Hydroxyphenylpropanoate compounds, compositions thereof, and their use for tumor immunotherapy

Abstract

This application relates to the use of hydroxyphenylpropanoate compounds of formula (I), (II), (III), (IV), or (V), or pharmaceutically acceptable salts, solvates, and / or ester prodrugs thereof, compositions comprising the same, and their use in, for example, cancer immunotherapy, for increasing antitumor immunity. More particularly, this application relates to compounds useful in the treatment of diseases, disorders, or conditions that can be treated by increasing intracellular antitumor immunity, such as cancer.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority from concurrently pending U.S. Patent Provisional Application No. 63 / 453947, filed on 22 March 2023, the contents of which are incorporated herein by reference as a whole.

[0002] This application relates to hydroxyphenylpropanoate compounds that enhance antitumor immunity, compositions containing these compounds, and their use in, for example, therapy. More particularly, this application relates to compounds useful in the treatment of diseases, disorders, or conditions that can be treated by enhancing antitumor immunity, such as cancer. [Background technology]

[0003] The microbiome has a significant impact on immune health and the response to immunostimulatory therapies, including cancer immunotherapy. Cancer immune surveillance relies on dynamic, stepwise interactions between immunogenic tumors and cells of the innate and adaptive immune systems (Non-Patent Literature 1; Non-Patent Literature 2). Myeloid cells within the tumor microenvironment (TME) are central to this complex interrelationship, detecting cancer and alerting cytotoxic T cells to its presence (Non-Patent Literature 1; Non-Patent Literature 2). However, myeloid cells are also generally polarized towards immunosuppression within the TME and actively participate in cancer immune evasion (Non-Patent Literature 3; Non-Patent Literature 4). The molecular mechanisms governing myeloid cell function within the TME are being elucidated, and recent studies have demonstrated significant contributions from commensal microorganisms and their derived metabolites (Non-Patent Literature 5; Non-Patent Literature 6; Non-Patent Literature 7; Non-Patent Literature 8). This has prompted the clinical evaluation of fecal transplantation as an intervention to improve cancer outcomes and responses to immunotherapy, and the initial results appear promising (Non-Patent Document 9; Non-Patent Document 4; Non-Patent Document 10; Non-Patent Document 11).

[0004] One initial study showed that desaminotyrosine (3,4-HPP) derived from the microbiome primes the amplification loop of type I interferon signaling and protects against influenza virus pathology (Non-Patent Literature 12). In contrast, other studies have reported that specific isomers of HPP metabolites may exhibit anti-inflammatory or antioxidant properties under certain circumstances (Non-Patent Literature 13; Non-Patent Literature 14; Non-Patent Literature 15). However, the molecular mechanisms by which commensal microorganisms influence cancer immunology remain largely unknown. Furthermore, previous studies have not demonstrated antitumor activity for these compounds. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Immunity 39, 1-10 (2013) [Non-Patent Document 2] Nature 541,321-330(2017) [Non-Patent Document 3] Journal for ImmunoTherapy of Cancer 7,(2019) [Non-Patent Document 4] Nat.Med.24,(2018) [Non-Patent Document 5] Frontiers in Immunology 6,(2015) [Non-Patent Document 6] Cell Death Differ. 26, (2019) [Non-Patent Document 7] J. Leukoc. Biol. 100, (2016) [Non-Patent Document 8] Nature Reviews Cancer 17,(2017) [Non-Patent Document 9] Oncoimmunology 9,1-8(2020) [Non-Patent Document 10] Davar,D.et al.Science 371,(2021) [Non-Patent Document 11] Baruch,EN.Et al.Science 371,(2021) [Non-Patent Document 12] Science 357, 498-502 (2017) [Non-Patent Document 13] FASEB J.34,16117-16128(2020) [Non-Patent Document 14] J.Cell.Physiol.235,(2020) [Non-Patent Document 15] J.Agric.Food Chem.69,(2021) [Overview of the Initiative] [Means for solving the problem]

[0006] The applicants investigated the interactions between commensal microorganisms, their derived metabolites, and anti-cancer immunity by performing deep multi-omics profiling on genetically identical mice with diverse microbiomes. A microbiome-derived metabolite called hydroxyphenylpropanoate (HPP) has been identified to enhance cancer immune surveillance, for example, by enhancing cytokine secretion to TME through gasdermin D (GSDMD) pores on bone marrow cells.

[0007] HPP molecules can bind to GSDMD and enhance the interferon regulator (IRF) pathway and the NF-κB pathway in a GSDMD-independent manner. The presence of multiple molecular targets in mammals demonstrates a complex process of natural selection in which microbiome-derived metabolites can dramatically influence antitumor immunity. Accelerated GSDMD cleavage is shown herein to lead to enhanced IL-1α and IL-1β release, as well as tumor-specific CD8 T cell accumulation. The results indicate that HPP compounds are useful in treating cancer / enhancing antitumor immunity. Accordingly, this application relates to a method for enhancing intracellular antitumor immunity in either a biological sample or subject, comprising the step of administering an effective amount of one or more of the compounds of this application to cells, wherein the compounds of this application are compounds of formula (I), or pharmaceutically acceptable salts, solvates, and / or ester prodrugs thereof:

[0008] [ka] During the ceremony, R 1 , R 2 , and R 3 Under the condition that at least one of them is OH, R 1 , R 2 , and R 3 These are independently OH or H. Includes methods selected from.

[0009] In some embodiments, the present application relates to a method for improving intracellular antitumor immunity in any biological sample or subject, comprising the step of administering an effective amount of one or more compounds of the present application to cells, wherein the compounds of the present application are compounds of formulas (II), (III), and (IV), or pharmaceutically acceptable salts, solvates, and / or ester prodrugs thereof:

[0010] [ka] During the ceremony, R 4is halogen, NR 5 R 6 and C 1~3 selected from alkyl; R 5 and R 6 are independently selected from H and C 1~3 alkyl;

[0011]

Chemical formula

[0012]

Chemical formula

[0013] In some embodiments, the present application is a method for improving antitumor immunity in cells in either a biological sample or a subject, comprising administering an effective amount of one or more compounds of the present application to the cells, wherein the compound of the present application is a compound of formula (V), or a pharmaceutically acceptable salt, solvate, and / or ester prodrug thereof:

[0014]

Chemical formula

[0015] This application also includes a method for improving antitumor immunity, comprising the step of administering a therapeutically effective amount of one or more compounds of this application to a subject in need thereof.

[0016] This application also includes a method for treating intracellular cancer in any biological sample or subject that requires such treatment, comprising the step of administering an effective amount of one or more of the compounds of this application to cells.

[0017] This application also includes a method for treating cancer, comprising the step of administering a therapeutically effective amount of one or more compounds of this application to a subject in need thereof.

[0018] This application also includes a method for treating cancer, comprising the step of administering a therapeutically effective amount of one or more of the compounds of this application to a subject in need, in combination with another known active substance or another known cancer therapy for treating cancer.

[0019] This application also includes pharmaceutical compositions comprising one or more of the compounds of this application and a pharmaceutically acceptable carrier, wherein one or more of the compounds of this application are present in the composition in an amount effective for increasing antitumor immunity or treating cancer.

[0020] In some embodiments, the pharmaceutical composition further comprises one or more additional anticancer agents.

[0021] This application further includes a method for enhancing immune surveillance in any biological sample or subject by increasing GSDMD cleavage and promoting the release of pro-inflammatory cytokines such as IL-1α and IL-1β within cells, while partially protecting against cell death, thereby enhancing the NF-κB pathway, comprising the step of administering an effective amount of one or more of the compounds of this application to cells.

[0022] This application includes a method for increasing antitumor immunity in either a biological sample or subject by increasing tumor stromal IL-1β release and tumor-specific CD8 T cell accumulation in intracellular TMEs, comprising the step of administering an effective amount of one or more of the compounds of this application to cells.

[0023] This application also includes a method for treating a disease, disorder, or condition that can be treated by increasing GSDMD cleavage, comprising the step of administering a therapeutically effective amount of one or more of the compounds of this application to a subject in need thereof.

[0024] This application also includes a method for treating a disease, disorder, or condition that can be treated by increasing tumor stromal IL-1β release and tumor-specific CD8 T cell accumulation in the tumor mesenteric cell (TME), comprising the step of administering a therapeutically effective amount of one or more of the compounds of this application to a subject in need thereof.

[0025] This application also includes a method for treating a disease, disorder, or condition that is treatable by increasing GSDMD cleavage, comprising the step of administering a therapeutically effective amount of one or more of the compounds of this application to a subject in need, in combination with another known active substance useful for treating a disease, disorder, or condition that is treatable by accelerating GSDMD cleavage.

[0026] This application also includes a method for treating a disease, disorder, or condition that can be treated by increasing tumor stromal IL-1β release and tumor-specific CD8 T cell accumulation in the tumor mesenteric area (TME), comprising the step of administering a therapeutically effective amount of one or more compounds of this application to a subject in need, in combination with another known active substance useful for treating a disease, disorder, or condition that can be treated by increasing tumor stromal IL-1β release and tumor-specific CD8 T cell accumulation in the TME.

[0027] In some embodiments, the disease, disorder, or condition that can be treated by increasing GSDMD cleavage is cancer. In some embodiments, the disease, disorder, or condition that can be treated by increasing tumor stromal IL-1β release and tumor-specific CD8 T cell accumulation in the tumor mesenteric cell (TME) is cancer.

[0028] This application also includes a method for increasing the potency of one or more additional active agents for the treatment and / or therapy of cancer, comprising the step of administering an effective amount of one or more of the compounds of this application to a subject in need, in combination with an effective amount of one or more additional active agents for the treatment and / or therapy of cancer.

[0029] Other features and advantages of this application will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while illustrating embodiments of this application, are given merely as illustrations, and the claims should not be limited by these embodiments, but rather given the broadest interpretation consistent with the overall description.

[0030] This application will be described in more detail with reference to the attached drawings and tables. [Brief explanation of the drawing]

[0031] [Figure 1a]Figure 1 shows the relationship between the host microbiome and its ability to monitor tumor immunity, and Figure 1a shows PcoA for CR, TAC, or JAX C57BL / 6 female mice before tumor transplantation (n=5 / group, 2 combined experiments, weighted UniFrac distance, pairwise PERMANOVA test); [Figure 1b] Figure 1 shows the relationship between the host microbiome and its ability to monitor tumor immunity, and Figure 1b shows the relative abundance of microbiomes at the family level in fecal microbiome samples from CR, TAC, or JAX C57BL / 6 female mice before tumor transplantation (n=5 / group, 2 combined experiments, weighted UniFrac distance, pairwise PERMANOVA test). [Figure 1c] Figure 1 shows the relationship between the host microbiome and its ability to provide tumor immune surveillance, and Figure 1c shows the titration survival curve (n=4-5 / group, single experiment) for M3-9-M tumor cells orthotopically transplanted into the gastrocnemius muscle of female C57BL / 6 mice procured from CR. [Figure 1d] Figure 1 shows the relationship between the host microbiome and its ability to monitor tumor immunity, and Figure 1d shows the titration survival curve (n=4-5 / group, single experiment) for M3-9-M tumor cells orthotopically transplanted into the gastrocnemius muscle of female C57BL / 6 mice procured from TAC. [Figure 1e] Figure 1 shows the relationship between the host microbiome and its ability to monitor tumor immunity, and Figure 1e shows the titration survival curve (n=4-5 / group, single experiment) for M3-9-M tumor cells orthotopically transplanted into the gastrocnemius muscle of female C57BL / 6 mice procured from JAX. [Figure 1f] Figure 1 shows the relationship between the host microbiome and its ability to provide tumor immune surveillance, and Figure 1f shows the overall survival (n=10-13 / group, two combined experiments, log-rank test) for M3-9-M tumors orthotopically transplanted into CR, TAC, and JAX C57BL / 6 female mice. [Figure 1g]Figure 1 illustrates the correlation between the host microbiome and its ability to monitor tumor immunity, and Figure 1g shows the effect of CD8 T cell depletion using an anti-CD8 (clone 2.43) neutralizing antibody on the overall survival of M3-9-M orthotopically transplanted into JAX C57BL / 6 female mice (n=4 / group, single experiment). [Figure 1h] Figure 1 shows the correlation between the host microbiome and its ability to monitor tumor immunity, and Figure 1h shows flow cytometry detection of CD8 T cell frequency within M3-9-M tumor mass 18 days after orthotopic transplantation (n=5 / group, 2 combined experiments, unpaired t-test). [Figure 1i] Figure 1 shows how the host microbiome is associated with its ability to monitor tumor immunity, and Figure 1i shows the number of qPCR cycles (n=6 / group, 2 combined experiments, unpaired t-test) for 16S gene microbial DNA in the fecal matter of mice that received ABX-supplemented or unsupplemented drinking water for 2 weeks. [Figure 1j] Figure 1 shows the relationship between the host microbiome and its ability to monitor tumor immunity, and Figure 1j shows the body weight (n=5 / group, two combined experiments) of mice that received drinking water supplemented with or not supplemented with ABX. [Figure 1k] Figure 1 shows the relationship between the host microbiome and its ability to monitor tumor immunity, and Figure 1k shows the overall survival (n=8-12 / group, two combined experiments, log-rank test) for M3-9-M tumors orthotopically transplanted into CR C57BL / 6 female mice that received either normal drinking water or drinking water supplemented with ABX. [Figure 1l] Figure 1 illustrates the correlation between the host microbiome and its ability to monitor tumor immunity. Figure 11 shows the overall survival rate (n=11-12 / group, two combined experiments, log-rank test) for orthotopically transplanted M3-9-M tumors in JAX C57BL / 6 female mice that received either normal drinking water or drinking water supplemented with ABX. [Figure 2a]Figure 2 illustrates how different microbiomes produce different capabilities for tumor immune surveillance in a genetically identical host, with Figure 2a showing the process followed for the production of IMDM mice. [Figure 2b] Figure 2 illustrates how different microbiomes produce different capabilities for tumor immune surveillance in genetically identical hosts, and Figure 2b shows the PcoA (Permanova test, weighted UniFrac method) for the fecal microbiome at the end of each production process in IMDM mice (circular dots represent each mouse). [Figure 2c] Figure 2 illustrates how different microbiomes produce different capabilities for tumor immune surveillance in genetically identical hosts, and Figure 2c shows the PcoA (Permanova test, weighted UniFrac method) for the fecal microbiome at the end of each production process in IMDM mice (circular dots represent each mouse). [Figure 2d] Figure 2 illustrates how different microbiomes produce different capabilities for tumor immune surveillance in genetically identical hosts, and Figure 2d shows the PcoA (Permanova test, weighted UniFrac method) for the fecal microbiome at the end of each production process in IMDM mice (circular dots represent each mouse). [Figure 2e] Figure 2 illustrates how different microbiomes produce different capabilities for tumor immune surveillance in genetically identical hosts, and Figure 2e shows the PcoA (Permanova test, weighted UniFrac method) of fecal microbiomes from 6-8 week old IMDM-CR vs. JAX mice, combining three sets of littermates across three generations. [Figure 2f] Figure 2 illustrates how different microbiomes contribute to different tumor immune surveillance capabilities in genetically identical hosts, and Figure 2f shows the relative abundance of fecal bacteria in IMDM mice, as identified by 16S amplicon sequencing. [Figure 2g]Figure 2 illustrates how different microbiomes contribute to different tumor immune surveillance capabilities in genetically identical hosts, and Figure 2g shows the relative abundance of fecal bacteria in IMDM mice, as identified by 16S amplicon sequencing. [Figure 2h] Figure 2 illustrates how different microbiomes generate different capabilities for tumor immune surveillance in genetically identical hosts, and Figure 2h shows the workflows related to Figures 2i-n. [Figure 2i] Figure 2 illustrates how different microbiomes produce different capabilities for tumor immune surveillance in genetically identical hosts, and Figure 2i shows tumor growth dynamics (n=10-12 / group, two combined experiments, two-way ANOVA for growth dynamics, log-rank test for overall survival) for M3-9-M tumors grown in IMDM-CR vs. JAX female mice. [Figure 2j] Figure 2 illustrates how different microbiomes produce different capabilities for tumor immune surveillance in genetically identical hosts, and Figure 2j shows the overall survival (n=10-12 / group, two combined experiments, two-way ANOVA for growth dynamics, log-rank test for overall survival) for M3-9-M tumors grown in IMDM-CR vs. JAX female mice. [Figure 2k] Figure 2 illustrates how different microbiomes produce different capabilities for tumor immune surveillance in genetically identical hosts, and Figure 2k shows tumor growth dynamics (n=7 / group, two combined experiments, two-way ANOVA for growth dynamics, log-rank test for overall survival) for M3-9-M tumors grown in IMDM-CR vs. JAX male mice. [Figure 2l] Figure 2 illustrates how different microbiomes produce different capabilities for tumor immune surveillance in genetically identical hosts, and Figure 2l shows the overall survival (n=7 / group, two combined experiments, two-way ANOVA for growth dynamics, log-rank test for overall survival) for M3-9-M tumors grown in IMDM-CR vs. JAX male mice. [Figure 2m]Figure 2 illustrates how different microbiomes produce different capabilities for tumor immune surveillance in genetically identical hosts. Figure 2m shows tumor growth dynamics (n=12-25 / group, two combined experiments, two-way ANOVA for growth dynamics, log-rank test for overall survival) for M3-9-MOVA tumors grown in IMDM-CR vs. JAX male mice. [Figure 2n] Figure 2 illustrates how different microbiomes produce different capabilities for tumor immune surveillance in genetically identical hosts, and Figure 2n shows the overall survival (n=12-25 / group, two combined experiments, two-way ANOVA for growth dynamics, log-rank test for overall survival) for M3-9-MOVA tumors grown in IMDM-CR vs. JAX male mice. [Figure 2o] Figure 2 illustrates how different microbiomes produce different capabilities for tumor immune surveillance in genetically identical hosts, and Figure 2o shows flow cytometry (n=6 / group, 2 combined experiments, unpaired t-test) for the detection of OVA-specific CD8 T cells in M3-9-MOVA tumors grown for 18 days. [Figure 3a] Figure 3 shows that various microbiomes induce unique metabolome profiles within genetically identical hosts, and that specific classes of metabolites are associated with superior host tumor immune surveillance capabilities. Figure 3a shows a PC plot (pooled samples from two different experiments, ellipses drawn with 95% confidence intervals) for fecal metabolome in IMDM-CR vs. JAX male mice. [Figure 3b] Figure 3 shows that different microbiomes induce unique metabolome profiles within genetically identical hosts, and that certain classes of metabolites are associated with superior host tumor immune surveillance capabilities. Figure 3b shows a Spearman correlation heatmap for fecal metabolomes of IMDM-CR vs. JAX male mice (pooled samples from two different experiments). [Figure 3c]Figure 3 shows that various microbiomes induce unique metabolome profiles within genetically identical hosts, and that specific classes of metabolites are associated with superior host tumor immune surveillance capabilities. Figure 3c shows PC plots (pooled samples from two different experiments, ellipses drawn with 95% confidence intervals) for orthotopically transplanted M3-9-MOVA tumor metabolomes in IMDM-CR vs. JAX male mice. [Figure 3d] Figure 3 shows that various microbiomes induce unique metabolome profiles within genetically identical hosts, and that specific classes of metabolites are associated with superior host tumor immune surveillance capabilities. Figure 3d shows a Spearman correlation heatmap (pooled samples from two different experiments) for orthotopically transplanted M3-9-MOVA tumor metabolomes in IMDM-CR vs. JAX male mice. [Figure 3e] Figure 3 shows that various microbiomes induce unique metabolome profiles within genetically identical hosts, and that specific classes of metabolites are associated with superior host tumor immune surveillance capabilities. Figure 3e shows PC plots (pooled samples from two different experiments, ellipses drawn with 95% confidence intervals) for fecal metabolome of IMDM-JAX female mice treated with normal water versus ABX-supplemented water. [Figure 3f] Figure 3 shows that various microbiomes induce unique metabolome profiles within genetically identical hosts, and that specific classes of metabolites are associated with superior host tumor immune surveillance capabilities. Figure 3f shows PC plots and Spearman correlation heatmaps (pooled samples from two different experiments) for fecal metabolomes of IMDM-JAX female mice treated with normal water versus ABX-supplemented water. [Figure 3g]Figure 3 shows that various microbiomes induce unique metabolome profiles within genetically identical hosts, and that specific classes of metabolites are associated with superior host tumor immune surveillance capabilities. Figure 3g shows PC plots (pooled samples from two different experiments, ellipses drawn with 95% confidence intervals) for orthotopically transplanted M3-9-M tumor metabolomes in IMDM-JAX female mice that received normal water or ABX-supplemented water. [Figure 3h] Figure 3 shows that various microbiomes induce unique metabolome profiles within genetically identical hosts, and that specific classes of metabolites are associated with superior host tumor immune surveillance capabilities. Figure 3h shows a Spearman correlation heatmap (pooled samples from two different experiments) for orthotopically transplanted M3-9-M tumor metabolomes in IMDM-JAX female mice that received normal water or ABX-supplemented water. [Figure 3i] Figure 3 shows that various microbiomes induce unique metabolome profiles within genetically identical hosts, and that specific classes of metabolites are associated with superior host tumor immune surveillance capabilities. Figure 3i shows volcano plots for feces from IMDM-CR vs. JAX male mice, respectively (pooled samples from two different experiments, adjusted for false positive rates). [Figure 3j] Figure 3 shows that diverse microbiomes induce unique metabolome profiles within genetically identical hosts, and that specific classes of metabolites are associated with superior host tumor immune surveillance capabilities. Figure 3j shows volcano plots for tumor metabolites in IMDM-CR vs. JAX male mice, respectively (pooled samples from two different experiments, adjusted for false positive rates). [Figure 3k]Figure 3 shows that diverse microbiomes induce unique metabolome profiles within genetically identical hosts, and that specific classes of metabolites are associated with superior host tumor immune surveillance capabilities. Figure 3k shows the relative levels of fecal samples from IMDM-CR vs. JAX male mice (pooled samples from two different experiments, unpaired t-test). Using the same metabolome dataset, different types of graphs were created for the same experimental group. [Figure 3l] Figure 3 shows how diverse microbiomes induce unique metabolome profiles within genetically identical hosts, and how specific classes of metabolites are associated with superior host tumor immune surveillance capabilities. Figure 31 shows the relative levels of selected metabolites in M3-9-MOVA tumors in IMDM-CR vs. JAX male mice (pooled samples from two different experiments, unpaired t-test). Using the same metabolome dataset, different types of graphs were created for the same experimental group. [Figure 3m] Figure 3 shows that diverse microbiomes induce unique metabolome profiles within genetically identical hosts, and that specific classes of metabolites are associated with superior host tumor immune surveillance capabilities. Figure 3m shows the relative levels of selected metabolites in feces and M3-9-MOVA tumors of IMDM-CR vs. JAX male mice (pooled samples from two different experiments, unpaired t-test). Different types of graphs were created for the same experimental group using the same metabolome dataset. [Figure 3n] Figure 3 shows how diverse microbiomes induce unique metabolome profiles within genetically identical hosts, and how specific classes of metabolites are associated with superior host tumor immune surveillance capabilities. Figure 3n shows the relative levels of selected metabolites in feces and M3-9-MOVA tumors of IMDM-CR vs. JAX male mice (pooled samples from two different experiments, unpaired t-test). Different types of graphs were created for the same experimental group using the same metabolome dataset. [Figure 4a]Figure 4 illustrates how diverse microbiomes induce unique metabolome profiles within genetically identical hosts, and the identification of metabolites associated with tumor immune surveillance capabilities. Figure 4a shows PC scores for fecal metabolome in IMDM-CR vs. JAX female mice (pooled samples from two different experiments, drawn as ellipses with 95% confidence intervals). [Figure 4b] Figure 4 illustrates how diverse microbiomes induce unique metabolome profiles within genetically identical hosts and the identification of metabolites associated with tumor immune surveillance capabilities, while Figure 4b shows a Spearman correlation heatmap for fecal metabolomes of IMDM-CR vs. JAX female mice (pooled samples from two different experiments). [Figure 4c] Figure 4 illustrates how diverse microbiomes induce unique metabolome profiles within genetically identical hosts and the identification of metabolites associated with tumor immune surveillance capabilities. Figure 4c shows PC scores (pooled samples from two different experiments, drawn as ellipses with 95% confidence intervals) for orthotopic M3-9-M RMS metabolomes transplanted into IMDM-CR vs. JAX female mice. [Figure 4d] Figure 4 illustrates how diverse microbiomes induce unique metabolome profiles within genetically identical hosts and the identification of metabolites associated with tumor immune surveillance capabilities. Figure 4d shows a Spearman correlation heatmap (pooled samples from two different experiments) for orthotopic M3-9-M RMS metabolomes transplanted into IMDM-CR vs. JAX female mice. [Figure 4e] Figure 4 illustrates how diverse microbiomes induce unique metabolome profiles within genetically identical hosts and the identification of metabolites associated with tumor immune surveillance capabilities. Figure 4e shows the results for fecal samples (pooled samples from two different experiments, FDR adjusted, logarithmically transformed p-values) from IMDM-CR vs. JAX female mice, selected by a volcano plot with a magnification change threshold (x)² and a t-test threshold (y) of 0.05. [Figure 4f] Figure 4 illustrates how diverse microbiomes induce unique metabolome profiles within genetically identical hosts and the identification of metabolites associated with tumor immune surveillance capabilities. Figure 4f shows the results for tumor metabolites from IMDM-CR vs. JAX female mice (pooled samples from two different experiments, FDR adjusted, log-transformed p-values) selected by a volcano plot with a magnification change threshold (x)² and a t-test threshold (y) of 0.05. [Figure 4g] Figure 4 illustrates how diverse microbiomes induce unique metabolome profiles within genetically identical hosts and the identification of metabolites associated with tumor immune surveillance capabilities. Figure 4g shows the relative levels of fecal samples from IMDM-CR vs. JAX female mice (pooled samples from two different experiments, unpaired t-test). [Figure 4h] Figure 4 illustrates how diverse microbiomes induce unique metabolome profiles within genetically identical hosts and the identification of metabolites associated with tumor immune surveillance capabilities. Figure 4h shows the relative levels of L-gln metabolites in M3-9-M RMS tumors in IMDM-CR vs. JAX female mice (pooled samples from two different experiments, unpaired t-test). [Figure 4i] Figure 4 illustrates how diverse microbiomes induce unique metabolome profiles within genetically identical hosts and the identification of metabolites associated with tumor immune surveillance capabilities. Figure 4i shows the relative levels of fecal samples from IMDM-CR vs. JAX female mice (pooled samples from two different experiments, unpaired t-test). [Figure 4j] Figure 4 illustrates how diverse microbiomes induce unique metabolome profiles within genetically identical hosts and the identification of metabolites associated with tumor immune surveillance capabilities. Figure 4j shows the relative levels of HPP metabolites in M3-9-M RMS in IMDM-CR vs. JAX female mice (pooled samples from two different experiments, unpaired t-test). [Figure 4k]Figure 4 illustrates how diverse microbiomes induce unique metabolome profiles within genetically identical hosts and the identification of metabolites associated with tumor immune surveillance capabilities. Figure 4k shows the relative levels of HPP metabolites in the feces of IMDM-JAX female mice treated with normal water versus ABX-supplemented water (pooled samples from two different experiments, unpaired t-test). Different types of graphs were created for the same experimental group using the same metabolome dataset. [Figure 4l] Figure 4 illustrates how diverse microbiomes induce unique metabolome profiles within genetically identical hosts and the identification of metabolites associated with tumor immune surveillance capabilities. Figure 41 shows the relative levels of HPP metabolites in M3-9-M RMS tumors in IMDM-JAX female mice treated with normal water versus ABX-supplemented water (pooled samples from two different experiments, unpaired t-test). Different types of graphs were created for the same experimental group using the same metabolome dataset. [Figure 5a] Figure 5 shows the HPP metabolite treatment strategy determination, and Figure 5a shows the structures of exemplary HPP isomers. [Figure 5b] Figure 5 shows the HPP metabolite processing strategy determination, and Figure 5b shows the workflow related to Figures 5c-d. [Figure 5c] Figure 5 shows the HPP metabolite treatment strategy, and Figure 5c shows the overall survival rate (single experiment) for intraperitoneal 3,2-HPP treatment at various doses. [Figure 5d] Figure 5 shows the HPP metabolite treatment strategy, and Figure 5d shows the change in body weight of mice treated with various doses of intraperitoneal 3,2-HPP (single experiment). [Figure 5e] Figure 5 shows the HPP metabolite processing strategy determination, and Figure 5e shows the workflow related to Figure 5f. [Figure 5f]Figure 5 shows the HPP metabolite treatment strategy determination, and Figure 5f shows the serum levels of 3,2-HPP metabolites at various time points after intraperitoneal delivery of 83 mg / kg body weight to mice (single experiment). [Figure 5g] Figure 5 shows the HPP metabolite processing strategy determination, and Figure 5g shows the workflow related to Figure 5h. [Figure 5h] Figure 5 shows the HPP metabolite treatment strategy determination, and Figure 5h shows the change in body weight of mice after receiving various metabolites (single experiment). [Figure 5i] Figure 5 shows the HPP metabolite processing strategy determination, and Figure 5i shows the workflow related to Figures 5j to k. [Figure 5j] Figure 5 shows the HPP metabolite treatment strategy determination, and Figure 5j shows the overall survival (two combined experiments, log-rank test) for B16.F10 orthotopically transplanted into IMDM-CR mice treated with vehicle or 3,2-HPP metabolite 7 days after tumor transplantation. [Figure 5k] Figure 5 shows the HPP metabolite treatment strategy determination, and Figure 5k shows the overall survival (two combined experiments, log-rank test) for M3-9-MOVA orthotopically transplanted into IMDM-CR mice treated with vehicle or 3,2-HPP metabolite 7 days after tumor transplantation. [Figure 5l] Figure 5 shows the HPP metabolite processing strategy determination and illustrates the workflow related to Figures 5l and 5m-p. [Figure 5m] Figure 5 shows the HPP metabolite treatment strategy determination, and Figure 5m shows flow cytometry to measure the efficacy of anti-CD8 neutralizing antibody (2.43 clone; ip) in mice, with ∫ showing the vehicle. [Figure 5n]Figure 5 shows the HPP metabolite treatment strategy determination, and Figure 5n shows flow cytometry to measure the efficacy of anti-CD8 neutralizing antibody (2.43 clone; ip) in mice, showing 3,2-HPP treatment. [Figure 5o] Figure 5 shows the HPP metabolite treatment strategy determination, and Figure 5o shows flow cytometry to measure the efficacy of anti-CD8 neutralizing antibody (2.43 clone; ip) in mice, indicating anti-CD8 antibody treatment. [Figure 5p] Figure 5 shows the HPP metabolite treatment strategy determination, and Figure 5p shows flow cytometry to measure the efficacy of anti-CD8 neutralizing antibody (2.43 clone; ip) in mice, indicating anti-CD8 antibody + 3,2-HPP treatment. [Figure 6a] Figure 6 shows an example of a microbiome-derived metabolite, 3,2-HPP, which enhances antitumor immunity, and Figure 6a shows the workflow related to Figures 6b-e. [Figure 6b] Figure 6 shows that the exemplary microbiome-derived metabolite 3,2-HPP enhances antitumor immunity, and Figure 6b shows the overall survival curves for immune surveillance of M3-9-M in females treated with the metabolite iP starting from day 1 (two combined experiments, log-rank test). [Figure 6c] Figure 6 shows that the exemplary microbiome-derived metabolite 3,2-HPP enhances antitumor immunity, and Figure 6c shows the overall survival curves for immune surveillance of M3-9-MOVA in males treated with ip treatment of the metabolite, initiated from day 1 (two combined experiments, log-rank test). [Figure 6d] Figure 6 shows that the exemplary microbiome-derived metabolite 3,2-HPP enhances antitumor immunity, and Figure 6d shows the overall survival curves (two combined experiments, log-rank test) for immune surveillance of M3-9-M in male IMDM-CR mice treated with the metabolite via ip starting from day 1. [Figure 6e]Figure 6 shows that the exemplary microbiome-derived metabolite 3,2-HPP enhances antitumor immunity, and Figure 6e shows the overall survival curves (two combined experiments, log-rank test) for immune surveillance in female B16.F10 treated with the metabolite iP starting from day 1. [Figure 6f] Figure 6 shows that the exemplary microbiome-derived metabolite 3,2-HPP enhances antitumor immunity, and Figure 6f shows the workflow related to Figures 6g-h. [Figure 6g] Figure 6 shows that the exemplary microbiome-derived metabolite 3,2-HPP enhances antitumor immunity, and Figure 6g shows the effect of ABX on the overall survival curve for M3-9-M immune surveillance in IMDM-JAX female mice treated with ip-3,2-HPP (two combined experiments, log-rank test). [Figure 6h] Figure 6 shows that the exemplary microbiome-derived metabolite 3,2-HPP enhances antitumor immunity, and Figure 6h shows the effect of ABX on the overall survival curve for M3-9-M immune surveillance in IMDM-CR female mice treated with ip-3,2-HPP (two combined experiments, log-rank test). [Figure 6i] Figure 6 shows an example of a microbiome-derived metabolite, 3,2-HPP, that enhances antitumor immunity, and Figure 6i shows the workflow related to Figures 6j-k. [Figure 6j] Figure 6 shows that the exemplary microbiome-derived metabolite 3,2-HPP enhances antitumor immunity, and Figure 6j shows flow cytometry (two combined experiments, unpaired t-test) for detection of OVA-specific CD8 T cells in the TME of M3-9-MOVA in IMDM-CR male mice treated with 3,2-HPP 18 days after tumor transplantation. [Figure 6k]Figure 6 shows that the exemplary microbiome-derived metabolite 3,2-HPP enhances antitumor immunity, and Figure 6k shows the expression of exhaustion markers on this cell type of OVA-specific CD8 T cells within the TME of M3-9-MOVA in IMDM-CR male mice 18 days after tumor transplantation, treated with 3,2-HPP (two combined experiments, unpaired t-tests). [Figure 6l] Figure 6 shows an example of a microbiome-derived metabolite 3,2-HPP that enhances antitumor immunity, and Figure 6l shows the workflow related to Figure 6m. [Figure 6m] Figure 6 shows that the exemplary microbiome-derived metabolite 3,2-HPP enhances antitumor immunity, and Figure 6m shows the effect of CD8 T cell neutralization (log-rank test, single experiment) on the overall survival curve for M3-9-M immune surveillance in IMDM-CR female mice treated with ip-3,2-HPP. [Figure 6n] Figure 6 shows an example of a microbiome-derived metabolite 3,2-HPP that enhances antitumor immunity, and Figure 6n shows the workflow related to Figures 6o-p. [Figure 6o] Figure 6 shows that the exemplary microbiome-derived metabolite 3,2-HPP enhances antitumor immunity, and Figure 6o shows the effects of anti-PD-1 and antibody PD-L1 therapy on overall survival curves for immune surveillance of M3-9-M in IMDM-CR female mice treated with ip-3,2-HPP (two combined experiments, log-rank test). [Figure 6p] Figure 6 shows that the exemplary microbiome-derived metabolite 3,2-HPP enhances antitumor immunity, and Figure 6p shows the effects of anti-PD-1 and antibody PD-L1 therapy on the overall survival curves for immune surveillance in B16.F10 in IMDM-CR female mice treated with ip-3,2-HPP (two combined experiments, log-rank test). [Figure 7a]Figure 7 shows an exemplary HPP enhancing the cancer immune signaling pathway in TME, and Figure 7a shows the workflow of the animal experiment used for scRNAseq. [Figure 7b] Figure 7 shows exemplary HPP enhancing cancer immune signaling pathways in the TME, and Figure 7b shows immune cell types within the mouse TME identified by scRNAseq of CD45+ cells isolated from orthotopically transplanted M3-9-MOVA RMS. [Figure 7c] Figure 7 shows an exemplary HPP enhancing the cancer immune signaling pathway in TME, and Figure 7c shows the number of cells used for statistical analysis in the scRNAseq experiment. [Figure 7d] Figure 7 shows an example of HPP enhancing cancer immune signaling pathways in TME, and Figure 7d shows the number of significantly affected genes obtained from pairwise comparisons (Wilcoxon rank-sum test). [Figure 7e] Figure 7 shows an exemplary HPP enhancing cancer immune signaling pathways in TME, and Figure 7e shows a plot of all significantly affected genes obtained from pairwise comparisons that overlap with each other through identical or shared pathways. [Figure 7f] Figure 7 shows an exemplary HPP enhancing cancer immune signaling pathways in TME, and Figure 7f is a bar graph of transcriptional regulators of genes affected by 3,2-HPP treatment. [Figure 7g] Figure 7 illustrates how exemplary HPP enhances cancer immune signaling pathways in TME, and Figure 7g shows NF-κB and IRF induction in mouse RAW-Dual® cells treated with LPS in the presence of HPP metabolites (three combined experiments, one-way ANOVA test). [Figure 7h]Figure 7 shows exemplary HPP enhancing cancer immune signaling pathways in TME, and Figure 7h shows NF-κB and IRF induction in mouse RAW-Dual® cells treated with VSV in the presence of HPP metabolites (three combined experiments, one-way ANOVA test). [Figure 7i] Figure 7 shows an exemplary HPP enhancing the cancer immune signaling pathway in TME, and Figure 7i shows the workflow for the animal experiments used in Figures 7j-l. [Figure 7j] Figure 7 shows exemplary HPP enhancing cancer immune signaling pathways in TME, and Figure 7j shows overall survival curves (two combined experiments; log-rank test) illustrating immune surveillance in male M3-9-M after ip treatment with HPP metabolites initiated from day 1. [Figure 7k] Figure 7 shows exemplary HPP enhancing cancer immune signaling pathways in TME, and Figure 7k shows overall survival curves (two combined experiments; log-rank test) illustrating immune surveillance of M3-9-MOVA in males treated with ip treatment of HPP metabolites initiated from day 1. [Figure 7l] Figure 7 shows exemplary HPP enhancing cancer immune signaling pathways in TME, and Figure 7l shows overall survival curves (two combined experiments; log-rank test) illustrating immune surveillance of B16.F10 in female IMDM-CR mice treated with ip treatment of HPP metabolites initiated from day 1. [Figure 8a] Figure 8 shows how exemplary 3,2-HPP metabolite treatments affect gene expression in various immune cell subsets in TME, and Figure 8a shows a general plot (Wilcoxon rank-sum test) showing duplicate genes that were statistically significantly affected in pairwise comparisons between IMDM-CR vs. JAX and IMDM-CR vs. IMDM-CR-HPP. [Figure 8b]Figure 8 shows how exemplary 3,2-HPP metabolite treatments affect gene expression in various immune cell subsets in TME, and Figure 8b shows a general plot (Wilcoxon rank-sum test) showing duplicate genes that were statistically significantly affected in pairwise comparisons between IMDM-CR vs. JAX and IMDM-CR vs. IMDM-CR-HPP. [Figure 8c] Figure 8 shows how exemplary 3,2-HPP metabolite treatments affect gene expression in various immune cell subsets in TME, and Figure 8c shows a general plot (Wilcoxon rank-sum test) showing duplicate genes that were statistically significantly affected in pairwise comparisons between IMDM-CR vs. JAX and IMDM-CR vs. IMDM-CR-HPP. [Figure 8d] Figure 8 shows how exemplary 3,2-HPP metabolite treatments affect gene expression in various immune cell subsets in TME, and Figure 8d shows a general plot (Wilcoxon rank-sum test) showing duplicate genes that were statistically significantly affected in pairwise comparisons between IMDM-CR vs. JAX and IMDM-CR vs. IMDM-CR-HPP. [Figure 8e] Figure 8 shows how exemplary 3,2-HPP metabolite treatments affect gene expression in various immune cell subsets in TME, and Figure 8e shows a general plot (Wilcoxon rank-sum test) showing duplicate genes that were statistically significantly affected in pairwise comparisons between IMDM-CR vs. JAX and IMDM-CR vs. IMDM-CR-HPP. [Figure 8f] Figure 8 shows how exemplary 3,2-HPP metabolite treatments affect gene expression in various immune cell subsets in TME, and Figure 8f shows a general plot (Wilcoxon rank-sum test) showing duplicate genes that were statistically significantly affected in pairwise comparisons between IMDM-CR vs. JAX and IMDM-CR vs. IMDM-CR-HPP. [Figure 9a] Figure 9 shows exemplary HPP isomers (I-1, I-2, and I-3) enhancing the NF-κB and IRF signaling pathways in bone marrow cells, with Figure 9a showing THP1-Dual® cells (two combined experiments) treated with LPS for 0–24 hours in the presence of a vehicle or HPP metabolite for NF-κB pathway induction. [Figure 9b] Figure 9 shows exemplary HPP isomers (I-1, I-2, and I-3) enhancing NF-κB and IRF signaling pathways in bone marrow cells, and Figure 9b shows THP1-Dual® cells treated for 16 hours with various NF-κB pathway inducers in the presence or absence of HPP isomers (three combined experiments, one-way ANOVA test). [Figure 9c] Figure 9 shows exemplary HPP isomers (I-1, I-2, and I-3) enhancing NF-κB and IRF signaling pathways in bone marrow cells, and Figure 9c shows THP1-Dual® cells treated with various NF-κB pathway inducers for 16 hours in the presence or absence of HPP isomers (three combined experiments, one-way ANOVA test). [Figure 9d] Figure 9 shows exemplary HPP isomers (I-1, I-2, and I-3) enhancing NF-κB and IRF signaling pathways in bone marrow cells, and Figure 9d shows THP1-Dual® cells treated for 16 hours with various NF-κB pathway inducers in the presence or absence of HPP isomers (three combined experiments, one-way ANOVA test). [Figure 9e] Figure 9 shows exemplary HPP isomers (I-1, I-2, and I-3) enhancing NF-κB and IRF signaling pathways in bone marrow cells, and Figure 9e shows THP1-Dual® cells treated for 16 hours with various NF-κB pathway inducers in the presence or absence of HPP isomers (three combined experiments, one-way ANOVA test). [Figure 9f]Figure 9 shows exemplary HPP isomers (I-1, I-2, and I-3) enhancing NF-κB and IRF signaling pathways in bone marrow cells, and Figure 9f shows THP1-Dual® cells treated for 16 hours with various NF-κB pathway inducers in the presence or absence of HPP isomers (three combined experiments, one-way ANOVA test). [Figure 9g] Figure 9 shows exemplary HPP isomers (I-1, I-2, and I-3) enhancing NF-κB and IRF signaling pathways in bone marrow cells, and Figure 9g shows THP1-Dual® cells treated for 16 hours with various IRF pathway inducers in the presence or absence of HPP (three combined experiments, one-way ANOVA test). [Figure 9h] Figure 9 shows exemplary HPP isomers (I-1, I-2, and I-3) enhancing NF-κB and IRF signaling pathways in bone marrow cells, and Figure 9h shows THP1-Dual® cells treated for 16 hours with various IRF pathway inducers in the presence or absence of HPP (three combined experiments, one-way ANOVA test). [Figure 9i] Figure 9 shows exemplary HPP isomers (I-1, I-2, and I-3) enhancing NF-κB and IRF signaling pathways in bone marrow cells, and Figure 9i shows THP1-Dual® cells treated for 16 hours with various IRF pathway inducers in the presence or absence of HPP (three combined experiments, one-way ANOVA test). [Figure 9j] Figure 9 shows exemplary HPP isomers (I-1, I-2, and I-3) enhancing NF-κB and IRF signaling pathways in bone marrow cells, and Figure 9j shows THP1-Dual® cells treated for 16 hours with various IRF pathway inducers in the presence or absence of HPP (three combined experiments, one-way ANOVA test). [Figure 9k]Figure 9 shows exemplary HPP isomers (I-1, I-2, and I-3) enhancing NF-κB and IRF signaling pathways in bone marrow cells, and Figure 9k shows THP1-Dual® cells treated for 16 hours with various IRF pathway inducers in the presence or absence of HPP (three combined experiments, one-way ANOVA test). [Figure 9l] Figure 9 shows exemplary HPP isomers (I-1, I-2, and I-3) enhancing NF-κB and IRF signaling pathways in bone marrow cells, and Figure 9l shows THP1-Dual® cells treated for 16 hours with various IRF pathway inducers in the presence or absence of HPP (three combined experiments, one-way ANOVA test). [Figure 9m] Figure 9 shows exemplary HPP isomers (I-1, I-2, and I-3) enhancing NF-κB and IRF signaling pathways in bone marrow cells, and Figure 9m shows THP1-Dual® cells treated for 16 hours with various IRF pathway inducers in the presence or absence of HPP (three combined experiments, one-way ANOVA test). [Figure 9n] Figure 9 shows exemplary HPP isomers (I-1, I-2, and I-3) enhancing the NF-κB and IRF signaling pathways in bone marrow cells, and Figure 9n shows THP1-Dual® cells (three combined experiments) treated for 16 hours with LPS, which induces NF-κB, and VSV, which induces the IRF pathway, in the presence or absence of L-gln. [Figure 9o] Figure 9 shows exemplary HPP isomers (I-1, I-2, and I-3) enhancing the NF-κB and IRF signaling pathways in bone marrow cells, and Figure 9o shows THP1-Dual® cells (three combined experiments) treated for 16 hours with LPS, which induces NF-κB, and VSV, which induces the IRF pathway, in or without L-gln. [Figure 10a]Figure 10 shows exemplary HPP isomers (I-1, I-2, and I-3) enhancing NF-κB signaling and antitumor immunity by binding to GSDMD, and Figure 10a shows NF-κB induction in human THP1-Dual® reporter cells treated with LPS and HPP molecules for 16 hours (six combined experiments, one-way ANOVA test). [Figure 10b] Figure 10 shows exemplary HPP isomers (I-1, I-2, and I-3) enhancing NF-κB signaling and antitumor immunity by binding to GSDMD, and Figure 10b shows the identification of potential HPP targets in THP1-Dual™ cells by performing the TPP technique (two combined experiments, proteome coverage: 4301, NPARC test). [Figure 10c] Figure 10 shows exemplary HPP isomers (I-1, I-2, and I-3) enhancing NF-κB signaling and antitumor immunity by binding to GSDMD, and Figure 10c shows the protein-protein interaction network between HPP target hits (red) and transcriptional regulators (yellow) of genes affected by 3,2-HPP treatment; [Figure 10d] Figure 10 shows exemplary HPP isomers (I-1, I-2, and I-3) enhancing NF-κB signaling and antitumor immunity by binding to GSDMD, and Figure 10d shows GSDMD protein denaturation curves affected by 3,2-HPP treatment (two combined experiments, NPARC test). [Figure 10e] Figure 10 shows exemplary HPP isomers (I-1, I-2, and I-3) enhancing NF-κB signaling and antitumor immunity by binding to GSDMD, and Figure 10e shows the workflow for f-g. [Figure 10f]Figure 10 shows that exemplary HPP isomers (I-1, I-2, and I-3) enhance NF-κB signaling and antitumor immunity by binding to GSDMD, and Figure 10f shows tumor growth dynamics (n=8 / group; two combined experiments, two-way ANOVA test for growth dynamics, log-rank test for overall survival) for M3-9-MOVA RMS orthotopically transplanted into male WT vs GSDMD-KO mice treated with vehicle or 3,2-HPP metabolites. [Figure 10g] Figure 10 shows that exemplary HPP isomers (I-1, I-2, and I-3) enhance NF-κB signaling and antitumor immunity by binding to GSDMD, and Figure 10g shows overall survival (n=8 / group; two combined experiments, two-way ANOVA test for growth dynamics, log-rank test for overall survival) for M3-9-MOVA RMS orthotopically transplanted into male WT vs GSDMD-KO mice treated with vehicle or 3,2-HPP metabolites. [Figure 10h] Figure 10 shows exemplary HPP isomers (I-1, I-2, and I-3) enhancing NF-κB signaling and antitumor immunity by binding to GSDMD, and Figure 10h shows the response of HEK-Blue® IL-1β cells to supernatant of THP1-WT vs. GSDMD-KO cells treated with LPS and HPP molecules (NF-κB / AP-1 induction by IL-1 receptor signaling) (three combined experiments, one-way ANOVA test). [Figure 10i] Figure 10 shows exemplary HPP isomers (I-1, I-2, and I-3) enhancing NF-κB signaling and antitumor immunity by binding to GSDMD, and Figure 10i shows the response of HEK-Blue™ IL-1β cells to the supernatant of THP1-WT vs. GSDMD-KO cells treated with LPS and HPP molecules, followed by NG addition 3 hours later (three combined experiments, one-way ANOVA test). [Figure 10j]Figure 10 shows exemplary HPP isomers (I-1, I-2, and I-3) enhancing NF-κB signaling and antitumor immunity by binding to GSDMD, and Figure 10j shows Western blots of THP-1 cells treated under various conditions (representative of two experiments). [Figure 10k] Figure 10 shows exemplary HPP isomers (I-1, I-2, and I-3) enhancing NF-κB signaling and antitumor immunity by binding to GSDMD, and Figure 10k shows the quantification of GSDMD peptide (R, n=40 and NR, n=27) from publicly available proteomic profiles of stage IV melanoma patients receiving anti-PD-1 therapy. [Figure 10l] Figure 10 shows that exemplary HPP isomers (I-1, I-2, and I-3) enhance NF-κB signaling and antitumor immunity by binding to GSDMD. Figure 101 shows the supernatant of huPBMCs treated with LPS and HPP molecules, followed by NG addition 3 hours later, tested for HEK-Blue® IL-1β cell response (three combined experiments, one-way ANOVA test). [Figure 10m] Figure 10 shows that exemplary HPP isomers (I-1, I-2, and I-3) enhance NF-κB signaling and antitumor immunity by binding to GSDMD. Figure 10m shows the supernatant of huPBMCs treated with LPS and HPP molecules, followed by NG addition 3 hours later, tested for released LDH levels (three combined experiments, one-way ANOVA test). [Figure 11] Figure 11 shows the expression levels of genes involved in the inflammasome pathway, as identified by scRNAseq. [Figure 12]Figure 12 illustrates how exemplary 3,2-HPP metabolite treatment significantly alters the cytokine environment in the tumor mesenteric neuropathy (TME), showing cytokine levels in tumor stromal fluid isolated from orthotopic M3-9-MOVA tumors transplanted into male JAX C57BL / 6 mice 14 days after 3,2-HPP metabolite treatment was initiated 1 day after tumor transplantation. (Two combined experiments; one-way ANOVA test). [Figure 13a] Figure 13 illustrates how exemplary HPP isomers (I-1, I-2, and I-3) accelerate immune signaling pathways by promoting gasdermin D cleavage. Figures 13a–e show THP1-WT vs. GSDMD-KO cells treated with LPS in or without HPP metabolites. Where necessary, IgG1 isotype (clone T8E5) or anti-IL-1α (clone 7D4) and anti-IL-1β (clone 4H5) antibodies were added to neutralize secreted IL-1 cytokines. Figure 13a shows the supernatant, NF-κB induction by THP1-Dual® cells (three combined experiments, one-way ANOVA). [Figure 13b] Figure 13 illustrates how exemplary HPP isomers (I-1, I-2, and I-3) accelerate immune signaling pathways by promoting gasdermin D cleavage. Figures 13a–e show THP1-WT vs. GSDMD-KO cells treated with LPS in or without HPP metabolites. Where necessary, IgG1 isotype (clone T8E5) or anti-IL-1α (clone 7D4) and anti-IL-1β (clone 4H5) antibodies were added to neutralize secreted IL-1 cytokines. Figure 13b shows the response of HEK-Blue® IL-1β cells to NF-κB / AP-1 induction via IL-1β signaling after 4 hours (three combined experiments, one-way ANOVA). [Figure 13c]Figure 13 illustrates how exemplary HPP isomers (I-1, I-2, and I-3) accelerate immune signaling pathways by promoting gasdermin D cleavage. Figures 13a–e show THP1-WT vs. GSDMD-KO cells treated with LPS in or without HPP metabolites. Where necessary, IgG1 isotype (clone T8E5) or anti-IL-1α (clone 7D4) and anti-IL-1β (clone 4H5) antibodies were added to neutralize secreted IL-1 cytokines. Figure 13c shows the levels of secreted IL-1α and IL-1β after 16 hours (two combined experiments, one-way ANOVA test). [Figure 13d] Figure 13 illustrates how exemplary HPP isomers (I-1, I-2, and I-3) accelerate immune signaling pathways by promoting gasdermin D cleavage. Figures 13a–e show THP1-WT vs. GSDMD-KO cells treated with LPS in or without HPP metabolites. Where necessary, IgG1 isotype (clone T8E5) or anti-IL-1α (clone 7D4) and anti-IL-1β (clone 4H5) antibodies were added to neutralize secreted IL-1 cytokines. Figure 13d shows the levels of secreted IL-1α and IL-1β after 16 hours (two combined experiments, one-way ANOVA test). [Figure 13e] Figure 13 illustrates how exemplary HPP isomers (I-1, I-2, and I-3) accelerate immune signaling pathways by promoting gasdermin D cleavage. Figures 13a–e show THP1-WT vs. GSDMD-KO cells treated with LPS in or without HPP metabolites. Where necessary, IgG1 isotype (clone T8E5) or anti-IL-1α (clone 7D4) and anti-IL-1β (clone 4H5) antibodies were added to neutralize secreted IL-1 cytokines. Figure 13e shows the levels of released LDH after 16 hours (three combined experiments, one-way ANOVA test); f–h show the supernatants of THP1-WT vs. GSDMD-KO cells treated with LPS in or without HPP metabolites, followed by NG addition after 3 hours. [Figure 13f] Figure 13 shows how exemplary HPP isomers (I-1, I-2, and I-3) accelerate immune signaling pathways by promoting gasdermin D cleavage, and Figure 13f shows the response of HEK-Blue® IL-1β cells to NF-κB / AP-1 induction by IL-1β signaling after 4 hours (three combined experiments, one-way ANOVA test). [Figure 13g] Figure 13 shows that exemplary HPP isomers (I-1, I-2, and I-3) accelerate immune signaling pathways by promoting gasdermin D cleavage, and Figure 13g was tested for the level of released LDH after 4 hours (three combined experiments, one-way ANOVA test). [Figure 13h] Figure 13 shows that exemplary HPP isomers (I-1, I-2, and I-3) accelerate immune signaling pathways by promoting gasdermin D cleavage, and Figure 13h tests the levels of released LDH after 16 hours (three combined experiments, one-way ANOVA test). [Figure 13i] Figure 13 illustrates how exemplary HPP isomers (I-1, I-2, and I-3) accelerate immune signaling pathways by promoting gasdermin D cleavage, and Figure 13i shows the response of HEK-Blue® IFN-α / β cells (IRF induction by type I IFN signaling) to the supernatant of THP1-WT vs. GSDMD-KO cells treated with VSV in or without HPP metabolites. [Figure 13j] Figure 13 shows how exemplary HPP isomers (I-1, I-2, and I-3) accelerate immune signaling pathways by promoting gasdermin D cleavage, and Figure 13j shows the levels of secreted IL-1α in human PBMC supernatants treated with LPS in or without HPP metabolites, followed by NG addition 3 hours later (two combined experiments, one-way ANOVA test). [Figure 13k]Figure 13 shows how exemplary HPP isomers (I-1, I-2, and I-3) accelerate immune signaling pathways by promoting gasdermin D cleavage, and Figure 13k shows the levels of secreted IL-1β in the supernatant of human PBMCs treated with LPS in or without HPP metabolites, followed by NG addition 3 hours later (two combined experiments, one-way ANOVA test). [Figure 14] Figure 14 is a schematic diagram illustrating how exemplary microbiome-derived metabolites, such as HPPs, contribute to the antitumor immune response. [Figure 15] Figure 15 shows THP1-Dual® cells treated with LPS for 16 hours in the presence of a vehicle or exemplary compounds II-4, II-3, II-2, II-1, and I-3 for NF-κB pathway induction. [Figure 16] Figure 16 shows THP1-Dual® cells treated with LPS for 16 hours in the presence of a vehicle or exemplary compounds I-7, I-6, I-5, I-4, and I-3 for NF-κB pathway induction. [Figure 17] Figure 17 shows THP1-Dual® cells treated with LPS for 16 hours in the presence of a vehicle or exemplary compounds III-2, III-1, and I-3 for NF-κB pathway induction. [Figure 18] Figure 18 shows THP1-Dual® cells treated with LPS for 16 hours in the presence of a vehicle or exemplary compounds IV and I-3 for NF-κB pathway induction. [Modes for carrying out the invention]

[0032] Detailed explanation I. Definition Unless otherwise indicated, the definitions and embodiments set forth in this section and other sections are intended to be applicable to all embodiments and aspects of the present application described herein, as they are appropriate, as will be understood by those skilled in the art.

[0033] All features disclosed herein, including the claims, abstract, and drawings, and all steps in any disclosed method or process, may be combined in any combination, except for any combination in which at least part of such features and / or steps are mutually exclusive. Each feature disclosed herein, including the claims, abstract, and drawings, may be replaced by an alternative feature serving the same, equivalent, or similar purpose, unless otherwise expressly stated.

[0034] As used herein, the terms “the compound of the application” or “the compound of the present application” and similar terms refer to one or more compounds of formula (I), (II), (III), or (IV), including their pharmaceutically acceptable salts, solvates, and / or ester prodrugs.

[0035] As used herein, the terms “the composition of the application” or “the composition of the present application” and similar terms refer to a composition comprising one or more compounds of the application and at least one additional component.

[0036] As used herein, the term "and / or" means that the listed items exist or are used individually or in combination. In practice, the term means that "at least one" or "one or more" of the listed items are used or are present. With respect to its pharmaceutically acceptable salts and / or solvates, the term "and / or" means that the compounds of this application exist as individual salts and hydrates, as well as, for example, combinations of solvates of salts of the compounds of this application.

[0037] In this application, the singular forms "a," "an," and "the" include plural references unless otherwise clearly indicated in the context. For example, an embodiment containing "a compound" should be understood to present a particular embodiment having one compound or two or more additional compounds.

[0038] In embodiments including an additional or second compound, or other “additional” or “second” component, the second component used herein is chemically different from the other component or the first component. The “third” component is different from the other, first, and second components, and the further listed or “additional” components are similarly different.

[0039] As used herein, the words “comprising” (and any form of “comprise,” such as “comprises,” and “comprises”), “having” (and any form of “have,” such as “have,” and “has”), “including” (and any form of “include,” such as “includes,” and “includes”), or “containing” (and any form of “contain,” such as “contains,” and “contains”) are comprehensive or unrestrictive and do not exclude additional unlisted elements or processes / methods.

[0040] As used herein, the word "consisting of" and its derivatives are intended to be closed terms that specify the existence of specified characteristics, elements, components, groups, integers, and / or processes, while also excluding the existence of other unspecified characteristics, elements, components, groups, integers, and / or processes.

[0041] As used herein, the term “essentially derived from” is intended to specify the presence of the specified properties, elements, components, groups, integers, and / or processes, as well as those that do not significantly affect the fundamental and novel characteristics of these properties, elements, components, groups, integers, and / or processes.

[0042] As used herein, terms of degree such as “substantially,” “about,” and “approximately” mean a reasonable deviation of the modified term such that the final result does not change substantially. These terms of degree should be understood to include a deviation of at least ±5% of the modified term, provided that this deviation does not negate the meaning of the word it modifies.

[0043] As used herein, the term “appropriate” means that the selection of a particular compound or conditions will depend on the specific synthetic operation to be carried out, the nature of the molecule to be converted, and / or the specific use of the compound, but the selection will be well within the scope of the skill of a person skilled in the art. All process / method steps described herein should be carried out under conditions sufficient to provide the indicated product. A person skilled in the art will understand that the yield of the desired product can be optimized by varying all reaction conditions, including, for example, the reaction solvent, reaction time, reaction temperature, reaction pressure, reactant ratio, and whether the reaction should be carried out under an anhydrous or inert atmosphere, and that this is within the scope of their skill to do so.

[0044] This application refers to a number of chemical terms and abbreviations used by those skilled in the art. Nevertheless, for clarity and consistency, definitions of selected terms are provided.

[0045] As used herein, the term “cell” refers to a single cell or a group of cells, including cells under cell culture or within a subject.

[0046] As used herein, the term “subject” includes all members of the animal kingdom, including mammals. Therefore, the methods and uses described herein are applicable to both human therapeutic and veterinary applications.

[0047] The term "pharmaceutically acceptable" means that it is suitable for the treatment of the subject.

[0048] The term "pharmaceutically acceptable carrier" means a non-toxic solvent, dispersant, excipient, adjuvant, or other material that is mixed with an active ingredient (e.g., one or more compounds of this application) to enable the formation of a pharmaceutical composition, i.e., a dosage form that can be administered to a subject.

[0049] The term "pharmaceutically acceptable salt" means either an acid addition salt or a base addition salt that is appropriate or suitable for the treatment of the subject.

[0050] The acid addition salts appropriate or suitable for the treatment of the subject are any non-toxic organic or inorganic acid addition salts of any basic compound.

[0051] The base addition salt appropriate or suitable for the treatment of the subject is any non-toxic organic or inorganic base addition salt of any acidic compound.

[0052] As used herein, the term “prodrug” means a compound that is converted to an active drug after administration, or a salt and / or solvate of such a compound.

[0053] As used herein, the term “solvate” means a compound in which molecules of a suitable solvent are incorporated into the crystal lattice, or a salt or prodrug of such a compound.

[0054] In this specification, the term "N" as used, for example, in "4N," refers to the unit symbol of a specified degree, representing "eq / L."

[0055] In this specification, the term "M" as used, for example, in "4M," refers to the unit symbol for molar concentration, which represents "moles / L."

[0056] As used herein, the term "DMSO" refers to dimethyl sulfoxide.

[0057] As used herein, the term "HCl" refers to hydrochloric acid.

[0058] As used herein, the term "PBS" refers to a phosphate-based buffer.

[0059] As used herein, the term "RT" refers to room temperature.

[0060] As used herein, the term "HPLC" refers to high-performance liquid chromatography.

[0061] As used herein, the term "EDTA" refers to ethylenediaminetetraacetic acid.

[0062] As used herein, the term "FBS" refers to fetal bovine serum.

[0063] As used herein, the term "HPP" refers to hydroxyphenylpropanoate.

[0064] As used herein, the term "ICB" refers to immune checkpoint blockade.

[0065] As used herein, the term "GSDMD" refers to gasdermin D.

[0066] As used herein, the term "TME" refers to the tumor microenvironment.

[0067] As used herein, the term "PCoA" refers to principal coordinate analysis.

[0068] As used herein, the term "IMDM" refers to genetically identical mice that have been established at birth with a diverse and complex microbiome.

[0069] As used herein, the term "ABX" refers to antibiotics.

[0070] As used herein, the term "UHPLC-MS" refers to ultra-high-performance liquid chromatography-mass spectrometry.

[0071] As used herein, the term "Treg" refers to regulatory T cells.

[0072] As used herein, the term "pDc" refers to plasmacytoid dendritic cells.

[0073] As used herein, the term "TPP" refers to thermal proteome profiling.

[0074] As used herein, the term "ASV" refers to an amplicon sequence variant.

[0075] As used herein, the term "RBC" refers to a red blood cell.

[0076] As used herein, the term "PVDF" refers to polyvinylidene fluoride.

[0077] As used herein and as well understood in the art, the terms “to treat” or “to treat” mean a method for obtaining beneficial or desired outcomes, including clinical outcomes. Beneficial or desired clinical outcomes may include, but are not limited to, the reduction or improvement of one or more symptoms or conditions, whether detectable or undetectable; a reduction in the severity of a disease, disorder, or condition; a stable (i.e., non-worsening) state of a disease, disorder, or condition; the cessation of the spread of a disease, disorder, or condition; the delay or slowing of the progression of a disease, disorder, or condition; the improvement or mitigation of the state of a disease, disorder, or condition; the reduction of relapses of a disease, disorder, or condition; and remission (whether partial or complete). “To treat” and “to treat” may also mean extending survival compared to the expected survival without treatment. As used herein, “to treat” and “to treat” also include prophylactic treatment.

[0078] To “alleviate” a disease, disorder, or condition means that, compared to not treating the disease, disorder, or condition, the severity and / or undesirable clinical signs of the disease, disorder, or condition are reduced and / or the time course of its progression is slowed or prolonged.

[0079] As used herein, the terms “prevention” or “inhibition,” or their synonyms, refer to a reduction in the risk or likelihood of an object suffering from a disease, disorder, or medical condition, or exhibiting symptoms associated with such a disease, disorder, or medical condition.

[0080] As used herein, the terms “effective dose” or “therapeutic dose” mean the amount of one or more compounds that is effective in the dosage and duration required to achieve the desired result.

[0081] As used herein, the expression "accelerates GSDMD cleavage" refers to promoting the cleavage of the intracellular pore-forming protein gasdermin D (GSDMD). Increased cleavage leads to therapeutic effects within the cell.

[0082] The terms “increase” or “to increase,” or any synonyms thereof, including “to improve,” “to accelerate,” and similar terms, mean any detectable increase in the function or quantity of the target substance in the presence of one or more of the compounds of this application, compared to otherwise identical conditions, except in the absence of one or more of the compounds of this application.

[0083] The terms “decrease” or “to reduce,” or any synonyms thereof including “to lower,” “to decrease,” and similar terms, mean any detectable reduction in the function or quantity of the target substance in the presence of one or more of the compounds of this application, compared to otherwise identical conditions, except in the absence of one or more of the compounds of this application.

[0084] As used herein, the term “administered” means the administration of a therapeutically effective amount of one or more compounds or compositions of this application to a cell or subject.

[0085] II. Method and Use of the Application The compounds described in this application have been shown to enhance antitumor immunity and synergistically interact with immune checkpoint blockade (ICB) therapy. HPP molecules act as broad-spectrum enhancers of innate immune signaling pathways in tumor-associated myeloid cells by promoting the cleavage of the pore-forming protein gasdermin D (GSDMD), an effector of standard and non-standard inflammasome signaling. Increased secretion of pro-inflammatory cytokines, including IL-1β, from HPP-treated myeloid cells promotes NF-κB activity in tumor-infiltrating leukocytes. This leads to improved anti-cancer CD8 T cell function, significant tumor regression, and better long-term cancer control with immune checkpoint therapy in mice. Human peripheral blood mononuclear cells respond similarly to HPP treatment. GSDMD cleavage is also associated with a favorable response to ICB therapy in patients with advanced melanoma. In summary, this application reveals previously unknown mechanisms of microbiome-mediated modulation of host antitumor immunity that are modifiable and can be utilized to enhance the efficacy of cancer immunotherapy.

[0086] Accordingly, this application includes a method for increasing intracellular antitumor immunity in either a biological sample or subject, comprising the step of administering an effective amount of one or more of the compounds of this application to cells that require it.

[0087] In some embodiments, the compounds of this application are compounds of formula (I), or pharmaceutically acceptable salts, solvates, and / or ester prodrugs thereof:

[0088] [ka] During the ceremony, R 1 , R 2 , and R 3 Under the condition that at least one of them is OH, R 1 , R 2 , and R 3 These are independently OH or H. That is the case.

[0089] In some embodiments, the compounds of this application are

[0090] [Table 1] or selected from its pharmaceutically acceptable salts, solvates, and / or ester prodrugs.

[0091] In some embodiments, the compounds of this application are

[0092] [Table 2] Selected from.

[0093] In some embodiments, the compounds of this application are compounds of formula (II), (III), or (IV), or pharmaceutically acceptable salts, solvates, and / or ester prodrugs thereof:

[0094] [ka] During the ceremony, R 4 Halogen, NR 5 R 6 , and C 1~3 Selected from alkyl groups; R 5 and R 6 H and C are independent of each other. 1~3 Selected from alkyl groups;

[0095] [ka] During the ceremony, R 7 is OH; n is 3 to 5; or

[0096] [ka] That is the case.

[0097] In some embodiments, the compound of formula (II), or a pharmaceutically acceptable salt, solvate, and / or ester prodrug thereof, has the following structure:

[0098]

Chemical formula

[0099] In some embodiments, R 4 is selected from F, Cl, NH2, and CH3.

[0100] In some embodiments, the compound of formula (III), or a pharmaceutically acceptable salt, solvate, and / or ester prodrug thereof, has the following structure:

[0101]

Chemical formula

[0102] In some embodiments, the compound of formula (IV), or a pharmaceutically acceptable salt, solvate, and / or ester prodrug thereof, has the following structure:

[0103]

Chemical formula

[0104] In some embodiments, the compounds of the present application are

[0105]

Table 3

[0106] In some embodiments, the compounds of the present application are compounds of formula (V), or pharmaceutically acceptable salts, solvates, and / or ester prodrugs thereof:

[0107]

Chemical formula

[0108] In some embodiments, R 8 、R​​​​​​​​​​​​​​​is H. In some embodiments, R 9 is OH, and R 8 and R 10 are H. In some embodiments, R 8 is OH, and R 9 and R 10 are H.

[0109] In some embodiments, R 8 is selected from halogen, NR 13 R 14 , and C 1~3 alkyl, and R 9 and R 10 are H. In some embodiments, R 8 is selected from F, Cl, NH2, and CH3, and R 9 and R 10 are H.

[0110] In some embodiments, R 11 is H. In some embodiments, R 11 is =O.

[0111] ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​OH is R 8 and R 10 H is R 11 H is R 12 H is and p is 1.

[0115] In some embodiments, R 10 OH is R 8 and R 9 H is R 11 H is R 12 H is and p is 1.

[0116] In some embodiments, R 8 OH is R 9 and R 10 H is R 11 H is R 12 The compound is selected from H, CH2-CH3, CH3, and succinimide, and p is 1.

[0117] In some embodiments, R 8 R is selected from F, Cl, NH2, and CH3. 9 and R 10 H is R 11 H is R 12 H is and p is 1.

[0118] In some embodiments, R 8 OH is R 9 and R 10 H is R 11 H is R 12 H is H, and p is 2 or 3.

[0119] In some embodiments, R 8 OH is R 9 and R 10 H is R 11 is C(O) and p is 2.

[0120] In some embodiments, the pharmaceutically acceptable salt is a base addition salt. The selection of a suitable salt can be made by those skilled in the art (see, for example, SMBerge, et a., “Pharmaceutical Salts,” J. Pharm. Sci. 1977, 66, 1-19).

[0121] In some embodiments, a base addition salt suitable or appropriate for the treatment of the subject is any non-toxic organic or inorganic base addition salt of the compound of formula (I). Exemplary inorganic bases that form a suitable salt include lithium hydroxide, sodium, potassium, calcium, magnesium, or barium, as well as ammonia. Exemplary organic bases that form a suitable salt include aliphatic, alicyclic, or aromatic organic amines such as isopropylamine, methylamine, trimethylamine, picoline, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydravamin, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins. Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. In some embodiments, the pharmaceutically acceptable salt is a sodium salt.

[0122] Solvates of the compound of formula (I), or salts or ester prodrugs thereof, include, for example, those prepared using pharmaceutically acceptable solvents. Examples of such solvents include water (the resulting solvate is called a hydrate) and ethanol, etc. A suitable solvent is physiologically tolerable at the administered dose.

[0123] The prodrugs of the compounds of this application include, for example, conventional esters formed using available hydroxy and / or carboxyl groups. Some common esters used as prodrugs are phenyl esters, aliphatic (C1-C) esters.24 ) They are esters, acyloxymethyl esters, carbamates, and amino acid esters. In some embodiments, the prodrug is a methyl ester, an ethyl ester, or an N-hydroxysuccinimide ester.

[0124] The compounds of the present application may further exist in various polymorphic forms, and any polymorph formed thereby or a mixture thereof is intended to be included within the scope of the present application.

[0125] The present application also includes the use of one or more compounds of the present application for increasing anti-tumor immunity in cells, and the use of one or more compounds of the present application for the preparation of a medicament for increasing anti-tumor immunity in cells. The present application further includes one or more compounds of the present application for use in increasing anti-tumor immunity in cells.

[0126] Since the compounds of the present application have been shown to increase anti-tumor immunity, the compounds of the present application are useful for treating a disease, disorder, or medical condition by increasing anti-tumor immunity in cells, either in a biological sample or in a subject. [[ID=!5]]

[0127] Therefore, the present application also includes a method for treating a disease, disorder, or medical condition that is treatable by increasing anti-tumor immunity in cells, either in a biological sample or in a subject, the method comprising administering a therapeutically effective amount of one or more compounds of the present application to the cells.

[0128] The present application also includes the use of one or more compounds of the present application for the treatment of a disease, disorder, or medical condition that is treatable by increasing anti-tumor immunity in cells, and the use of one or more compounds of the present application for the preparation of a medicament for the treatment of a disease, disorder, or medical condition that is treatable by increasing anti-tumor immunity in cells. The present application further includes one or more compounds of the present application for use in treating a disease, disorder, or medical condition that is treatable by increasing anti-tumor immunity in cells.

[0129] In some embodiments, the application also includes the use of one or more compounds of the application for the treatment of diseases, disorders, or conditions that can be treated by increasing intracellular GSDMD cleavage, as well as the use of one or more compounds of the application for the preparation of pharmaceuticals for the treatment of diseases, disorders, or conditions that can be treated by increasing intracellular GSDMD cleavage. The application further includes one or more compounds of the application for use in treating diseases, disorders, or conditions that can be treated by increasing intracellular GSDMD cleavage.

[0130] In some embodiments, the application also includes the use of one or more of the compounds of this application for the treatment of diseases, disorders, or conditions that can be treated by increasing tumor stromal IL-1β release and tumor-specific CD8 T cell accumulation in the tumor mesenteric area (TME), as well as the use of one or more of the compounds of this application for the preparation of pharmaceuticals for the treatment of diseases, disorders, or conditions that can be treated by increasing tumor stromal IL-1β release and tumor-specific CD8 T cell accumulation in the TME. The application further includes one or more of the compounds of this application for use in treating diseases, disorders, or conditions that can be treated by increasing tumor stromal IL-1β release and tumor-specific CD8 T cell accumulation in the TME.

[0131] The compounds of this application have been demonstrated to increase antitumor immunity in cancer cells. In some embodiments, the disease, disorder, or condition that can be treated by increasing antitumor immunity is cancer.

[0132] Accordingly, this application also includes a method for treating cancer, comprising the step of administering a therapeutically effective amount of one or more compounds of this application to a subject in need thereof. This application also includes the use of one or more compounds of this application for the treatment of cancer, and the use of one or more compounds of this application for the preparation of pharmaceuticals for the treatment of cancer. This application further includes one or more compounds of this application for use in treating cancer.

[0133] In some embodiments, cancer is not limited to these, but includes acute lymphoblastic leukemia, adults; acute lymphoblastic leukemia, children; acute myeloid leukemia, adults; adrenocortical carcinoma; adrenocortical carcinoma, children; AIDS-associated lymphoma; AIDS-associated malignant tumor; anal cancer; astrocytoma, pediatric cerebellar; astrocytoma, pediatric cerebral; cholangiocarcinoma, extrahepatic; bladder cancer; bladder cancer, children; bone cancer, osteosarcoma / malignant fibrous histiocytoma; brainstem glioma, children; brain tumor, adults; brain tumor, brainstem glioma, children; brain tumor, cerebellar astrocytoma, children; brain tumor, cerebral astrocytoma / malignant glioma, children; brain tumor, ependymoma, children; brain tumor, Medulloblastoma, pediatric; Brain tumor, supratentorial primitive neuroectodermal tumor, pediatric; Brain tumor, visual tract and hypothalamic glioma, pediatric; Brain tumor, pediatric (other); Breast cancer; Breast cancer and pregnancy; Breast cancer, pediatric; Breast cancer, male; Bronchial adenoma / carcinoid, pediatric; Carcinoid tumor, pediatric; Carcinoid tumor, gastrointestinal; Carcinoma, adrenal cortex; Carcinoma, pancreatic islet cell; Carcinoma of unknown primary origin; Central nervous system lymphoma, primary; Cerebellar astrocytoma, pediatric; Cerebral astrocytoma / malignant glioma, pediatric; Cervical cancer; Childhood cancer; Chronic lymphocytic leukemia; Chronic myeloleukemia; Chronic myeloproliferative disorder; Clear cell sarcoma of the tendon sheath; Colon cancer; Colorectal cancer, pediatric; cutaneous T-cell lymphoma; endometrial cancer; ependymoma, pediatric; epithelial cancer, ovarian; esophageal cancer; esophageal cancer, pediatric; Ewing family tumors; extracranial germ cell tumors, pediatric; extragonadal germ cell tumors; extrahepatic bile duct cancer; eye cancer, intraocular melanoma; eye cancer, retinoblastoma; gallbladder cancer; stomach cancer; stomach cancer, pediatric; gastrointestinal carcinoid tumors; germ cell tumors, extracranial, pediatric; germ cell tumors, extragonadal; germ cell tumors, ovarian; gestational trophoblastic neoplasm; glioma, pediatric brainstem; glioma, pediatric visual tract and hypothalamus; hair cell leukemia; head and neck cancer; hepatocellular carcinoma, adult (primary) Primary hepatocellular carcinoma (liver cancer), childhood; Hodgkin lymphoma, adult; Hodgkin lymphoma, childhood; Hodgkin lymphoma during pregnancy; Hypopharyngeal cancer; Hypothalamic and optic tract glioma, childhood; Intraocular melanoma; Islet cell carcinoma (endocrine pancreas); Kaposi's sarcoma; Kidney cancer; Laryngeal cancer; Laryngeal cancer, childhood; Leukemia, acute lymphoblastic, adult; Leukemia, acute lymphoblastic, childhood; Leukemia, acute myeloid, adult; Leukemia, acute myeloid, childhood; Leukemia, chronic lymphocytic; Leukemia, chronic myeloid; Leukemia, hair cell; Lip and oral cancer; Liver cancer, adult (primary); Liver cancer, childhood (primary);Lung cancer, non-small cell; lung cancer, small cell; lymphoblastic leukemia, adult acute; lymphoblastic leukemia, pediatric acute; lymphocytic leukemia, chronic; lymphoma, AIDS-related; lymphoma, central nervous system (primary); lymphoma, cutaneous T cell; lymphoma, Hodgkin, adult; lymphoma, Hodgkin, pediatric; lymphoma, Hodgkin during pregnancy; lymphoma, non-Hodgkin, adult; lymphoma, non-Hodgkin, pediatric; lymphoma, non-Hodgkin during pregnancy; lymphoma, primary central nervous system; macroglobulinemia, Waldenström; male breast cancer; malignant mesothelioma, adult; malignant mesothelioma, pediatric; malignant thymoma; medulloblastoma Tumors, pediatric; melanoma; intraocular melanoma; Merkel cell carcinoma; malignant mesothelioma; metastatic squamous cell carcinoma of unknown primary origin; multiple endocrine neoplasia syndrome, pediatric; multiple myeloma / plasmacytic neoplasm; mycosis fungoides; myelodysplastic syndrome; chronic myeloid leukemia; acute myeloid leukemia, pediatric; multiple myeloma; chronic myeloproliferative disorders; nasal and paranasal sinus cancer; nasopharyngeal cancer; nasopharyngeal cancer, pediatric; neuroblastoma; non-Hodgkin lymphoma, adult; non-Hodgkin lymphoma, pediatric; non-Hodgkin lymphoma during pregnancy; non-small cell lung cancer; oral cancer, pediatric; oral and lip cancer; oropharyngeal cancer; osteosarcoma / malignant fibrous bone Histiocytoma; ovarian cancer, pediatric; ovarian epithelial carcinoma; ovarian germ cell tumor; low-grade ovarian tumor; pancreatic cancer; pancreatic cancer, pediatric; pancreatic cancer, islet cell; sinus and nasal cavity cancer; parathyroid cancer; penile cancer; pheochromocytoma; pineal and supratentorial primitive neuroectodermal tumors, pediatric; pituitary tumors; plasma cell neoplasms / multiple myeloma; pleuropulmonary blastoma; pregnancy and breast cancer; pregnancy and Hodgkin lymphoma; pregnancy and non-Hodgkin lymphoma; primary central nervous system lymphoma; primary liver cancer, adult; primary liver cancer, pediatric; prostate cancer; rectal cancer; renal cell (kidney) carcinoma; renal cell carcinoma, pediatric; renal pelvis and Ureteral transitional cell carcinoma; retinoblastoma; rhabdomyosarcoma, pediatric; salivary gland cancer; salivary gland cancer, pediatric; sarcoma, Ewing family tumor; sarcoma, Kaposi's sarcoma; sarcoma (osteosarcoma) / malignant fibrous histiocytoma of bone; sarcoma, rhabdomyosarcoma, pediatric; sarcoma, soft tissue, adult; sarcoma, soft tissue, pediatric; Sézary syndrome; skin cancer; skin cancer, pediatric; skin cancer (melanoma); skin cancer, Merkel cells; small cell lung cancer; small intestine cancer; soft tissue sarcoma, adult; soft tissue sarcoma, pediatric; squamous cell carcinoma of unknown primary origin, metastatic; gastric cancer; gastric cancer, pediatric; supratentorial primitive neuroectodermal tumor, pediatric;A selection of cancers may be treated from the following: T-cell lymphoma, cutaneous; testicular cancer; thymoma, pediatric; malignant thymoma; thyroid cancer; thyroid cancer, pediatric; transitional cell carcinoma of the renal pelvis and ureter; gestational trophoblastic neoplasm; cancer of unknown primary site, pediatric; rare pediatric cancer; transitional cell carcinoma of the ureter and renal pelvis; urethral cancer; uterine sarcoma; vaginal cancer; glioma of the visual pathway and hypothalamus, pediatric; vulvar cancer; Waldenström macroglobulinemia; and Wilms' tumor. Metastases of the aforementioned cancers may also be treated according to the methods described herein.

[0134] In some embodiments, the cancer is selected from one or more of the following: solid tumors, breast cancer, colon cancer, bladder cancer, skin cancer, head and neck cancer, liver cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, bone cancer, and glioblastoma. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is skin cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is colorectal cancer (CRC). In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is glioblastoma. In some embodiments, the cancer is osteosarcoma.

[0135] This application also includes a method for treating a disease, disorder, or condition treatable by increasing intracellular antitumor immunity in any biological sample or subject, comprising the step of administering a therapeutically effective amount of one or more compounds of this application to cells in combination with another active agent useful for treating a disease, disorder, or condition treatable by increasing antitumor immunity. This application also includes the use of one or more compounds of this application in combination with another active agent useful for treating a disease, disorder, or condition treatable by increasing antitumor immunity, and the use of one or more compounds of this application in combination with another active agent useful for treating a disease, disorder, or condition treatable by increasing antitumor immunity for the preparation of a medicament for the treatment of a disease, disorder, or condition treatable by increasing antitumor immunity. This application further includes one or more compounds of this application in combination with another active agent useful for treating a disease, disorder, or condition treatable by increasing antitumor immunity for use in treating a disease, disorder, or condition treatable by increasing antitumor immunity.

[0136] In some embodiments, the GSDMD is cut in the use and method of this application.

[0137] In one embodiment, the subject is a subject having a disease, disability, or medical condition.

[0138] In one embodiment, the subject is a mammal. In another embodiment, the subject is a human.

[0139] In some embodiments, the disease, disorder, or condition that can be treated by increasing antitumor immunity is cancer, and one or more of the compounds of this application are administered or used in combination with one or more additional active agents for treating and / or treating cancer.

[0140] This application also includes a method for increasing the potency of one or more additional active agents for treating and / or treating cancer, comprising the step of administering an effective amount of one or more of the compounds of this application to a subject in need, in combination with an effective amount of one or more additional active agents for treating and / or treating cancer.

[0141] This application also includes the use of one or more compounds of this application in combination with one or more additional active substances for treating and / or treating cancer, for the purpose of treating cancer, to increase the potency of one or more additional active substances for treating and / or treating cancer, and the use of one or more compounds of this application in combination with one or more additional active substances for treating and / or treating cancer, for the purpose of increasing the potency of one or more additional active substances for treating and / or treating cancer. This application further includes the use of one or more compounds of this application in combination with one or more additional active substances for treating and / or treating cancer, for use in increasing the potency of one or more additional active substances for treating and / or treating cancer.

[0142] In some embodiments, one or more additional agents for treating cancer are small molecule chemotherapy agents such as cisplatin, tyrosine kinase inhibitors, glutaminase inhibitors (e.g., glutaminase-1 (GLS1) inhibitors), and asparagine synthase (ASNS) inhibitors. In some embodiments, cancer therapy is, for example, radiotherapy, targeted therapy such as antibody therapy (including anti-PD-1 and / or anti-PD-L1 antibodies), immunotherapy, hormone therapy, and anti-angiogenic therapy.

[0143] In some embodiments, the immunotherapy is immune checkpoint blockade therapy. In some embodiments, the immune checkpoint blockade therapy is a PD-1 inhibitor, including one or more of pembrolizumab, nivolumab, and semiprimab, or a PD-L1 inhibitor, including one or more of atezolizumab, avelumab, and durvalumab. In some embodiments, the immune checkpoint blockade therapy is a CTLA-4 inhibitor, including ipilimumab and / or tremelimumab. In some embodiments, the immune checkpoint blockade therapy is a LAG-3 inhibitor, including relatrimab and / or Opdualag.

[0144] In some embodiments, chemotherapy is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is cisplatin. Therefore, in some embodiments, the disease, disorder, or condition treatable by increasing antitumor immunity is cancer, and one or more compounds of this application are administered or used in combination with cisplatin. In some embodiments, the chemotherapeutic agent is L-asparaginase (L-ASNase). Therefore, in some embodiments, the disease, disorder, or condition treatable by increasing antitumor immunity is cancer, and one or more compounds of this application are administered or used in combination with L-asparaginase (L-ASNase).

[0145] In some embodiments, the small molecule therapy is a glutaminase (e.g., glutaminase-1, (GLS1)) inhibitor or an asparagine synthase (ASNS) inhibitor. Therefore, in some embodiments, the disease, disorder, or condition that can be treated by increasing antitumor immunity is cancer, and one or more compounds of this application are administered or used in combination with one or more glutaminase inhibitors (e.g., GLS1 inhibitors) and / or asparagine synthase (ASNS) inhibitors.

[0146] In some embodiments, the disease, disorder, or condition that can be treated by increasing antitumor immunity is cancer, and one or more compounds of the present application are administered or used in combination with one or more glutaminase inhibitors (e.g., GLS1 inhibitors) and / or asparagine synthase (ASNS) inhibitors and / or L-asparaginase (L-ASNase).

[0147] When used in combination with other active ingredients useful in treating diseases, disorders, or conditions, or cancer, that can be treated by increasing antitumor immunity, this is an embodiment in which the compounds of this application are administered simultaneously with such active ingredients. As used herein, “simultaneous administration” of two substances to a subject means that they provide each of the two substances so that both are biologically active in the subject at the same time. The exact details of the administration are considered to depend on the pharmacokinetics of the two substances in the presence of each other, and may include administering the two substances within a few hours of each other, or further administering one substance within 24 hours of the other administration, provided that the pharmacokinetics are appropriate. Designing a suitable dosing regimen is a routine task for those skilled in the art. In certain embodiments, the two substances will be administered substantially simultaneously, i.e., within a few minutes of each other, or in a single composition containing both substances. This is a further embodiment of this application in which the combination of active ingredients is administered to the subject in a non-simultaneous form. In some embodiments, the compounds of this application are administered with another therapeutic agent, either simultaneously or sequentially in separate unit dosage forms, or together in a single unit dosage form. Accordingly, this application provides a single unit dosage form comprising one or more of the compounds of this application, an additional therapeutic agent, and a pharmaceutically acceptable carrier.

[0148] The treatment method comprises administering one or more therapeutically effective doses of the compounds of this application to a subject, optionally consisting of a single dose or alternatively comprising a series of doses, and optionally including concurrent administration or use of one or more other therapeutic agents. For example, in some embodiments, the compounds of this application may be administered at least once a week. In some embodiments, the compounds may be administered to a subject about once every two or three weeks or about once a week to about once a day for a given treatment. In other embodiments, the compounds are administered two, three, four, five, or six times a day. The length of the treatment period depends on a variety of factors, such as the severity of the disease, disorder, or condition, the age of the subject, the concentration and / or activity of the compounds of this application, and / or combinations thereof. It will also be understood that the effective dose of the compound used for treatment may increase or decrease over the course of a particular treatment regime. Changes in dosage may occur and may be revealed by standard diagnostic assays known in the art. In some cases, chronic administration may be required. For example, the compound is administered to the subject in an amount and duration sufficient to treat the subject. In some embodiments, the treatment includes prophylactic treatment. For example, a subject with early-stage cancer may be treated to halt progression, or alternatively, a subject in remission may be treated with the compounds or compositions of this application to prevent recurrence.

[0149] The dosage of the compounds in this application will vary depending on many factors, including the pharmacodynamic properties of the compounds, the mode of administration, the age, health status, and weight of the recipient, the nature and severity of symptoms, the frequency of treatment, the type of parallel treatment if any, and the clearance rate of the compounds within the target to be treated. Those skilled in the art will be able to determine an appropriate dosage based on the above factors. The compounds in this application may be administered initially at an appropriate dosage, which may be adjusted as needed in response to the clinical response. The dosage will generally be selected to maintain serum levels of the compounds in this application at approximately 0.01 μg / cc to approximately 1000 μg / cc, or approximately 0.1 μg / cc to approximately 100 μg / cc. As a typical example, oral dosages of one or more of the compounds in this application for adults may range from approximately 0.05 mg to approximately 1000 mg per day, appropriately from approximately 0.1 mg to approximately 500 mg per day, and more appropriately from approximately 1 mg to approximately 200 mg per day. For parenteral administration, typical doses are approximately 0.001 mg / kg to 10 mg / kg, 0.01 mg / kg to 10 mg / kg, 0.01 mg / kg to 1 mg / kg, or 0.1 mg / kg to 1 mg / kg. For oral administration, typical doses are approximately 0.001 mg / kg to 10 mg / kg, 0.1 mg / kg to 10 mg / kg, 0.01 mg / kg to 1 mg / kg, or 0.1 mg / kg to 1 mg / kg. For administration in suppository form, typical doses are approximately 0.1 mg / kg to 10 mg / kg or 0.1 mg / kg to 1 mg / kg. The compound of this application may be administered once daily, once weekly, or once monthly, or the total daily dose may be divided into two, three, or four daily doses.

[0150] In one embodiment, the effective dose varies according to factors such as disease state, age, sex, and / or weight of the subject. In further embodiments, the amount of one or more compounds that would constitute an effective dose would vary according to factors such as a given drug or compound, pharmaceutical formulation, route of administration, condition, disease, or disorder, and the characteristics of the subject being treated, but can nevertheless be routinely determined by those skilled in the art.

[0151] For clarity, the term “a compound” above includes embodiments in which one or more compounds are referred to. Similarly, the term “compounds of this application” includes embodiments in which only one compound is referred to.

[0152] The compounds of this application are appropriately formulated into pharmaceutical compositions for administration to a subject in a biocompatible form suitable for in vivo administration. Accordingly, this application further includes pharmaceutical compositions comprising one or more of the compounds of this application and a pharmaceutically acceptable carrier. In embodiments of this application, the pharmaceutical composition is used in the treatment of any of the diseases, disorders, or medical conditions described herein, and one or more of the compounds of this application are present in the composition in an amount effective to increase antitumor immunity or to treat cancer.

[0153] The compounds of this application are administered to subjects in a variety of forms depending on the chosen route of administration, as will be understood by those skilled in the art. For example, the compounds of this application are administered orally, by inhalation, parenterally, buccally, sublingually, nasally, rectally, vaginally, by patch, pump, minipump, topically, or transdermally, and the pharmaceutical compositions are formulated accordingly. In some embodiments, administration is by pump for periodic or continuous delivery. Conventional procedures and components for the selection and preparation of suitable compositions are described, for example, in Remington's Pharmaceutical Sciences (2000 - 20th edition) and The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.

[0154] Parenteral administration includes systemic delivery routes other than the gastrointestinal tract (GI), and includes, for example, intravenous, intra-arterial, intraperitoneal, subcutaneous, intramuscular, transepithelial, transnasal, intrapulmonary (e.g., by the use of aerosols), intrathecal, rectal, and topical (including the use of patches or other transdermal delivery devices). Parenteral administration may also be by continuous infusion over a selected period.

[0155] In some embodiments, the compounds of this application are administered orally, for example, with an inert diluent or with an absorbable food carrier, or they are encapsulated in hard or soft-shell gelatin capsules, or they are compressed into tablets, or they are incorporated directly into food for meals. In some embodiments, the compounds are incorporated with excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, caplets, pellets, granules, lozenges, chewing gum, powders, syrups, elixirs, wafers, aqueous solutions, and suspensions. In the case of tablets, the carriers used include salts of lactose, corn starch, sodium citrate, and phosphoric acid. Pharmaceutically acceptable excipients include binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). In embodiments, tablets are coated by methods well known in the art. For tablets, capsules, caplets, pellets, or granules for oral administration, pH-sensitive enteric coatings such as Eudragits®, designed to control the release of the active ingredient, are optionally used. Oral dosage forms also include modified release formulations, such as immediate-release and time-release formulations. Examples of modified release formulations are employed, for example, in the form of coated tablets, osmotic delivery devices, coated capsules, microencapsulated microspheres, aggregated particles, such as molecular sieve-type particles, or fine hollow permeable fiber bundles, or shredded hollow permeable fibers aggregated or held within fibrous packets, and include sustained release (SR), extended release (ER, XR, or XL), time-release or time-delayed release, controlled release (CR), or continuous release (CR or Contin). Time-delayed release compositions are formulated, for example, as liposomes, or in which the active compound is protected by a differentially degradable coating, such as microencapsulation or multiple coating.Liposome delivery systems include, for example, small monolayer vesicles, large monolayer vesicles, and multilayer vesicles. In some embodiments, liposomes are formed from a variety of phospholipids such as cholesterol, stearylamine, or phosphatidylcholine. For oral administration in capsule form, useful carriers or diluents include lactose and dried corn starch.

[0156] In some embodiments, liquid preparations for oral administration may take the form of solutions, syrups, or suspensions, or these may be appropriately presented as dried products for composition using water or other suitable vehicles before use. When aqueous suspensions and / or emulsions are administered orally, the compounds of this application are appropriately suspended or dissolved in an oil phase combined with emulsifiers and / or suspending agents. Certain sweeteners and / or flavoring agents and / or coloring agents are added, if desired. Such liquid preparations for oral administration are prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methylcellulose, or hydrogenated edible oils); emulsifiers (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoate, or sorbic acid). Useful diluents include lactose and high molecular weight polyethylene glycol.

[0157] The compound of this application can also be freeze-dried, and the resulting freeze-dried product can be used, for example, in the preparation of injectable products.

[0158] In some embodiments, the compounds of this application are administered parenterally. For example, a solution of the compounds of this application is prepared in water appropriately mixed with a surfactant such as hydroxypropylcellulose. In some embodiments, dispersions are prepared in glycerol, liquid polyethylene glycol, DMSO, and mixtures thereof with or without alcohol, as well as in oil. Under normal storage and use conditions, these preparations contain preservatives to inhibit microbial growth. Those skilled in the art will know how to prepare suitable formulations. With regard to parenteral administration, sterile solutions of the compounds of this application are usually prepared, and the pH of the solution is appropriately adjusted and buffered. With regard to intravenous use, the total concentration of the solute should be controlled to make the preparation isotonic. With regard to ophthalmic administration, an ointment or droppable liquid is delivered by an ophthalmic delivery system known in the art, such as an applicator or eyedropper. In some embodiments, such compositions include a mucus mimic such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose, or polyvinyl alcohol, a preservative such as sorbic acid, EDTA, or benzyl chromium chloride, and a typical amount of diluent or carrier. For pulmonary administration, a diluent or carrier suitable for enabling aerosol formation will be selected.

[0159] In some embodiments, the compounds of this application are formulated for parenteral administration by injection, including using conventional catheter techniques or infusions. The formulations for injection are presented in unit dosage forms, for example, ampoules or multi-dose containers, with, for example, added preservatives. In some embodiments, the compositions take the form of a sterile suspension, solution, or emulsion in an oily or aqueous vehicle and contain formulation agents such as suspending agents, stabilizers, and / or dispersants. In all cases, the form must be sterile and fluid enough to allow for easy syringability. Alternatively, the compounds of this application are preferably in the form of a sterile powder for reconstitution using a suitable vehicle, such as sterile pyrogen-free water, before use.

[0160] In some embodiments, compositions for nasal administration are conveniently formulated as aerosols, infusions, gels, and powders. With regard to intranasal or inhalation administration, the compounds of this application are conveniently delivered in the form of solutions, dry powder formulations, or suspensions from a pump spray container pushed in or dispensed by the patient, or as aerosol spray presentations from a pressurized container or nebulizer. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multiple dose quantities in a sealed container, typically in the form of a cartridge or refill for use with a spray device, for example. Alternatively, the sealed container is a unit dispensing device, such as a single-dose nasal inhaler or an aerosol dispenser with a metering valve, intended for disposal after use. If the dosage form involves an aerosol dispenser, it will contain a propellant, such as a compressed gas, such as compressed air, or an organic propellant, such as a fluorochloro hydrocarbon. Suitable propellants include, but are not limited to, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, heptafluoroalkane, carbon dioxide, or another suitable gas. In the case of pressurized aerosols, the dosage unit is appropriately determined by providing a valve for delivering a metered amount. In some embodiments, the pressurized vessel or nebulizer contains a solution or suspension of the active compound. Capsules and cartridges for use in inhalers or blowers (e.g., made from gelatin) are formulated to contain, for example, a powder mix of the compound of this application and a suitable powder base such as lactose or starch. The aerosol dosage form may also take the form of a pump atomizer.

[0161] Compositions suitable for cheek or sublingual administration include tablets, lozenges, and pastilles, and the compounds of this application are formulated with carriers such as sugars, acacia, tragacanth, or gelatin and glycerin. Compositions for rectal administration are conveniently in the form of suppositories containing conventional suppository bases such as cocoa butter.

[0162] The suppository forms of the compounds of this application are useful for vaginal, urethral, ​​and rectal administration. Such suppositories would generally be constructed from a mixture of substances that are solid at room temperature but melt at body temperature. Substances commonly used to produce such vehicles include, but are not limited to, theobroma oil (also known as cocoa butter), glycerin-gelatinized, other glycerides, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights, and fatty acid esters of polyethylene glycol. For further consideration of suppository forms, see, for example, Remington's Pharmaceutical Sciences, 16th Ed., Mack Publishing, Easton, PA, 1980, pp. 1530–1533.

[0163] In some embodiments, the compounds of this application are conjugated with soluble polymers as targetable drug carriers. Such polymers include, for example, polyvinylpyrrolidone, pyran copolymers, polyhydroxypropyl methacrylamide-phenol, polyhydroxyethyl aspartamido-phenol, or polyethylene oxide-polylysine substituted with palmitoyl residues. Furthermore, in some embodiments, the compounds of this application are conjugated with a class of biodegradable polymers useful in achieving controlled drug release, such as polylactic acid, polyglycolic acid, copolymers of polylactic acid and polyglycolic acid, polyepsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and crosslinked or amphiphilic block copolymers of hydrogels.

[0164] In some embodiments, the compounds of this application may be conjugated with a viral vector, a nonviral vector, or other vector. Viral vectors may include retroviruses, lentiviruses, adenoviruses, herpesviruses, poxviruses, alphaviruses, vaccinia viruses, or adeno-associated viruses. Nonviral vectors may include nanoparticles, cationic lipids, cationic polymers, metal nanoparticles, nanorods, liposomes, micelles, microbubbles, cell-permeable peptides, or lipospheres. Nanoparticles may include silica, lipids, carbohydrates, or other pharmaceutically acceptable polymers.

[0165] While the compounds of this application are suitable for use alone, they will generally be administered in the form of pharmaceutical compositions in which one or more of the compounds of this application (active ingredients) are bound to a pharmaceutically acceptable carrier. Depending on the mode of administration, the pharmaceutical composition is thought to contain about 0.05 wt% to about 99 wt% or about 0.10 wt% to about 70 wt% of the active ingredient and about 1 wt% to about 99.95 wt% or about 30 wt% to about 99.90 wt% of the pharmaceutically acceptable carrier, all weight percentages based on the entire composition.

[0166] In some embodiments, the pharmaceutical composition of this application further comprises one or more additional active ingredients for treating cancer.

[0167] In some embodiments, one or more additional agents for treating cancer are, for example, cisplatin, tyrosine kinase inhibitors, glutaminase inhibitors (e.g., glutaminase-1 (GLS1) inhibitors), asparagine synthase (ASNS) inhibitors, immune checkpoint blockers, or antibody therapies (including anti-PD-1 and / or anti-PD-L1 antibodies).

[0168] In some embodiments, one or more additional agents for treating cancer are, for example, immune checkpoint blockers. In some embodiments, the immune checkpoint blocker is a PD-1 inhibitor, including one or more of pembrolizumab, nivolumab, and semiprimab, or a PD-L1 inhibitor, including one or more of atezolizumab, avelumab, and durvalumab. In some embodiments, the immune checkpoint blocker is a CTLA-4 inhibitor, including ipilimumab and / or tremelimumab. In some embodiments, the immune checkpoint blocker is a LAG-3 inhibitor, including relatrimab and / or Opduralag.

[0169] V. Method for preparing the compound of this application The compounds of this application may be prepared using known synthesis processes from starting materials that are commercially available or obtainable from commercial chemical suppliers. The selection of a particular process for preparing a given compound of this application is within the scope of those skilled in the art.

[0170] Salts of the compounds of this application are generally formed by dissolving a neutral compound in an inert organic solvent, adding either the desired acid or base, and isolating the resulting salt by filtration or any other known means.

[0171] The formation of the desired compound salt is achieved using standard techniques. For example, a neutral compound is treated with an acid or base in a suitable solvent, and the resulting salt is isolated by filtration, extraction, or any other suitable method.

[0172] The formation of solvates will vary depending on the compound and the solvate. Generally, solvates are formed by dissolving the compound in a suitable solvent and then cooling or isolating the solvate using a poor solvent (antisolvent). Solvates are typically dried or azeotropically formed under ambient conditions. The selection of appropriate conditions for forming a particular solvate can be made by those skilled in the art. Examples of suitable solvents are ethanol, water, etc. When water is the solvent, the molecule is referred to as a "hydrate". The formation of solvates of the compounds of this application will vary depending on the compound and the solvate. Generally, solvates are formed by dissolving the compound in a suitable solvent and then cooling or isolating the solvate using a poor solvent. Solvates are typically dried or azeotropically formed under ambient conditions. The selection of appropriate conditions for forming a particular solvate can be made by those skilled in the art.

[0173] The prodrugs of the compounds of this application may be, for example, conventional esters formed with available hydroxyl or carboxyl groups. For example, available hydroxyl groups may be acylated using an activating acid in the presence of a base and optionally in an inert solvent (e.g., an acid chloride in pyridine), and available carboxyl groups may be converted to esters by activating an acid, for example, to an acid chloride, or by reacting an activating acid with a suitable nucleophile, generally in the presence of a non-nucleophilic base, using a known acid conjugate.

[0174] The products of the process of this application may be isolated according to known methods, for example, the compounds may be isolated by evaporation of the solvent, filtration, centrifugation, chromatography, or other suitable methods.

[0175] Those skilled in the art will recognize that, if the reaction steps of this application are carried out in a variety of solvents or solvent systems, the reaction steps may also be carried out in a suitable mixture of solvents or solvent systems. [Examples]

[0176] A class of microbiome-derived metabolites called hydroxyphenylpropanoates (HPPs) has been found to enhance tumor immune surveillance in mice and synergistically interact with immune checkpoint block (ICB) therapy. HPP molecules act as broad-spectrum enhancers of innate immune signaling pathways in tumor-associated myeloid cells by promoting the cleavage of the pore-forming protein gasdermin D (GSDMD), a critical effector of standard and non-standard inflammasome signaling. Increased secretion of pro-inflammatory cytokines, including IL-1β, from HPP-treated myeloid cells promotes NF-κB activity in tumor-infiltrating leukocytes. This leads to improved anti-cancer CD8 T cell function, significant tumor regression, and better long-term cancer control with immune checkpoint therapy in mice. Human peripheral blood mononuclear cells also respond similarly to HPP treatment. GSDMD cleavage is also associated with a favorable response to ICB therapy in patients with advanced melanoma. In summary, we have uncovered previously unknown mechanisms of microbiome-mediated modulation of host antitumor immunity that are modifiable and can be used to enhance the efficacy of cancer immunotherapy.

[0177] The following non-limiting embodiments are illustrative of the present application.

[0178] Various microbiomes give rise to diverse capabilities of tumor immune surveillance in genetically identical hosts. The study showed that inbred C57BL / 6 mice obtained from different laboratories possess diverse microbiomes and various capabilities for cancer immune surveillance (JCI insight 3, e94952 (2018); Science 350, 1084-1089 (2015)). To confirm these observations, mice were obtained from Jackson Laboratories (JAX), Charles River Laboratories (CR), and Taconic Farms (TAC). As expected, principal coordinate analysis (PCoA) and relative abundance plots of 16S amplicon-sequenced fecal samples showed that C57BL / 6 mice obtained from these different repositories were colonized with diverse microbiomes (Figure 1a, b). Consistent with previous studies (JCI insight 3,e94952(2018); Science 350,1084-1089(2015)), Muribaculaceae, previously known as Bacteroidales S24-7, was the most abundant bacterial family in the feces of mice obtained from JAX. In contrast, Bacteroidaceae was the most abundant bacterial family in mice obtained from CR and TAC. To test the impact on cancer immune surveillance, M3-9-M rhabdomyosarcoma (RMS) cells were orthotopically transplanted into the gastrocnemius muscle of female mice, and the size of these tumors was monitored over time. Compared to CR or TAC mice, JAX mice showed delayed tumor growth and extended overall survival (Figure 1c-f). This effect was dependent on CD8 T cells, which were more abundant in tumors isolated from JAX mice compared to CR mice (Figure 1h) (Figure 1g). In a complementary approach, broad-spectrum antibiotics (ABX) were used to induce gut microbiota abnormalities with a drug regimen that significantly reduced fecal bacteria abundance without affecting body weight (Figure 1i, j). As expected, ABX treatment suppressed M3-9-M growth in CR mice but enhanced tumor growth in JAX mice (Figure 1k, l). These data suggest that CR and TAC mice are colonized with microorganisms that suppress anti-cancer immunity, while JAX mice have a microbiota that promotes CD8 T cell activity against cancer.

[0179] However, data and previous reports using similar methods have several important limitations, including the fact that C57BL / 6 mice sourced from different repositories are clearly genetically distinct due to polymorphism resulting from decades of isolated inbreeding (Genome Biol. 14, 1-22 (2013); Nature 477, 289-294 (2011); Exp. Anim. 58, 141-149 (2009)), and that ABX treatment can directly affect host biology, apart from microbial depletion (Gut 64, 1732-1743 (2015)). To address these caveats, we created colonies of genetically identical C57Bl / 6 mice with JAX or CR microbial communities by vertical transfer. These colonies were called genetically identical mice (IMDM) with diverse and complex microbial communities established at birth (Figure 2a). PCoA in 16S amplicon-sequenced fecal samples showed efficient, stable, and generally representative microbial transfer from donor mice to IMDM hosts (Figures 2b-e). Muribaculaceae were a relatively abundant bacterial family that colonized IMDM-JAX mice, while Bacteroidaceae were relatively abundant in IMDM-CR mice (Figures 2f, g). Once established, M3-9-M cells were orthotopically transplanted into female IMDM-CR and -JAX mice, and tumor growth was monitored over time (Figure 2h). Consistent with the results in donor mice (Figure 1), M3-9-M tumor growth was delayed and overall survival was extended in IMDM-JAX mice compared to IMDM-CR mice (Figures 2i, j). The M3-9-M cell line is not immunogenic unless manipulated using a model antigen (ovalbumin, OVA) (Sci. Transl. Med. 6, (2014)) (Figure 2m, n). This difference was not observed in male mice (Figure 2k, l). In summary, these results provide direct support that diverse and complex microbiomes give rise to various capabilities of host tumor immune surveillance, which have a significant impact on overall tumor control. Consistent with this, the M3-9-M cell line established in IMDM-JAX was used against IMDM-CR mice. OVA Tumors showed a greater abundance of OVA-specific CD8 T cells (Figure 2o).

[0180] Diverse microbiomes produce unique metabolome profiles within the TME of a genetically identical host. We hypothesized that diverse compositions of the microbiome create unique metabolome profiles within host tumors, which modulate innate antitumor immunity. To test this, we transplanted M3-9-M and M3-9-M OVA RMS tumors were orthotopically excised from female and male IMDM mice 18 days after tumor transplantation. Fecal samples were collected before tumor transplantation. Non-targeted ultrafast liquid chromatography-mass spectrometry (UHPLC-MS) was performed to detect mouse fecal and tumor metabolites using 120 metabolite standards. The fecal and tumor metabolomes of the IMDM-CR mouse cluster were found to be different from those of IMDM-JAX (Figures 3a-d; 4a-d), suggesting that these microbiomes give rise to unique metabolomes in the feces and tumors. Additionally, ABX was provided to IMDM-JAX female mice with drinking water two weeks prior to tumor transplantation and continued throughout the experiment. Exposure to ABX altered the clustering patterns of the fecal and tumor metabolomes (Figures 3e-h), suggesting that the overall metabolome composition is modifiable.

[0181] Further examination of the metabolome datasets was conducted to identify whether specific metabolites were associated with the ability to monitor tumor immunity. Volcano plots for fecal and tumor metabolomes showed that some metabolites were significantly upregulated in the fecal and tumor metabolomes of IMDM-JAX mice (Figure 3i, j, and Figure 4e, f). The study focused on L-glutamine (L-gln) and hydroxyphenylpropanoate (HPP) because the appearance of these two metabolites was consistent across the metabolome datasets. Relative levels of L-gln were higher in fecal and tumor samples from IMDM-CR mice (Figure 4g, h; Figure 3k, l). On the other hand, relative levels of HPP were higher in fecal and tumor samples from IMDM-JAX mice, suggesting that the HPP metabolite was associated with a superior ability to monitor tumor immunity (Figure 3m, n; Figure 4i, j). ABX was also found to significantly reduce the relative levels of HPP metabolites in fecal and tumor samples from IMDM-JAX mice (Figure 4k, l), suggesting that ABX-sensitive bacteria produce this metabolite. Next, we explored which members of the gut microbiota produce HPP metabolites. Fecal samples from IMDM mice were equally divided into two halves: one half for non-targeted UHPLC-MS metabolomics and the other half for 16S amplicon sequencing. Metabolome and metagenomic datasets were analyzed using MelonnPan, a computational method for predicting metabolite composition from microbiota sequencing data. 20 The tools were trained. Through this method, we identified 77 gut microbiota amplicon sequence variants (ASVs) positively associated with HPP metabolite production, including members of the Muribaculaceae family previously linked to enhanced antitumor immunity (JCI insight 3, e94952 (2018)). These results, collectively, demonstrate that diverse microbiomes induce modifiable and distinctive metabolome profiles in distant host tumors, and that specific metabolites are associated with antitumor immunity.

[0182] Microbiota-derived metabolites HPP enhance the ability of tumor immune surveillance. The effect of HPP treatment on tumor outcomes was evaluated. Immunogenic and non-immunogenic tumors were treated with commercially available purified HPP metabolites. There are three positional isomers of HPP: 3,2-HPP, 3,3-HPP, and 3,4-HPP, which are present in animal microbiome samples, including human samples. Since analyses using various techniques did not detect any specific HPP isomers abundant in the IMDM-JAX mouse specimens, all three HPP isomers were tested, starting with 3,2-HPP for in vivo experiments. L-gln was used as a control metabolite where necessary. Endotoxin levels of the metabolite solutions used were confirmed to be within acceptable limits. Experiments showed that a dose of 83 mg / kg body weight given every 48 hours was appropriate for treating mice without toxicity or weight loss (Figures 5b-h).

[0183] To assess the effect of 3,2-HPP on antitumor immunity, orthotopic immunogenic and non-immunogenic tumors were treated in IMDM-CR mice using metabolites or vehicle controls from day 1 post-transplant (Figure 6a). 3,2-HPP treatment affected M3-9-M tumors in females and M3-9-M tumors in male mice. OVA It was found to enhance immune surveillance of tumors (Figure 6b, c). Conversely, non-immunogenic M3-9-M tumors in male mice did not respond to 3,2-HPP treatment (Figure 6d). The study was expanded to include subcutaneous B16.F10 melanoma tumors in female IMDM-CR mice, which have the potential to elicit an immune response due to the presence of HY-specific antigens (Cell Reports Methods 2, (2022)). 3,2-HPP treatment similarly increased the overall survival of these mice (Figure 6e). Additionally, initiation of 3,2-HPP treatment 7 days after transplantation of immunogenic tumors remained effective in IMDM-CR mice (Figure 5i-k). These findings suggest that 3,2-HPP metabolites have the potential to enhance antitumor immunity in hosts with an "immunosuppressive" microbiome.

[0184] Next, we tested the effect of 3,2-HPP on antitumor immunity in hosts with a dysbiotic gut microbiota. To achieve this, mice were administered ABX-supplemented drinking water starting two weeks before tumor transplantation and continuing until the end of the experiment (Figure 6f). As expected, ABX reduced immune surveillance of M3-9-M tumors in female IMDM-JAX mice, while it increased it in female IMDM-CR mice (Figure 6g, h). Interestingly, 3,2-HPP treatment increased overall survival in IMDM-JAX mice and attenuated the negative effect of ABX (Figure 6g). Conversely, the combination of ABX and 3,2-HPP treatment resulted in even higher overall survival in IMDM-CR mice than either treatment alone (Figure 6h). These findings indicate that 3,2-HPP metabolites enhance antitumor immunity in hosts with a dysbiotic gut microbiota.

[0185] We hypothesized that treatment with HPP metabolites would enhance tumor-specific CD8 T cells within the TME, thereby improving anti-tumor immunity. To test this hypothesis, we used M3-9-M in male IMDM-CR mice. OVA The effect of 3,2-HPP treatment on OVA-specific CD8 T cells within tumors was evaluated (Figure 6i). Eighteen days after transplantation, 3,2-HPP treatment was found to increase the frequency of OVA-specific CD8 T cells within tumors (Figure 6j), indicating that tumor-specific CD8 T cell activity is associated with the therapeutic efficacy of 3,2-HPP. However, there were no significant changes in the expression of exhaustion markers such as PD1, TIM3, and LAG3 on these T cells (Figure 6k). To assess the dependence of 3,2-HPP therapeutic outcomes on CD8 T cell activity, CD8 T cells were depleted using an anti-CD8 neutralizing antibody (Figure 6l, Figures 5l-p). CD8 T cell depletion neutralized the efficacy of 3,2-HPP treatment (Figure 6m), indicating that CD8 T cell effector function is relevant to the therapeutic outcomes of metabolites.

[0186] Due to this dependence on CD8 T cell activity, we tested the efficacy of 3,2-HPP in combination with clinically approved ICBs such as anti-PD-1 and anti-PD-L1. The investigation built upon previous reports (Sci. Transl. Med. 6, (2014); Mol. Ther. 25, (2017)) showing that M3-9-M tumors respond to anti-PD-1 and B16.F10 tumors respond to anti-PD-L1 therapy. Using IMDM-CR female mice transplanted with M3-9-M or B16.F10 tumors, we found that while anti-PD-1 and anti-PD-L1 therapy alone increased overall survival in mice, the combination of 3,2-HPP with these ICB therapies resulted in further improvement (Figure 6n~p). This demonstrates the potential of 3,2-HPP to enhance the potency of ICB therapy.

[0187] The findings described above demonstrate that microbiome-derived HPP metabolites enhance the host's ability to monitor tumor immunity. Combining these metabolites with other cancer therapies has the potential to be a promising strategy for enhancing treatment outcomes.

[0188] HPP enhances anti-tumor immunity by enhancing cancer immune signaling pathways in TMEs. We hypothesized that HPP metabolites enhance antitumor immunity by modulating cancer immune signaling pathways in TMEs. To test this, we used single-cell RNA sequencing (scRNAseq) to study M3-9-M grown in male IMDM-JAX, IMDM-CR, and 3,2-HPP-treated IMDM-CR mice (IMDM-CR-HPP). OVAGene expression in immune cells isolated from RMS tumors was analyzed. Fourteen distinct immune cell clusters were identified, including B cells, regulatory T cells (Treg), plasmacytoid dendritic cells (pDCs), CD8 T cells, CD4 T cells, natural killer (NK) cells, NKT cells, CD34+ cells, classical dendritic cells (cDCs), atypical antigen-presenting cells (aAPCs), M1-like and M2-like macrophages, monocytic myeloid suppressor cells (M-MDSCs), and polymorphonuclear MDSCs (Figure 7a-c).

[0189] The Wilcoxon rank-sum test was performed to compare gene expression between IMDM-CR and IMDM-JAX, as well as between IMDM-CR and IMDM-CR-HPP. The findings showed that 3,2-HPP treatment had similar effects on the JAX microbiome and gene expression levels across multiple immune cell types (Figure 7d). Circos plots for CD8 T cells, PMN-MDSCs, M-MDSCs, M1-like macrophages, aAPCs, and NKT cells suggest that many of the genes affected by 3,2-HPP treatment are also involved in the same pathways as those affected by the JAX microbiome (Figure 8). A composite circos plot (Figure 7e) further highlights the similarity between the effects of 3,2-HPP treatment and the JAX microbiome. The Metascape TRRUST database was used to predict transcriptional regulators that influence the expression of 3,2-HPP-affected genes. Several key regulators involved in cancer immune pathways, such as NF-κB and IRF, were identified (Figure 7f).

[0190] The effects of all three HPP metabolites on the activation of two cancer immune signaling pathways, NF-κB and IRF, were investigated in murine macrophage-like RAW-Dual® reporter cells and human monocyte THP1-Dual® reporter cells. The results showed that HPP metabolites alone did not activate NF-κB and IRF signaling in either cell type. However, when combined with PRR agonists such as lipopolysaccharide (LPS), vesicular stomatitis virus (VSV), or others, HPP metabolites significantly enhanced the activation of these pathways (Figure 7g; Figures 9a-m). In contrast, another metabolite, L-gln, had no effect on the NF-κB or IRF pathways (Figure 9n, o). These findings suggest that HPP metabolites act as broad accelerators of NF-κB and IRF signaling when these pathways are initiated by an immune response.

[0191] Since variable effects of HPP metabolites on the NF-κB and IRF pathways were observed in cell culture, we tested these effects on tumor immune surveillance in mice. After administering an HPP treatment regimen one day after orthotopic tumor transplantation in IMDM-CR mice, non-immunogenic M3-9-M was found to be unresponsive to any HPP metabolite (Figure 7j). However, all three HPP metabolites did not affect M3-9-M. OVA We enhanced immune surveillance for RMS and B16.F10 melanoma (Figure 7k, l). In summary, these results indicate that HPP metabolites enhance anti-tumor immunity by enhancing cancer immune signaling pathways initiated by innate tumor immune surveillance.

[0192] The effects of analogues of the HPP metabolites described in this application on the activation of the NF-κB pathway in human monocyte THP1-Dual® cells were also investigated. When treated with LPS, exemplary compounds II-1, II-2, II-3, II-4, I-4, I-5, I-6, I-7, III-1, III-2, and IV significantly enhanced the activation of the NF-κB pathway (Figures 15-18). These findings further support the idea that HPP metabolites and their analogues act as broad accelerators of NF-κB signaling when this pathway is initiated by an immune response.

[0193] HPP enhances NF-κB signaling and anti-tumor immunity by binding to GSDMD. We hypothesized that HPP metabolites enhance cancer immunity by targeting key signaling proteins involved in the NF-κB pathway. Since HPP was found to enhance NF-κB induction in human THP1-Dual® cells when induced by LPS (Figure 10a), we performed thermal proteomic profiling (TPP) to determine the molecular targets of the metabolites. Nonparametric analysis of response curves (NPARC) (Mol. Cell. Proteomics 18, (2019)) identified 647 potential protein targets of 3,2-HPP metabolites (Figure 10b). To narrow down the focus of the study, we concentrated on GSDMD, a cell membrane pore-forming protein that plays a crucial role in the inflammasome pathway, IL-1β release, and tumor immunity (Figure 10c, d) (Cell Rep.34,(2021);Cell Res.25,1285-98(2015);J.Immunother.Cancer 10,(2022);Cell Rep.41,(2022);Int.Immunopharmacol.74,(2019);J.Dig.Dis.19,(2018);Int.J.Biol.Sci.17,(2021);Nat.Commun.13,1-20(2022);BMC Cancer 20,(2020);Oncogene 41,5092-5106(2022)). We observed the correlation between the expression of inflammasome genes, GSDMD, and NF-κB1 in tumor-infiltrating immune cells (Figure 11). To determine whether 3,2-HPP targets GSDMD-mediated antitumor immunity, we injected M3-9-M into the gastrocnemius muscle of male C57BL / 6NJ (WT) and C57BL / 6N-Gsdmdem4Fcw / J (KO) mice. OVA The effects were verified by orthotopic transplantation of RMS (Figure 10e). Treatment with 3,2-HPP reduced tumor growth and increased overall survival in WT mice, but had no effect on tumors transplanted in KO mice (Figure 10f, g). 3,2-HPP treatment was also found to affect the secretion of several GSDMD regulatory cytokines, including a significant increase in IL-1β release (Figure 12).

[0194] We hypothesized that HPP-GSDMD interaction helps stimulate the secretion of specific cytokines such as IL-1β, which in turn induces NF-κB induction via IL-1 receptor signaling. To test this, wild-type (WT) and GSDMD knockout (KO) THP1 cells were treated with LPS in the presence of HPP molecules and an IL-1 neutralizing antibody. After 16 hours, supernatants were collected from KO and WT cells and treated with the IL-1 neutralizing antibody, which was found to inhibit NF-κB induction in THP1-Dual® reporter cells (Figure 13a). In addition, the effect of LPS-treated supernatants from WT and KO THP1 cells was evaluated on IL-1 receptor signaling using HEK-Blue® IL-1β cells in the presence of either a vehicle or HPP molecules. Supernatants collected from WT cells after 16 hours were treated in the presence of HPP molecules and enhanced IL-1 receptor signaling in HEK-Blue® IL-1β cells (Figure 13b; Figure 10h). Further examination of the supernatant revealed that the presence of HPP molecules increased the release of IL-1α and IL-1β from WT cells (Figure 13c-d). Assessment of lactate dehydrogenase (LDH) release as a measure of cell death did not show a significant change in response to LPS or HPP treatment alone or in combination (Figure 13e).

[0195] In another complementary approach, HPP-GSDMD interactions in THP1 cells were investigated using LPS and nigericin (NG) stimulation. The results showed that HEK-Blue® IL-1β cells responded to the initial supernatant from LPS+NG-treated WT cells but not to the supernatant from GSDMD-KO cells, indicating that initial IL-1 secretion is dependent on GSDMD activity (Figure 13f). However, late supernatants from both WT and KO cells elicited similar responses, suggesting that long-term LPS+NG-induced IL-1 secretion is independent of GSDMD activity (Figure 10i). HPP treatment enhanced IL-1 release from WT cells while protecting against LDH release in this context (Figure 10i; Figures 13g, h). Additionally, HPP isomers alone did not induce GSDMD cleavage, but enhanced it in the presence of LPS or LPS+NG stimulation (Figure 10j). These results indicate that the HPP molecule enhances GSDMD activity in a unique way, promoting the release of specific NF-κB-inducible cytokines while protecting against cell death. The effect of HPP-GSDMD interaction on IRF pathway induction was also evaluated. WT and KO cells were treated with VSV in the presence of the HPP molecule, and the supernatant was collected after 16 hours to assess IRF induction via IFN-α / β receptor signaling using HEK-Blue® IFN-α / β cells. Supernatants from both HPP-treated WT and KO cells enhanced the response of HEK-Blue® IFN-α / β cells (Figure 13i), suggesting that HPP promotes this pathway in a GSDMD-independent manner.

[0196] The clinical relevance of GSDMD cleavage was examined by peptide quantification from publicly available proteomic profiles of stage IV melanoma patients receiving either tumor-infiltrating lymphocyte (TIL)-based therapy or anti-PD-1 therapy (Cell 179, (2019)). Although data from the TIL cohort were not useful in assessing protein cleavage, uncleaved peptides were reduced in responders to anti-PD-1 therapy. Caspases cleave the human GSDMD protein at residue D275 after the FLTD motif, resulting in membrane localization and pore formation of the N-terminal domain (Nature 526, (2015); Proc. Natl. Acad. Sci. 115, (2018)). Therefore, the reduction in uncleaved peptides in responders suggests that enhanced GSDMD cleavage is associated with a favorable response to anti-PD-1 therapy (Figure 10k). To transfer the effects of HPP metabolites to human primary peripheral blood mononuclear cells (huPBMCs), huPBMCs were treated with LPS followed by NG for 16 hours in the presence of HPP molecules, and the supernatant was collected for cytokine assessment. HPP molecules enhanced the release of IL-1α and IL-1β (Figure 13j, k). Furthermore, the supernatant of HPP-treated huPBMCs enhanced the response of HEK-Blue® IL-1β reporter cells (Figure 10k) and protected huPBMCs from LDH release (Figure 10m). In summary, the results suggest that HPP metabolites have a unique effect on GSDMD activity in bone marrow cells. These enhance the release of certain cytokines that activate the NF-κB pathway, leading to improved antitumor immunity (Figure 10).

[0197] Consideration This study investigated the impact of complex microbiomes on tumor immune surveillance in genetically identical hosts. To do this, we used an in-house inbred mouse model called IMDM, which mimics the natural processes of complex microbiome establishment and stability in genetically identical hosts. This allowed us to demonstrate the causal effects of the microbiome. The findings suggest that the establishment of various complex microbiomes can produce distinct metabolome profiles in genetically identical hosts, leading to diverse capabilities of tumor immune surveillance. Various microbiome-derived metabolites have been associated with enhancing antitumor immune responses and immunotherapies (Science 369,1481-1489(2020);Nat.Metab.2,(2020);Cell Metab.34,(2022);Cancer Discov.12,(2022);Sci.Immunol.7,(2022);Sci.Immunol.8,(2023);Cell 184,(2021)). For example, specific microbiome-derived metabolites have been shown to induce or enhance the type 1 interferon pathway in mononuclear phagocytes, leading to improved antitumor immune responses (Sci.Immunol.7,(2022);Sci.Immunol.8,(2023);Cell 184,(2021)). Combining microbiome-derived HPP metabolites with ICB has been demonstrated to improve therapeutic outcomes in hosts with diverse microbiomes, including those with dysbiotic balance of gut microbiota. Furthermore, HPP has been shown to broadly enhance innate immune signaling, including the NF-κB and IRF pathways in bone marrow cells, and to enhance CD8 T cell-dependent tumor clearance.

[0198] Like inflammation, the GSDMD protein likely acts as a double-edged sword in tumor immunity. Some reports indicate that GSDMDs limit antitumor immunity during ICB therapy (J.Immunother.Cancer 10,(2022);Cell Rep.41,(2022)), while others counter that activation of GSDMDs may enhance antitumor immunity and effector CD8 T cell responses (Int.Immunopharmacol.74,(2019);J.Dig.Dis.19,(2018);Int.J.Biol.Sci.17,(2021);Nat.Commun.13,1-20(2022);BMC Cancer 20,(2020);Oncogene 41,5092-5106(2022)). Various molecular functions have been associated with the GSDMD protein, including the release of pro-inflammatory cytokines that may or may not induce cell death (Nature 526, (2015); Immunity 48, (2018)), mucin secretion from intestinal goblet cells (Sci.Immunol.7, (2022)), and nuclear translocations that interfere with the DNA damage repair ability of PARP-133. Enhanced GSDMD cleavage has been found to be associated with a favorable response to ICB therapy in a cohort of patients with advanced melanoma. Additionally, HPP molecules have been shown to enhance the NF-κB pathway by accelerating GSDMD cleavage and promoting the release of pro-inflammatory cytokines such as IL-1α and IL-1β, while partially protecting against cell death. IL-1β has been shown to enhance CD8 T cell enlargement, function, and antitumor immunity (Sci.Immunol.6, (2021); J.Exp.Med.210,491-502(2013)). HPP metabolites were found to enhance tumor stromal IL-1β release and tumor-specific CD8 T cell accumulation in the tumor mesenteric area (TME), thereby improving anti-tumor immunity. In conclusion, microbiome-derived HPP metabolites were demonstrated to enhance the host tumor immune surveillance capability and can be used as therapeutic adjuvants in various settings. Furthermore, some of these findings were validated in human cells.

[0199] material and method cell culture M3-9-M cells were provided by Crystal MacKall (Stanford, CA, USA). M3-9-M cells were transfected with the super piggyBac transposase expression vector (System Biosciences; Palo Alto, CA, USA). OVA Cells were generated. B16.F10 (CRL-6475) and THP-1 (TIB-202™) cells were supplied by the American Cell Culture Lineage Preservation Center (ATCC; Manassas, VA, USA). RAW-Dual™ cells, THP1-Dual™ cells, HEK-Blue™ IL-1β cells, THP1-Null2 cells, and THP1-KO-GSDMD cells were supplied by InvivoGen (San Diego, CA, USA). M3-9-M and M3-9-M OVA Cells were grown in RPMI 1640 (Life Technologies, Carlsbad, CA, USA) supplemented with 10% thermo-inactivated fetal bovine serum (FBS; Life Technologies) and 50 μM 2-mercaptoethanol (Gibco™). Transgenic M3-9-M OVA To maintain the cells, 1 μg / mL puromycin dihydrochloride (Gibco™) was added to the culture medium at each cell culture passage interval. B16.F10 (CRL-6475) cells were grown in DMEM (Life Technologies) containing 10% thermally inactivated FBS (Life Technologies).

[0200] RAW-Dual® cells and HEK-Blue® IL-1β cells were grown in DMEM (Life Technologies), 4.5 g / l glucose, 2 mM L-glutamine, 10% thermoactivated FBS, 100 μg / ml Normocin®, and penicillin-streptomycin (100 U / mL-100 μg / mL). To maintain the transgenic cell lines, 200 μg / mL and 100 μg / mL of Zeocin® were added to the growth medium at each passage. THP-1 (TIB-202™), THP1-Null2 cells, THP1-Dual™, and THP1-KO-GSDMD cells were grown in RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, 10% thermally inactivated FBS, 100 μg / mL Normocin™, and Pen-Strep (100 U / ml-100 μg / mL). To maintain THP1-Dual™ cells, 10 μg / mL blastosidine and 100 μg / mL Zeocin™ were added to the growth medium at each passage. To maintain THP1-KO-GSDMD and THP1-HMGB1-Lucia™ cells, 100 μg / mL Zeocin™ was added to the growth medium at each passage. Batch of frozen stocks was prepared for each cell line. The cells were stored by resuspending them in growth medium containing 20% ​​FBS and 10% DMSO. All cell lines were screened for mycoplasma every 8–12 weeks using a PCR mycoplasma detection kit from Thermo Scientific.

[0201] Animal models All animal experiments were conducted in accordance with the Canadian Council on Animal Care guidelines and the University of Calgary (Canada) Health Sciences Animal Care Committee approved protocols (AC20-0208 and AC20-0146). Animal experiments were performed using complex microbiomes at the University of Calgary Biohazard Facility. SPF C57BL / 6J, C57BL / 6NCr, and C57BL / 6NTac mice were obtained from three different commercial suppliers: Jackson Laboratories (room number RB08), Charles River Laboratory Inc. (room C62), and Taconic Farm (room IBU1501C). GF mice were supplied by the International Microbiota Centre (IMC; University of Calgary) to produce genetically identical mice (IMDM) with various complex microbiomes established at birth. IMDM models were produced through cohabitation and rearing in isolated cages within the biohazard facility. Aseptic techniques were followed to avoid cross-contamination of the microbiome. Microbial coherence was confirmed experimentally using 16S amplicon sequencing of fecal samples. Age- and sex-matched mice were randomly selected for each experimental group. GSDMD knockout (KO) and wild-type control (WT) mice were obtained from Jackson Laboratories.

[0202] cancer model Tumor cells were orthotopically transplanted into 6-8 week old mice. Prior to transplantation, representative tumor cell lines were tested and found to be negative for common experimental animal pathogens (Charles River). For experiments using a rhabdomyosarcoma model, mice were injected with 1.5 × 10⁴ cells in 50 μL of PBS into the right hind limb gastrocnemius muscle using an insulin syringe. 5 Individual M3-9-M or M3-9-M OVA Cells were transplanted orthotopic. The RMS tumor volume was calculated by subtracting the volume of the calf muscle before tumor transplantation from the volume after tumor transplantation (length × width × height). Using an insulin syringe, 5 × 10¹⁴ cells were injected into the right flank of the mouse in 50 μL of PBS. 5 Individual B16.F10 melanoma tumor cells were transplanted. The transplantation site area (length × width) 2The melanoma tumor volume was determined by measuring (×0.5). Tumor measurements were performed twice a week using a dendritic graft from the transplantation date until any experimental or humane endpoint.

[0203] Microbiota composition analysis Mice were gently restrained, and fecal samples were collected directly into tubes for a miniature centrifuge. The fecal samples were stored at -80°C for later use. Microbial DNA from the fecal samples was extracted and purified using the DNeasy PowerSoil Pro Kit (Qiagen®). The DNA concentration of all samples was adjusted to 25 ng / μL and sent to the Health Genomics and Informatics Center (CHGI; University of Calgary) for sequencing. At CHGI, the following was performed: 16S V3-V4 rRNA gene amplicon library preparation; Kapa qPCR library quantification assay (Roche); and 600-cycle MiSeq v3 sequencing (Illumina). Sequencing files were processed using the DADA2 R package (Nat. Methods 13,581 (2016)) to identify amplicon sequence variants (ASVs). Classifications were assigned to the ASVs using the SILVA53 version 138 reference database maintained by DADA2. Bacterial abundance, density, distance, and location were determined using the phyloseq R package (PLoS One 8, (2013)). For multivariate analysis, permutational ANOVA was performed using the "pairwise.adonis" function (R package version 0.4, (2020)) with 999 permutations and Bonferroni correction.

[0204] UHPLC-MS non-targeted metabolomics After collection, flash-frozen fecal, serum, and tumor samples were stored at -80°C. On the day of metabolite extraction, 20 mg of fecal or tumor mass or 20 μL of serum sample was thawed on ice, followed by the addition of ice-cold 50% methanol for extraction (1:50 dilution). Fecal and tumor samples were homogenized by bead beating, incubated on ice for 30 minutes, and centrifuged at 21,000 × g for 10 minutes at 4°C. Half of the supernatant was collected and centrifuged again. Finally, 250 μL of supernatant was collected from each sample for analysis by ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) using a Q Exactive® HF mass spectrometer (Thermo Scientific) in negative ion full scan mode (50–750 m / z) with a resolution of 240,000 at the Calgary Metabolomics Research Facility (CMRF; University of Calgary). To separate metabolites by UHPLC, a two-component solvent mixture of LC-MS grade 20 mM ammonium formate in pH 3.0 water (solvent A) and LC-MS grade 0.1% formic acid (%v / v) in acetonitrile (solvent B) was used in combination with a Syncronis® column (Thermo Fisher Scientific). Samples were run at a flow rate of 600 μL / min using the following gradients: 0–2 min, 100% B; 2–7 min, 100–80% B; 7–10 min, 80–5% B; 10–12 min, 5% B; 12–13 min, 5–100% B; 13–15 min, 100% B. For all runs, the sample injection volume was 2 μL. XCMS and MAVEN software packages (Anal.Chem.84,5035(2012); Curr.Protoc.Bioinformatics 14,(2012)) were used for primary analysis, and metabolites were identified by matching m / z signals and retention times with commercial reference materials. For statistical significance testing, the data were further analyzed with the MetaboAnalyst v.5.0 tool (Nat.Protoc.17,1735-1761(2022)).

[0205] Identify specific metabolite-producing bacteria within the same biological specimen. Fecal samples were collected from six IMDM-CR and six IMDM-JAX female C57BL / 6 mice. Each sample was split into two parts: one for UHPLC-MS non-targeted metabolomics assays in CMRF and the other for 16S amplicon sequencing in CHGI. After filtering initial metagenomic and metabolome data, HPP-producing bacteria were identified using the MelonnPan R package (Nat.Commun.10,1-11(2019)). A table of paired sequence characteristics and microbial community metabolite abundances was used as input to the "melonnpan.train" function, enabling unbiased identification of specific bacteria producing the metabolite within the same specimen.

[0206] Antibiotic-mediated imbalance of intestinal flora symbiosis A broad-spectrum antibiotic cocktail consisting of ampicillin (1 mg / mL), neomycin (1 mg / mL), vancomycin (0.5 mg / mL), and metronidazole (1 mg / mL) was added to the drinking water of mice. The concentration of metronidazole was gradually increased, reaching 1 mg / mL on day 9. Antibiotic treatment was initiated two weeks before tumor inoculation and continued throughout the experiment, with the antibiotic-supplemented drinking water being replaced every 3-4 days. The well-being of the mice was monitored by measuring their body weight. To evaluate the efficacy of the antibiotics, universal oligonucleotide primers specific to the conserved region of the eubacterial 16S rRNA gene (forward primer, 5') were used. 1320 -CCATGAAGTCGGAATCGCTAG- 1341 3'; reverse primer, 5' 1431 -ACTCCCATGGTGTGACGG- 1413Real-time polymerase chain reaction (RT-PCR) was performed on fecal microbial DNA using method 3'). Fecal microbial DNA was extracted using the DNeasy PowerSoil Pro Kit (Qiagen®), and 20 μL of PCR reaction mixture was prepared using iQ® SYBR® Green supermix (Bio-Rad), 300 nM primers, 3 ng of DNA template, and UltraPure® DNase / RNase-Free Distilled Water (Invitrogen®). The Bio-Rad® CFX96® RT-PCR system was thermally cycled using a protocol of polymerase activation and denaturation at 95°C for 5 minutes, followed by 35 cycles of denaturation at 95°C for 15 seconds and annealing and extension at 60°C for 1 minute.

[0207] Flow cytometry Tumors were isolated from mice, and single-cell suspensions were prepared using a mouse tumor dissociation kit (Miltenyi Biotec) according to the manufacturer's instructions. The isolated tumors were finely chopped into pieces approximately 2-4 mm in diameter using a sterile scalpel, and the tumor samples were homogenized for 1 minute in RPMI 1640 medium and enzyme mix. The homogenates were incubated at 37°C for 40 minutes with gentle continuous shaking. The homogenates were then filtered through a 70 μm cell strainer, followed by centrifugation of the single-cell suspension. RBCs were removed using erythrocyte (RBC) lysis buffer. Leukocyte separation was performed based on a Percoll gradient, followed by live and dead cell staining using Zombie Aqua dye (BioLegend). T lymphocytes were stained with cell surface markers using anti-CD4-FITC (clone RM4-4, BioLegend), anti-CD8-PE.Cy7 (clone 53-6.7, BioLegend), anti-CD3-BV421 (clone 145-2C11, BioLegend), anti-CD45-APC.Cy7 (clone 30-F11, BioLegend), anti-PD-1-FITC (clone 29F.1A12, BioLegend), anti-TIM3-PE (clone RMT3-23, BioLegend), and anti-LAG3-PerCP / Cy5.5 (clone C9B7W, BioLegend). For OVA-specific CD8 T cell detection, H-2K(b) chicken egg 257-264 human B2M SIINFEKL Alexa 647 labeled tetramers were used. After staining, the cells were washed with FACS buffer and quantified using an Attune NxT flow cytometer (Thermo Fisher Scientific).

[0208] Mouse processing regimen Metabolite compounds were obtained from Sigma-Aldrich and prepared as a metabolite stock solution by dissolving them in UltraPure™ DNase / RNase-Free Distilled Water (Invitrogen™) containing 1.0N NaOH (Sigma). The pH of the stock solution was adjusted to 7.4-7.6, and the endotoxin level of the solution was determined by the horseshoe crab slime cell lysate (LAL) test. First, the maximum tolerated dose and pharmacokinetics of the metabolites for animal experiments were established. To test the effect of the metabolites on antitumor immunity, 100 μL of the metabolite at a dose of 83 mg / kg body weight per mouse was administered intraperitoneally every 48 hours for seven doses. For immune checkpoint blockade (ICB) experiments, anti-PD-1 (clone RMP1-14, BioXcell) and anti-PD-L1 (clone 10F.9G2, BioXcell) antibodies were used. Three doses of 250 μg each of either anti-PD-1 or anti-PD-L1 were administered intraperitoneally on days 10, 13, and 16 post-tumor transplantation. Anti-CD8 (clone 2.43; BioXcell) monoclonal antibody was used to neutralize CD8 T cells in mice. 250 μg of anti-CD8 antibody in PBS was intraperitoneally injected per mouse 7 and 1 day prior to tumor transplantation, followed by administration of 100 μg of anti-CD8 in PBS on days 3, 7, and 10. The efficiency of T cell depletion was monitored by flow cytometry using anti-CD8-PE.Cy7 antibody (clone 53-6.7, BioLegend).

[0209] NF-κB and IRF activity reporter assay NF-κB and IRF induction assays were performed using mouse RAW-Dual® (InvivoGen) and human THP1-Dual® (InvivoGen) reporter cell lines. Cells were divided into 1.0 × 10⁶ cells for RAW-Dual®. 6 Individual cells / mL, and 5.0 × 10 for THP1-Dual®. 5Cells were resuspended in freshly prepared test medium at a concentration of individual cells / mL. 180 μL of cells were seeded per well in a standard 96-well plate, and the concentrations of several PRR agonists that induce NF-κB and IRF were optimized. To test the metabolite effects on these pathways, 10 μL of the optimized receptor agonist and 10 μL of 2 mM metabolite solution or vehicle were added to each well to a final volume of 200 μL. To neutralize IL-1 receptor signaling, anti-IL-1α (clone 7D4, InvivoGen) and anti-IL-1β (clone 4H5, InvivoGen) antibodies were added as recommended by the manufacturer, and IgG1 isotype (clone T8E5, InvivoGen) was used as a control. NF-κB activity was determined by measuring secreted embryonic alkaline phosphatase (SEAP) levels using QUANTI-Blue® solution (InvivoGen). 20 μL of cell culture supernatant was divided equally into 180 μL of QUANTI-Blue®, and the color conversion to blue was monitored. SEAP levels were determined using a Molecular Devices SpectraMax i3 at 650 nm. To assess IRF induction, secreted lucial ciferase levels were measured in the culture medium using QUANTI-Luc® solution (InvivoGen). 20 μL of cell culture supernatant was divided equally into 50 μL of QUANTI-Luc® solution, and luminescence readings were immediately recorded using a SpectraMax i3.

[0210] Cell viability and cytotoxicity assays Cell viability and cytotoxicity assays were performed using alamarBlue® (Invitrogen®) and Roche's Cytotoxicity Detection Kit PLUS (LDH). For cell viability assays, 10 μL of alamarBlue® and 90 μL of cell culture medium were mixed per well in a 96-well plate, incubated at 37°C with 5% CO2, and fluorescence readings were performed using SpectraMax i3. Cell-containing culture medium was used as the positive control, and cell-free medium was used as the negative control. For the LDH release assay, 46 μL of reaction mixture and 46 μL of cell culture supernatant were mixed in a 96-well plate and incubated in the dark for 20 minutes. Readings were performed using SpectraMax i3. The positive control was the supernatant of cells treated with lysis solution, and the negative control was the cell-free culture medium.

[0211] Cytokine assay Place the cells in a 96-well plate in a 0.5-1.0 × 10⁶ size. 6 Cells were seeded at individual cell / well density and treated with PRR agonists, either in combination with or without metabolites. Culture supernatants were collected at specific time points to measure cytokine levels. Samples were prepared according to the protocol mentioned by Kim et al. (Nat. Commun. 8, (2017)) to check cytokine levels in tumor stroma. Intracellular IL-1 levels were determined by incubating HEK-Blue® IL-1β cells (Invitrogen®) with the collected culture supernatant and measuring SEAP levels in the culture medium using QUANTI-Blue® solution (Invitrogen®). Multiplex quantification of cytokines, chemokines, and growth factors was also performed by Eve Technologies Corp. (Calgary, Alberta) using the Luminex® 200 system (Luminex, Austin, TX, USA).

[0212] Single-cell RNA sequencing (scRNAseq) M3-9-M OVA RMS cells were orthotopically inoculated into male IMDM mice, and a 3,2-HPP treatment regimen was initiated one day after tumor transplantation. The final dose was administered 6 hours before tumor excision to assess its effect on initial gene expression in the context of overall survival experiments. After 14 days, the tumors were excised and finely chopped into small pieces (approximately 2-4 mm in diameter) using a sterile scalpel, followed by the preparation of single-cell suspensions using a tumor dissociation kit (Miltenyi Biotec). Viable cell density was enriched using a dead cell removal kit (Miltenyi Biotec), and CD45+ tumor-infiltrating immune cells were isolated using mouse CD45 (TIL) microbeads (Miltenyi Biotec). Flow cytometry was performed to verify the purity of the isolated CD45+ cell population (average 95%), and cell health was confirmed by microscopy. Two samples were then pooled from each experimental group to enrich cell numbers for scRNA library construction. We constructed scRNA libraries using the Chromium Single Cell 3' GEM (Library and Gel Bead Kit v3) and the Chromium Controller platform, aiming for an estimated 5,000 cells per library. The libraries were then sequenced using the NovaSeq® 6000 platform (Illumina) at CHGI (University of Calgary), with a target of 100,000 reads per cell per cartridge.

[0213] scRNAseq data analysis Raw sequencing files were processed using Cell Ranger 6.0.2 (10x Genomics). Raw base call (BCL) files were demultiplexed into FASTQ files using the "cellranger mkfastq" command (sample indices: IMDM-CR=SI-GA-A3, IMDM-CR-HPP=SI-GA-B3, and IMDM-JAX=SI-GA-C3). Sequence alignment, filtering, barcode counting, UMI counting, and mapping to a mouse reference genome (mm10, GENCODE vM23 / Ensembl 98, July 7, 2020) preconstructed by 10x Genomics were performed using the "cellranger count" pipeline. For analysis in Loupe Browser 5.1.0, libraries were collected to equal sequencing depths using the "cellranger aggr" pipeline. Data were also analyzed using Seurat R packages 60-62, and the results were consistent with the Loupe Browser analysis. Seurat was used to filter data, consolidate samples, normalize genes, reduce dimensionality, and visualize data. Cells with low or high trait counts (<200 or >7500) and high mitochondrial gene frequencies (>5%) were removed. All samples were consolidated into a single Seurat object, data were scaled, linear and nonlinear dimensionality reduction was performed, and cells were clustered. Clusters were manually annotated using cell type-specific expression markers (Nature 562,367-372(2018); Nat.Commun.11,(2020); Nucleic Acids Res.51,(2022); J.Immunother.Cancer 9,1-18(2021); J.Immunother.Cancer 10,1-14(2022); J.Exp.Med.218,(2021)). Differentially expressed genes were identified using the Wilcoxon rank-sum test.Metascape (Nat.Commun.10,(2019)) and Cytoscape (Genome Res.13,(2003)) were used for pathway identification and protein network visualization.

[0214] Thermal profiling using intact cells 24 mL of THP1-Dual® cells (density 1.5 × 10⁶ cells / mL) were treated with 2 mM metabolite or vehicle control for 16 hours. The cells were then centrifuged at 340 × g for 5 minutes at 4°C, resuspended in 20 mL of ice-cold PBS, and centrifuged again. The cells were resuspended in 1200 μL of PBS and divided into 10 aliquots of 100 μL each in 0.2 mL PCR tubes, and centrifuged at 325 × g for 2 minutes at 4°C. 80 μL of PBS supernatant was removed using a pipette, and the cells were resuspended by gently tapping the tubes. The metabolite and vehicle-treated cell tubes were then heated in parallel in a PCR instrument at temperatures ranging from 37°C to 67°C for 3 minutes. After incubating the cells at room temperature for 3 minutes, they were flash-frozen in liquid nitrogen for 1 minute. The cells were briefly thawed in a 25°C water bath, placed on ice, and resuspended using pipetting. The freeze-thaw cycle was repeated once, and then 30 μL of PBS was added to the sample. The entire contents were centrifuged at 100,000 × g for 20 minutes at 4°C, and the supernatant was taken out into a new tube. The protein concentration of the 37°C sample was measured. The lysate was reduced using 10 mM dithiothreitol (DTT) at room temperature for 30 minutes and alkylated using 50 mM chloroacetamide (CAA) in the dark for 30 minutes. The volume of lysate yielding 200 μg of protein at the 37°C temperature point was determined and taken out into a new tube for all other temperature points.

[0215] Protein purification and peptide digestion Proteins were purified and peptides digested according to previously described methods (Science 346, (2014); Nat. Protoc. 10, 1567-1593 (2015)). For each sample, the volume of lysate equivalent to 200 μg from a temperature point of 37°C was diluted to 190 μL with 50 mM HEPES-NaOH pH 7.3. SP3 beads (Sera-Mag SpeedBeads, GE Healthcare, catalog numbers 41552105050250 and 65152105050250) were washed twice with 180 μL of H2O, resuspended in 10 μL of H2O, and added to the sample with 200 μL of 100% ethanol. The sample was then incubated at room temperature for 10 minutes at 1000 rpm in a thermomixer. The samples were washed four times with 180 μL of 80% ethanol, and the beads were resuspended in 200 μL of 50 mM HEPES-NaOH pH 7.3 with 4 μg of trypsin Lys C (Promega). The samples were incubated overnight at 37°C on a 600 rpm shaking platform. The following day, the digested peptides were transferred to new Eppendorf® tubes, and the peptide concentrations were measured at a temperature of 37°C.

[0216] TMT labeling 80 μL of 100% acetonitrile was added to 0.8 mg of TMT reagent. An equivalent volume of 100 μg of peptide from a temperature point of 37°C was then selected and placed in a new Eppendorf tube for each sample. 10 μL of TMT label was added to each sample and incubated at room temperature for 30 minutes. This process was repeated using the addition of 10 μL of TMT label. TMT labeling was stopped with 10 μL of 1 M glycine. All 10 samples were then pooled and dried using a speedy vac. 50% of the samples were desalted using a Pierce Peptide Desalting Spin column (catalog no. 89852) and resuspended in high pH mobile phase A (20 mM ammonium formate). The peptides were fractionated using high pH C18 reversed-phase HPLC with mobile phase B (100% ACN) having a gradient (5%–80% B) at 0.2 mL / min for 54 minutes. The 54 fractions were ligated into 18 fractions and dried. Finally, the peptides were resuspended in 20 μL of 0.1% formic acid for analysis by mass spectrometry. The NPARC R package (Mol. Cell. Proteomics 18, (2019)) was used for data analysis.

[0217] Western blotting Cells were harvested and lysates were prepared using a complete lysis buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1% Triton X-100; 1% SDS). The supernatant was obtained by centrifugation at 10,000 RPM for 10 minutes at 4°C using a benchtop PCR centrifuge. Protein quantification was performed using the Bio-Rad DC Protein Assay Kit (Bio-Rad, NC, USA). Proteins were separated using a 12% SDS-PAGE gel and transferred to a PVDF membrane using a Bio-Rad Trans-turbo semi-dry transfer device at 25V / 1A for 1 hour. The membrane was blocked at room temperature for 30 minutes using Tris-buffered saline-Tween-20 (TBST) containing 5% skim milk. Next, the membranes were probed overnight at 4°C with rabbit monoclonal recombinant anti-GSDMD antibody (ab210070, Abcam), anti-cleavage N-terminal GSDMD antibody (ab215203, Abcam), and mouse anti-actin monoclonal antibody (MAB1501, MilliporeSigma) against human FL-GSDMD, CL-GSDMD, and actin. The following day, the membranes were washed three times with TBST and probed at room temperature for 1 hour with goat anti-rabbit (1706515, Bio-Rad) or goat anti-mouse (1706516, Bio-Rad) horseradish peroxidase conjugate IgG. The secondary antibody was diluted 1:5000 in TBST containing 5% (w / v) skim milk, the membrane was washed with TBST, and then the protein of interest was detected using Clarity® Western ECL Substrate (Bio-Rad) on a Chemidoc-IT Imager (UVP, Upland, CA, USA).

[0218] Sample size determination and statistical analysis Sample sizes were determined in advance for each experiment, and preliminary tests were conducted to get an estimate of the effect size. The power analysis tool from ClinCalc.com (https: / / clincalc.com / stats / samplesize.aspx) was used to estimate sample sizes for animal experiments. Random assignment of mice to experimental groups was ensured, and cage effects were eliminated by housing mice in multiple cages. Previous experience and expert consultation also helped determine sample sizes for specific techniques. All data points were included in the analysis, and tumor growth dynamics, animal survival rates, and in vitro experimental data were analyzed using GraphPad Prism version 7.0. Statistical significance tests were performed on TPP-TR, scRNAseq, and 16S amplicon sequencing data using the R software package. Shapiro-Wilk normality tests or D'Agostino-Pearson omnibus normality tests were performed to determine the type of data distribution. Parametric statistical tests were performed if the values ​​formed a Gaussian or normal distribution; otherwise, nonparametric statistical tests were used. A confidence interval of 95% was set, and the name of the statistical test and the p-value were reported in the legend of the corresponding figure and table.

[0219] While this application is written with reference to what is currently considered a preferred example, it should be understood that this application is not limited to the examples disclosed. On the contrary, this application is intended to cover a variety of modifications and equivalent configurations that fall within the spirit and scope of the attached claims.

[0220] All publications, patents, and patent applications are incorporated herein by reference as collectively as each individual publication, patent, or patent application is specifically and individually indicated to be incorporated herein by reference as a whole. Where it is found that a term in this application is defined differently in a document incorporated herein by reference, the definition provided herein shall serve as the definition for that term.

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Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526, (2015). Yang, J. et al. Mechanism of gasdermin D recognition by inflammatory caspases and their inhibition by a gasdermin D-derived peptide inhibitor. Proc. Natl. Acad. Sci. 115, (2018). Mager, L. F. et al. Microbiome-derived inosine modulates response to checkpoint inhibitor immunotherapy. Science 369, 1481-1489 (2020). Wang, T. et al. Inosine is an alternative carbon source for CD8+-T-cell function under glucose restriction. Nat. Metab. 2, (2020). Wang, H. et al. The microbial metabolite trimethylamine N-oxide promotes antitumor immunity in triple-negative breast cancer. Cell Metab. 34, (2022). Messaoudene, M. et al. A Natural Polyphenol Exerts Antitumor Activity and Circumvents Anti-PD-1 Resistance through Effects on the Gut Microbiota. Cancer Discov. 12, (2022). Mirji, G. et al. The microbiome-derived metabolite TMAO drives immune activation and boosts responses to immune checkpoint blockade in pancreatic cancer. Sci. Immunol. 7, (2022). Jneid, B. et al. Selective STING stimulation in dendritic cells primes antitumor T cell responses. Sci. Immunol. 8, (2023). Lam, K. C. et al. Microbiota triggers STING-type I IFN-dependent monocyte reprogramming of the tumor microenvironment. Cell 184, (2021). Steed, A. L. et al. The microbial metabolite desaminotyrosine protects from influenza through type I interferon. Science 357, 498-502 (2017). Wei, Y. et al. The intestinal microbial metabolite desaminotyrosine is an anti-inflammatory molecule that modulates local and systemic immune homeostasis. FASEB J. 34, 16117-16128 (2020). Zhao, H., Lazarenko, O. P. & Chen, J. R. Hippuric acid and 3-(3-hydroxyphenyl) propionic acid inhibit murine osteoclastogenesis through RANKL-RANK independent pathway. J. Cell. Physiol. 235, (2020). Cheng, D. et al. 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Claims

1. One or more compounds of formula (I), or pharmaceutically acceptable salts, solvates, and / or ester prodrugs thereof: 【Chemistry 1】 During the ceremony, R １ , R ２ , and R ３ Under the condition that at least one of them is OH, R １ , R ２ , and R ３ These are independently OH or H. A method for increasing intracellular antitumor immunity in either a biological sample or a patient, comprising the step of administering an effective amount of to cells.

2. One or more compounds of formula (I), or pharmaceutically acceptable salts, solvates, and / or ester prodrugs thereof: 【Chemistry 2】 During the ceremony, R １ 、R ２ 、and R ３ is OH, under the condition that at least one of R １ 、R ２ 、and R ３ is independently OH or H A method for increasing antitumor immunity, comprising the step of administering a therapeutically effective amount thereof to a subject in need.

3. One or more compounds of formula (I), or pharmaceutically acceptable salts, solvates, and / or ester prodrugs thereof: 【Transformation 3】 During the ceremony, R １ , R ２ , and R ３ Under the condition that at least one of them is OH, R １ , R ２ , and R ３ These are independently OH or H. A method for treating cancer, comprising the step of administering a therapeutically effective amount of [a substance] to a subject in need thereof.

4. The method according to any one of claims 1 to 3, wherein one or more compounds of formula (I) are selected from 3,2-hydroxyphenylpropanoate, 3,3-hydroxyphenylpropanoate, and 3,4-hydroxyphenylpropanoate, or pharmaceutically acceptable salts, solvates, and / or ester prodrugs thereof.

5. The method according to any one of claims 1 to 3, wherein one or more compounds of formula (I) are selected from sodium 3-(4-hydroxyphenyl)propionate, methyl 3-(4-hydroxyphenyl)propionate, ethyl 3-(4-hydroxyphenyl)propionate, and N-hydroxysuccinimide 3-(4-hydroxyphenyl)propionate.

6. One or more compounds of formula (II), (III), or (IV), or their pharmaceutically acceptable salts, solvates, and / or ester prodrugs: 【Chemistry 4】 During the ceremony, R ４ Halogen, NR ５ R ６ , and C １～３ Selected from alkyl groups; R ５ and R ６ H and C are independent of each other. １～３ Selected from alkyl groups; 【Transformation 5】 During the ceremony, R ７ is OH; n is between 3 and 5; or 【Transformation 6】 A method for increasing intracellular antitumor immunity in either a biological sample or a patient, comprising the step of administering an effective amount of to cells.

7. One or more compounds of formula (II), (III), or (IV), or their pharmaceutically acceptable salts, solvates, and / or ester prodrugs: 【Transformation 7】 During the ceremony, R ４ Halogen, NR ５ R ６ , and C １～３ Selected from alkyl groups; R ５ and R ６ H and C are independent of each other. １～３ Selected from alkyl groups; 【Transformation 8】 During the ceremony, R ７ is OH; n is between 3 and 5; or 【Chemistry 9】 A method for increasing antitumor immunity, comprising the step of administering a therapeutically effective amount thereof to a subject in need.

8. One or more compounds of formula (II), (III), or (IV), or their pharmaceutically acceptable salts, solvates, and / or ester prodrugs: 【Chemistry 10】 During the ceremony, R ４ Halogen, NR ５ R ６ , and C １～３ Selected from alkyl groups; R ５ and R ６ H and C are independent of each other. １～３ Selected from alkyl groups; 【Chemistry 11】 During the ceremony, R ７ is OH; n is between 3 and 5; or 【Chemistry 12】 A method for treating cancer, comprising the step of administering a therapeutically effective amount of [a substance] to a subject in need thereof.

9. The compound is Table 1 The method according to any one of claims 6 to 9, or selected from a pharmaceutically acceptable salt, solvate, and / or ester prodrug thereof.

10. One or more compounds of formula (V), or pharmaceutically acceptable salts, solvates, and / or ester prodrugs thereof: 【Chemistry 13】 During the ceremony, R ８ , R ９ , and R １０ Under the condition that at least one of them is OH, R ８ , R ９ , and R １０ These are independently OH or H, or R ８ , R ９ , and R １０ One of them is halogen, NR １３ R １４ , and C １～３ Selected from alkyl groups, R ８ , R ９ , and R １０ The other two of them are H; R １３ and R １４ H and C are independent of each other. １～３ Selected from alkyl groups; R １１ is selected from H and = O; R １２ H, C １～４ Selected from alkyl and succinimide; p is 1 to 4. A method for increasing intracellular antitumor immunity in either a biological sample or a patient, comprising the step of administering an effective amount of to cells.

11. One or more compounds of formula (V), or pharmaceutically acceptable salts, solvates, and / or ester prodrugs thereof: 【Chemistry 14】 During the ceremony, R ８ , R ９ , and R １０ Under the condition that at least one of them is OH, R ８ , R ９ , and R １０ These are independently OH or H, or R ８ , R ９ , and R １０ One of them is halogen, NR １３ R １４ , and C １～３ Selected from alkyl groups, R ８ , R ９ , and R １０ The other two of them are H; R １３ and R １４ H and C are independent of each other. １～３ Selected from alkyl groups; R １１ is selected from H and = O; R １２ H, C １～４ Selected from alkyl and succinimide; p is 1 to 4. A method for increasing antitumor immunity, comprising the step of administering a therapeutically effective amount thereof to a subject in need.

12. One or more compounds of formula (V), or pharmaceutically acceptable salts, solvates, and / or ester prodrugs thereof: 【Chemistry 15】 During the ceremony, R ８ 、 R ９ 、 and R １０ at least one of which is OH, R ８ 、 R ９ 、 and R １０ are independently OH or H, or R ８ 、 R ９ 、 and R １０ one of which is selected from halogen, NR １３ R １４ 、 and C １～３ alkyl, and the other two of R ８ 、 R ９ 、 and R １０ are H; R １３ and R １４ H and C are independent of each other. １～３ Selected from alkyl groups; R １１ is selected from H and = O; R １２ H, C １～４ Selected from alkyl and succinimide; p is 1 to 4. A method for treating cancer, comprising the step of administering a therapeutically effective amount of [a substance] to a subject in need thereof.

13. The method according to any one of claims 1 to 12, further comprising administering an effective amount of one or more additional active substances for the treatment and / or therapy of cancer.

14. The method according to claim 13, wherein one or more additional active agents for treating cancer are small molecule chemotherapy such as cisplatin, tyrosine kinase inhibitors, glutaminase inhibitors (e.g., glutaminase-1 (GLS1) inhibitors), and / or asparagine synthase (ASNS) inhibitors, and known cancer therapies are, for example, radiotherapy, targeted therapies such as antibody therapy (including anti-PD-1 and / or anti-PD-L1 antibodies), immunotherapy, hormone therapy, and / or anti-angiogenic therapy.

15. One or more compounds of formula (I), or pharmaceutically acceptable salts, solvates, and / or ester prodrugs thereof: 【Chemistry 16】 During the ceremony, R １ , R ２ , and R ３ Under the condition that at least one of them is OH, R １ , R ２ , and R ３ is independently OH or H; and Pharmacologically acceptable carriers A pharmaceutical composition comprising, wherein one or more compounds of formula (I), or pharmaceutically acceptable salts, solvates, and / or ester prodrugs thereof, are present in the composition in an amount effective for increasing antitumor immunity or treating cancer.

16. One or more compounds of formula (II), (III), or (IV), or their pharmaceutically acceptable salts, solvates, and / or ester prodrugs: 【Chemistry 17】 During the ceremony, R ４ Halogen, NR ５ R ６ , and C １～３ Selected from alkyl groups; R ５ and R ６ H and C are independent of each other. １～３ Selected from alkyl groups; [Chemistry 18] During the ceremony, R ７ is OH; n is between 3 and 5; or 【Chemistry 19】 ; and Pharmacologically acceptable carriers A pharmaceutical composition comprising, wherein one or more compounds of formula (II), (III), or (IV), or pharmaceutically acceptable salts, solvates, and / or ester prodrugs thereof, are present in the composition in an amount effective for increasing antitumor immunity or treating cancer.

17. A pharmaceutical composition according to claim 15 or 16 for use in the preparation of a pharmaceutical for the treatment of cancer.

18. The pharmaceutical composition for use according to claim 17, further comprising one or more additional anticancer agents.

19. The pharmaceutical composition according to claim 18, wherein one or more additional anticancer agents are selected from cisplatin, tyrosine kinase inhibitors, glutaminase inhibitors (e.g., glutaminase-1 (GLS1) inhibitors), asparagine synthase (ASNS) inhibitors, immune checkpoint blockers, and antibody therapies (including anti-PD-1 and / or anti-PD-L1 antibodies).

20. The pharmaceutical composition according to claim 19, wherein the immune checkpoint blocker is a PD-1 inhibitor comprising one or more of pembrolizumab, nivolumab, and semiprimab, or a PD-L1 inhibitor comprising one or more of atezolizumab, avelumab, and durvalumab.

21. The pharmaceutical composition according to claim 19, wherein the immune checkpoint blocker is a CTLA-4 inhibitor comprising ipilimumab and / or tremelimumab.

22. The pharmaceutical composition according to claim 21, wherein the immune checkpoint blocker is a LAG-3 inhibitor including relatrimab and / or opturalag.

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