy for enhancing tumor eradication and generating immunological memory. However, recent studies suggest the importance of other cell populations in generating antitumor immunity. Natural killer cells can mediate antitumor immunity through antibody-dependent cell-mediated cytotoxicity (64, 65). Vaccine-based immunotherapeutic strategies aim to stimulate B cell-mediated production of tumor-specific antibodies that can bind to tumor antigens in the blood or at the tumor site. Antibody binding can enable various responses, including neutralization of target protein function (66), direct tumor clearance via phagocytosis (67), complement-dependent cytotoxicity (68), or antibody-dependent cell-mediated cytotoxicity (69). In addition to their helper function, CD4 T cells have been shown to possess direct cytotoxic activity in preclinical models (70-73), with clinical validation in melanoma (74) and hepatocellular carcinoma (75).

[0147] Here, we report the generation of a CD8-independent systemic immune response that relies on both Th1 CD4 T cells and macrophages. Our data suggest that CD4 T cell polarization toward a Th1 phenotype is required for the generation of functional antitumor antibodies, predominantly of the IgG2c subclass. We demonstrate that these antibodies have the potential to enable macrophage-mediated tumor cell killing in vitro and in vivo only when macrophages are stimulated with MPL. Interestingly, loss of natural killer cells in vivo did not abolish the antitumor immune response, despite their ability to generate antibody-dependent cell-mediated cytotoxicity. This may be explained, at least in part, by the observation that NK cells express relatively low amounts of TLR4 compared to macrophages (76) and, based on our data, that functional antibody recognition in this model system requires MPL stimulation.

[0148] Previous studies have suggested the potential benefits of combining MPL with checkpoint blockade. In preclinical models, MPL has been shown to increase the antitumor efficacy of anti-PD-1 / anti-PD-L1 therapy through enhanced dendritic cell activation and antigen presentation (77, 78). Within tumor-draining lymph nodes, we observed a significant increase in dendritic cell activation after the combination of radiation and anti-CTLA-4, consistent with previous reports (7, 38, 39). However, the addition of MPL did not further increase dendritic cell activation, suggesting that the role of MPL in our treatment regimen extends beyond supporting antigen presentation.

[0149] As a general class of immune adjuvants, TLR4 agonists have generated interest in testing their potential to enhance conventional vaccine efficacy, namely by promoting Th1 polarization (79-81). In our model, a significant increase in Th1 polarization was observed when MPL was added to RT and anti-CTLA-4, but not as a single agent. Interestingly, the depth of response to treatment positively correlated with the magnitude of Th1 polarization, as measured by IgG2c class switching. The magnitude of IgG2c class switching was significantly increased in mice exhibiting a complete response, but only when MPL was added to RT and anti-CTLA-4. This contrasts with previous reports demonstrating that anti-CTLA-4 allows for the expansion of Th1-like CD4 T cell populations (82, 83). However, it is noteworthy that these studies were performed in the MC-38 colon cancer model, which has a relatively high immunogenicity. Furthermore, the authors confirmed these findings in the less immunogenic B16 melanoma model, the parent cell line of our B78 melanoma model, but required treatment with the GVAX tumor vaccine to enhance overall T cell infiltration to allow their analysis. This overall lack of T cell infiltration may at least partially explain the lack of Th1 polarization observed with the combination of RT and anti-CTLA-4 in our model.

[0150] In addition to serving as a potential biomarker of response to treatment, Th1 antitumor antibody class switching served a functional purpose and was critical for response to combination treatment in our model via Fcγ receptor-mediated recognition. Previous reports have described the importance of FcγR4 in mediating anti-CTLA-4-induced regulatory T cell depletion (84), which can be enhanced by upregulation of FcγR4 by TLR1 / 2 agonist treatment (85). These findings build on similar reports describing the importance of Fcγ receptor subclasses in the context of monoclonal antibody treatment (86-88).

[0151] However, to our knowledge, this is the first report documenting the dominance of endogenously generated antitumor antibody classes and the importance of recognition by macrophage Fcγ receptor binding in generating a successful antitumor immune response. Four individual Fcγ receptors have been identified in mice, and all four are expressed on macrophages. Receptors FcγR1 and FcγR4 have high affinity only for the antibody subclass IgG2c, whereas FcγR2 and FcγR3 have low affinity for both IgG2c and IgG1 (89). Given that our combined treatment results in an antibody population that significantly prefers IgG2c over IgG1, these antitumor antibodies selectively stimulate the activating receptors FcγR1 and FcγR4, the expression of which increases with MPL treatment and further increases with the addition of RT. These mechanisms may underlie the observed synergistic effect between MPL and the combination of RT and anti-CTLA-4.

[0152] We acknowledge several limitations of this study, including the use of syngeneic and heterotopic mouse tumor models that do not fully recapitulate the tumor heterogeneity or immune microenvironment observed in humans. While our findings in two separate syngeneic tumor models, melanoma and prostate cancer, suggest that the addition of MPL to the combination of RT and anti-CTLA-4 is a promising treatment strategy, it has previously been shown that syngeneic tumor models may have pre-existing immunity that is important for the response to RT and immune checkpoint blockade (90, 91). Therefore, further studies using this combination treatment strategy in spontaneously developing mouse tumor models will further support the potential for successful clinical translation. A similar limitation arises from the observation that anti-CTLA-4 generates superior immune responses compared with anti-PD-1 / L1 therapy in many mouse tumor models, which is likely due, at least in part, to regulatory T cell depletion that occurs through CTLA-4 blockade. This directly contrasts with clinical studies and further highlights important differences between mouse and human immunity. To overcome this limitation and test whether MPL could enhance antitumor immune responses to the combination of RT and anti-CTLA-4, we focused on utilizing a poorly immunogenic mouse tumor model that responds poorly to anti-CTLA-4 monotherapy. For RT treatment, we used a single fraction of 12 Gy based on our previous data in the B78 tumor model; however, we did not test other RT doses or fractionation schemes, which may affect treatment efficacy. Future studies dedicated to testing MPL in combination with other RT modalities, doses, and fractionation schemes will help fully determine the potential for MPL treatment in the context of RT. Finally, our treatment strategy involves intratumoral administration of MPL, which may limit its potential for exploration. However, several immunotherapeutic regimens using intratumoral approaches for delivery are currently under development (92). Furthermore, advances in image guidance in the field of interventional imaging may enable exploration of a broader range of cancer types.

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[0154] The embodiments illustratively described herein may suitably be practiced in the absence of any element or elements, or any limitation, not specifically disclosed herein. The terms and expressions used are used as terms of description, not of limitation, and the use of such terms and expressions is not intended to exclude equivalents of the features shown and described, or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed embodiments. Thus, while the present specification is specifically disclosed by embodiments, it should be understood that appropriate features, modifications, and variations of the concepts disclosed herein may be employed by those skilled in the art, and that such modifications and variations are considered to be within the scope of these embodiments as defined by the description and the appended claims. While several aspects of the present disclosure may be identified herein as particularly advantageous, it is not intended that the present disclosure be limited to these specific aspects of the present disclosure.

[0155] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more enumerated claims are introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as a list, for example, in Markush group format, each subgroup of elements is also disclosed, and any element can be removed from the group.

[0156] Generally, when the present disclosure or aspects of the present disclosure are referred to as comprising particular elements and / or features, it is to be understood that particular embodiments of the present disclosure or aspects of the present disclosure consist of or consist essentially of such elements and / or features, and for simplicity, these embodiments have not been specifically set forth in these terms herein.

Claims

1. 1. A method of treating cancer in a subject in need thereof, comprising: a) administering a therapeutically effective amount of an adjuvant to a tumor in said subject, wherein said adjuvant is administered intratumorally; b) administering a therapeutically effective amount of radiation therapy and / or local ablative therapy to said tumor; c) administering to said subject a therapeutically effective amount of an immune checkpoint inhibitor; and d) inducing an immune response against said cancer; A method comprising:

2. The method of claim 1 , wherein the adjuvant comprises a TLR4 agonist.

3. 3. The method of claim 2, wherein the TLR4 agonist comprises one or more of monophosphoryl lipid A, monophosphoryl lipid A-504, monophosphoryl triacyl lipid A, monophosphoryl 3-deacyl lipid A, monophosphoryl tetraacyl lipid A, monophosphoryl hexaacyl lipid A, 3-deacyl, D-(+)-trehalose 6,6'-dibehenate, and dimethyldioctadecylammonium (bromide salt).

4. 10. The method of claim 1, wherein the therapeutically effective amount of the adjuvant comprises about 20 μg to about 70 mg or about 0.5 to about 5 mg / kg.

5. 10. The method of claim 1, wherein the radiation therapy comprises external beam radiation therapy (EBRT) and / or internal radiation therapy.

6. 6. The method of claim 5, wherein the radiation therapy is EBRT.

7. 6. The method of claim 5, wherein the internal radiation therapy comprises brachytherapy and / or a radiopharmaceutical.

8. 10. The method of claim 1, wherein the therapeutically effective amount of radiation treatment is from about 2 to about 20 Gy.

9. 10. The method of claim 1, wherein the therapeutically effective amount of radiation therapy is administered at a gradient dose of about 2 Gy / min.

10. The method of claim 1 , wherein the local ablative treatment comprises radiofrequency ablation, microwave ablation, and / or cryoablation.

11. 10. The method of claim 1, wherein the immune checkpoint inhibitor comprises one or more therapeutic agents that inhibit CTLA-4, PD-1 and / or PD-L1.

12. 12. The method of claim 11, wherein the immune checkpoint inhibitor comprises an anti-CTLA-4 antibody.

13. 1. A composition for treating cancer, comprising: a) a therapeutically effective amount of an adjuvant; b) a therapeutically effective amount of an immune checkpoint inhibitor, and c) a pharmaceutically acceptable carrier or diluent A composition comprising:

14. The composition of claim 13 , wherein the adjuvant comprises a TLR4 agonist.

15. 15. The composition of claim 14, wherein the TLR4 agonist comprises one or more of monophosphoryl lipid A, monophosphoryl lipid A-504, monophosphoryl triacyl lipid A, monophosphoryl 3-deacyl lipid A, monophosphoryl tetraacyl lipid A, monophosphoryl hexaacyl lipid A, 3-deacyl, D-(+)-trehalose 6,6'-dibehenate, and dimethyldioctadecylammonium (bromide salt).

16. 14. The composition of claim 13, wherein the immune checkpoint inhibitor comprises one or more therapeutic agents that inhibit CTLA-4, PD-1 and / or PD-L1.

17. 17. The composition of claim 16, wherein the immune checkpoint inhibitor comprises an anti-CTLA-4 antibody.

18. 1. A method of treating cancer in a subject in need thereof, comprising: a) administering a therapeutically effective amount of a TLR4 agonist to a tumor in said subject, wherein said TLR4 agonist is administered intratumorally; b) administering a therapeutically effective amount of EBRT to the tumor; c) administering to said subject a therapeutically effective amount of an anti-CTLA-4 antibody; and d) at least one of decreasing tumor volume, increasing the overall survival of said subject, and increasing the complete response rate of said subject. A method comprising:

19. 19. The method of claim 18, wherein the TLR4 agonist comprises one or more of monophosphoryl lipid A, monophosphoryl lipid A-504, monophosphoryl triacyl lipid A, monophosphoryl 3-deacyl lipid A, monophosphoryl tetraacyl lipid A, monophosphoryl hexaacyl lipid A, 3-deacyl, D-(+)-trehalose 6,6'-dibehenate, and dimethyldioctadecylammonium (bromide salt).

20. 20. The method of claim 19, wherein the therapeutically effective amount of the TLR4 agonist comprises about 0.5 to about 5 mg / kg.

21. 19. The method of claim 18, wherein the therapeutically effective amount of EBRT is about 2 to about 20 Gy administered at a gradient dose of about 2 Gy / min.

22. 19. The method of claim 18, wherein the therapeutically effective amount of the anti-CTLA-4 antibody is about 10 mg / kg.

23. 23. The method of any one of claims 1-12 and 18-22, wherein the cancer comprises one or more solid tumors.

24. 24. The method of claim 23, wherein the cancer is melanoma or prostate cancer.

25. The method of any one of claims 1 to 12 and 18 to 22, further comprising the step of: e) increasing the production of Th1-associated IgG2c anti-tumor antibodies associated with said tumor.

26. The method of any one of claims 1 to 12 and 18 to 22, further comprising the step of e) inducing a systemic anti-tumor immune response.

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