A method to regulate regulatory T cells and inhibit tumor growth.

Abstract

This invention provides a method for reducing the number of regulatory T cells within a tumor in subjects where this is necessary. [Solution] A method comprising the step of administering an effective amount of a CD36 inhibitor to a target. Preferably, the method is one in which the CD36 inhibitor is an anti-CD36 antibody or a low-molecular-weight CD36 inhibitor, or a method comprising the step of administering an effective amount of a PPARβ inhibitor to a target. More preferably, the method is one in which the PPARβ inhibitor is an anti-PPARβ antibody or a low-molecular-weight PPARβ inhibitor.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 731,351, filed on 14 September 2018 under 35 U.S.C. §119(e). The aforementioned application is incorporated herein by reference in its entirety.

[0002] Technical field of inventions The present invention relates to a method for regulating intratumoral regulatory T (Treg) cells and inhibiting tumor growth in a subject, and more specifically, to a method for regulating intratumoral Treg cells and inhibiting tumor growth in a subject using a CD36 inhibitor or a PPARβ inhibitor. [Background technology]

[0003] Background of the Invention Regulatory T (Treg) cells are a unique population of T cells that modulate the immune system, maintain resistance to autoantigens, suppress the abnormal activation of autoreactive T cells, and prevent autoimmune diseases. Treg cells mediate their regulatory function through several mechanisms. First, Treg cells express anti-inflammatory cytokines, including IL-10, TGFβ, and IL-35. Another regulatory mechanism is through cell-to-cell contact. Cytotoxic T lymphocyte antigen-4 (CTLA-4), expressed on Treg cells, binds to the costimulatory B7 molecule on antigen-presenting cells (APCs) with approximately 10 times higher affinity than CD28, thereby preventing APCs from activating naive T cells. It has also been proposed that Treg cells inhibit the differentiation of effector T cells by consuming cytokines (e.g., IL-2, IL-4, IL-7) necessary for T cell activation and polarization (Ward-Hartstonge and Kemp. Clinical & Translational Immunology, 6:9 (2017) (Non-Patent Literature 1)).

[0004] One mechanism used by cancer cells to evade immune surveillance is the induction of Treg cells, which in turn suppress the organism's innate immune response. By inactivating Treg cells, it is possible to evade the suppression of the immune system, allowing the immune system to initiate a response to destroy the primary and metastatic tumors. It has been shown that depleting Treg cells deregulates antitumor immunity and prevents the formation of an immunosuppressive tumor microenvironment (TME). However, systemic elimination of Treg cells due to Treg depletion often leads to severe autoimmunity (Wang, H., et al. Trends Cancer 3, 583-592 (2017) (Non-patent Literature 2)).

[0005] Treg cells are frequently found in both mouse and human cancers (Roychoudhuri, R., Eil, RL & Restifo, NP The interplay of effector and regulatory T cells in cancer. Current opinion in immunology 33, 101-111 (2015) (Non-patent Literature 3); Delgoffe, GM et al. Nature 501, 252-256 (2013) (Non-patent Literature 4); Saito, T. et al. Nat Med 22, 679-684 (2016) (Non-patent Literature 5)), and therefore they represent a major barrier to antitumor immunotherapy and cancer immunotherapy (Rech, AJ et al. Sci Transl Med 4, 134ra162 (2012) (Non-patent Literature 6); Sutmuller, RP et al. J Exp Med 194, 823-832 (2001) (Non-patent Literature 7)). While strategies to deplete Tregs increase the antitumor response, the severe autoimmunity and undesirable depletion of effector T cells caused by systemic disappearance of Tregs limit the therapeutic potential of Treg-targeted approaches. In addition, systemic impairment of Treg suppression function during treatments targeting immune checkpoints expressed in Tregs, such as OX40, GITR, and CTLA-4, also hinders the application of Treg-targeted approaches in cancer treatment (Nishikawa, H. & Sakaguchi, S. International journal of cancer 127, 759-767 (2010) (Non-patent document 8); Simpson, TR et al. J Exp Med 210, 1695-1710 (2013) (Non-patent document 9); Curtin, JF et al. PLoS One 3, e1983 (2008) (Non-patent document 10)). To this day, the search for effective targeted approaches that selectively destroy intratumoral Tregs remains a challenge in cancer immunotherapy.

[0006] Therefore, there is an urgent need for methods to specifically target Treg cells within tumors in order to induce anti-tumor immunity without triggering an autoimmune response. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Ward-Hartstonge and Kemp. Clinical & Translational Immunology, 6:9 (2017) [Non-Patent Document 2] Wang, H., et al. Trends Cancer 3, 583-592 (2017) [Non-Patent Document 3] Roychoudhuri, R., Eil, RL & Restifo, NP The interplay of effector and regulatory T cells in cancer. Current opinion in immunology 33, 101-111 (2015) [Non-Patent Document 4] Delgoffe, GM et al. Nature 501, 252-256 (2013) [Non-Patent Document 5] Saito, T. et al. Nat Med 22, 679-684 (2016) [Non-Patent Document 6] Rech, AJ et al. Sci Transl Med 4, 134ra162 (2012) [Non-Patent Document 7] Sutmuller, RP et al. J Exp Med 194, 823-832 (2001) [Non-Patent Document 8] Nishikawa, H. & Sakaguchi, S. International journal of cancer 127, 759-767 (2010) [Non-Patent Document 9] Simpson, TR et al. J Exp Med 210, 1695-1710 (2013) [Non-Patent Document 10] Curtin, JF et al. PLoS One 3, e1983 (2008) [Overview of the project]

[0008] This disclosure describes methods for reducing the number of intratumor Treg cells in a subject and for increasing the number of intratumor cytotoxic T cells in a subject. These methods can be used to inhibit tumor growth in subjects with cancer.

[0009] In one aspect, the present disclosure provides a method for reducing the number of intratumoral Treg cells (e.g., CD4+ cells) in a subject. The method may include a step of administering an effective dose of a CD36 inhibitor to the subject. In some embodiments, the method may include a step of administering an effective dose of a PPARβ inhibitor to the subject.

[0010] In a second aspect, the disclosure provides a method for increasing the number of tumor-derived cytotoxic T cells (e.g., CD8+ cells) in a subject. The method may include a step of administering an effective dose of a CD36 inhibitor to the subject. In some embodiments, the method may include a step of administering an effective dose of a PPARβ inhibitor to the subject.

[0011] CD36 inhibitors may be anti-CD36 antibodies or small molecule CD36 inhibitors. Anti-CD36 antibodies may be human antibodies, humanized antibodies, chimeric antibodies, or bispecific antibodies. Non-limiting examples of small molecule CD36 inhibitors may include AP-5258, AP5055, EP-80317, MPE-002, CHEML1789142, CHEML1789302, CHEML1789297, CHEML1789141, CHEML1789270, and CHEML1789308. Similarly, PPARβ inhibitors may be anti-PPARβ antibodies or small molecule PPARβ inhibitors. Non-limiting examples of small molecule PPARβ inhibitors may include FH535, GSK0660, GSK3787, PT-S58, PT-S77, and ST-247.

[0012] In some embodiments, a method for reducing the number of intratumoral Treg cells in a subject may further include a step of administering an additional therapeutic agent to the subject. The additional therapeutic agent may be an antibody specific to immune checkpoint modulators, such as CTLA-4, PD-1, PD-L1, PD-L2, killer immunoglobulin receptor (KIR), LAG3, B7-H3, B7-H4, TIM3, A2aR, CD40L, CD27, OX40, 4-IBB, TCR, BTLA, ICOS, CD28, CD80, CD86, ICOS-L, B7-H4, HVEM, 4-1BBL, OX40L, CD70, CD40, and GALS.

[0013] CD36 inhibitors, PPARβ inhibitors, or additional therapeutic agents may be administered intratumor, intravenously, subcutaneously, intraosseously, orally, percutaneously, by sustained release, controlled release, delayed release, as suppositories, or sublingually.

[0014] In some aspects, the subjects may have cancer. Non-limiting examples of cancer may include oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, gastrointestinal cancer, cancer of the central or peripheral nervous system, endocrine or neuroendocrine cancer or hematopoietic cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, islet cell carcinoma, Lie-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma, multiple neuroendocrine neoplasia type I and II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, and skin cancer.

[0015] In a third aspect, the present disclosure provides a method for inhibiting tumor growth in subjects having cancer. The method may include the step of administering a therapeutically effective dose of a CD36 inhibitor to the subject alone or in combination with an additional therapeutic agent. In some embodiments, the method may include the step of administering a therapeutically effective dose of a PPARβ inhibitor to the subject alone or in combination with an additional therapeutic agent.

[0016] CD36 inhibitors may be anti-CD36 antibodies or small-molecule CD36 inhibitors. Anti-CD36 antibodies may be human antibodies, humanized antibodies, chimeric antibodies, or bispecific antibodies. Non-limiting examples of small-molecule CD36 inhibitors may include AP-5258, AP5055, EP-80317, MPE-002, CHEML1789142, CHEML1789302, CHEML1789297, CHEML1789141, CHEML1789270, and CHEML1789308. Similarly, PPARβ inhibitors may be anti-PPARβ antibodies or small-molecule PPARβ inhibitors. Examples of small molecule PPARβ inhibitors may, non-limitingly, include FH535, GSK0660, GSK3787, PT-S58, PT-S77, and ST-247.

[0017] The additional therapeutic agent may be an immune checkpoint modulator, such as an antibody specific to an immune checkpoint. Examples of immune checkpoints may include CTLA-4, PD-1, PD-L1, PD-L2, killer immunoglobulin receptor (KIR), LAG3, B7-H3, B7-H4, TIM3, A2aR, CD40L, CD27, OX40, 4-IBB, TCR, BTLA, ICOS, CD28, CD80, CD86, ICOS-L, B7-H4, HVEM, 4-1BBL, OX40L, CD70, CD40, and GALS. In some embodiments, the additional therapeutic agent includes a PD-1 inhibitor. In some embodiments, the additional therapeutic agent includes a CTLA-4 inhibitor. In some embodiments, the additional therapeutic agent includes both a PD-1 inhibitor and a CTLA-4 inhibitor.

[0018] CD36 inhibitors, PPARβ inhibitors, or additional therapeutic agents may be administered intratumor, intravenously, subcutaneously, intraosseously, orally, percutaneously, by sustained release, controlled release, delayed release, as suppositories, or sublingually.

[0019] [Invention 1001] A method for reducing the number of intratumoral regulatory T cells in a target in need, comprising the step of administering an effective dose of a CD36 inhibitor to the target. [Invention 1002] A method for reducing the number of intratumoral regulatory T cells in a target in need, comprising the step of administering an effective dose of a PPARβ inhibitor to the target. [Invention 1003] The method of the present invention 1001 or 1002, wherein the intratumor regulatory T cells are CD4+ cells. [Invention 1004] A method for increasing the number of intratumor cytotoxic T cells in a target in need, comprising the step of administering an effective dose of a CD36 inhibitor to the target. [Invention 1005] A method for increasing the number of intratumor cytotoxic T cells in a target in need, comprising the step of administering an effective dose of a PPARβ inhibitor to the target. [Invention 1006] The method of the present invention 1004 or 1005, wherein the intratumor cytotoxic T cells are CD8+ cells. [Invention 1007] The method of the present invention 1001 or 1004, wherein the CD36 inhibitor is an anti-CD36 antibody or a small molecule CD36 inhibitor. [Invention 1008] The method of the present invention 1007, wherein the anti-CD36 antibody is a human antibody, a humanized antibody, a chimeric antibody, or a bispecific antibody. [Invention 1009] The method of the present invention 1007, wherein a small molecule CD36 inhibitor is selected from the group consisting of AP-5258, AP5055, EP-80317, MPE-002, CHEML1789142, CHEML1789302, CHEML1789297, CHEML1789141, CHEML1789270, and CHEML1789308. [Invention 1010] A method of the present invention 1001, 1004, and 1007-1009, wherein a CD36 inhibitor is administered intratumor, intravenously, subcutaneously, intraosseously, orally, percutaneously, by sustained release, controlled release, delayed release, as a suppository, or sublingually. [Invention 1011] The method of the present invention 1002 or 1005, wherein the PPARβ inhibitor is an anti-PPARβ antibody or a small molecule PPARβ inhibitor. [Invention 1012] The method of the present invention 1011, wherein the anti-PPARβ antibody is a human antibody, a humanized antibody, a chimeric antibody, or a bispecific antibody. [Invention 1013] The method of the present invention 1011, wherein the small molecule PPARβ inhibitor is selected from the group consisting of FH535, GSK0660, GSK3787, PT-S58, PT-S77, and ST-247. [Invention 1014] A method of the present invention 1002, 1005, and any of 1011-1013, wherein a PPARβ inhibitor is administered intratumor, intravenously, subcutaneously, intraosseously, orally, percutaneously, by sustained release, controlled release, delayed release, as a suppository, or sublingually. [Invention 1015] Any method of the present invention further comprising the step of administering an additional therapeutic agent to the target. [Invention 1016] The method of the present invention 1015, wherein the additional therapeutic agent comprises an immune checkpoint modifier. [Invention 1017] The method of the present invention 1016, wherein the immune checkpoint regulator comprises antibodies specific to CTLA-4, PD-1, PD-L1, PD-L2, killer immunoglobulin receptor (KIR), LAG3, B7-H3, B7-H4, TIM3, A2aR, CD40L, CD27, OX40, 4-IBB, TCR, BTLA, ICOS, CD28, CD80, CD86, ICOS-L, B7-H4, HVEM, 4-1BBL, OX40L, CD70, CD40, and GALS. [Invention 1018] The method of the present invention 1015, wherein the additional therapeutic agent comprises an anti-PD-1 mAb, an anti-CTLA4 mAb, or a combination thereof. [Invention 1019] Any method of the present invention described above, wherein the subject has cancer. [Invention 1020] The method of the present invention 1019, wherein the cancer is selected from the group consisting of oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, digestive cancer, cancer of the central or peripheral nervous system, endocrine or neuroendocrine cancer or hematopoietic cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, islet cell carcinoma, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma, multiple neuroendocrine tumors type I and II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, and skin cancer. [Invention 1021] A method for inhibiting tumor growth in a subject with cancer, comprising the step of administering a therapeutically effective dose of a CD36 inhibitor to the subject alone or in combination with an additional therapeutic agent. [Invention 1022] A method for inhibiting tumor growth in a subject with cancer, comprising the step of administering a therapeutically effective dose of a PPARβ inhibitor to the subject, either alone or in combination with an additional therapeutic agent. [Invention 1023] The method of the present invention 1021, wherein the CD36 inhibitor is an anti-CD36 antibody or a small molecule CD36 inhibitor. [Invention 1024] The method of the present invention 1023, wherein the anti-CD36 antibody is a human antibody, a humanized antibody, a chimeric antibody, or a bispecific antibody. [Invention 1025] The method of the present invention 1023, wherein a small molecule CD36 inhibitor is selected from the group consisting of AP-5258, AP5055, EP-80317, MPE-002, CHEML1789142, CHEML1789302, CHEML1789297, CHEML1789141, CHEML1789270, and CHEML1789308. [Invention 1026] A method of the present invention 1021 and any of 1023-1025 wherein a CD36 inhibitor is administered intratumor, intravenously, subcutaneously, intraosseously, orally, percutaneously, by sustained release, controlled release, delayed release, as a suppository, or sublingually. [Invention 1027] The method of the present invention 1022, wherein the PPARβ inhibitor is an anti-PPARβ antibody or a small molecule PPARβ inhibitor. [Invention 1028] The method of the present invention 1027, wherein the anti-PPARβ antibody is a human antibody, a humanized antibody, a chimeric antibody, or a bispecific antibody. [Invention 1029] The method of the present invention 1027, wherein the small molecule PPARβ inhibitor is selected from the group consisting of FH535, GSK0660, GSK3787, PT-S58, PT-S77, and ST-247. [Invention 1030] A method according to any of the present invention 1022 and 1027-1030, wherein the CD3 inhibitor is administered intratumor, intravenously, subcutaneously, intraosseously, orally, percutaneously, by sustained release, controlled release, delayed release, as a suppository, or sublingually. [Invention 1031] A method according to any one of the present invention 1021 to 1030, wherein the additional therapeutic agent comprises an immune checkpoint modulator. [Invention 1032] The method of the present invention 1031, wherein the immune checkpoint regulator includes antibodies specific to CTLA-4, PD-1, PD-L1, PD-L2, killer immunoglobulin receptor (KIR), LAG3, B7-H3, B7-H4, TIM3, A2aR, CD40L, CD27, OX40, 4-IBB, TCR, BTLA, ICOS, CD28, CD80, CD86, ICOS-L, B7-H4, HVEM, 4-1BBL, OX40L, CD70, CD40, and antibodies specific to GALS. [Invention 1033] Any method of the present invention 1021 to 1032, wherein the cancer is selected from the group consisting of oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, digestive cancer, cancer of the central or peripheral nervous system, endocrine or neuroendocrine cancer or hematopoietic cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, islet cell carcinoma, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma, multiple neuroendocrine tumors type I and II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, and skin cancer. The above summary is not intended to define all aspects of this disclosure, and further aspects are described in other sections, such as the detailed description below. The entire application is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if combinations of features are not found together in the same sentence, paragraph, or section of the application. Other features and advantages of the present invention will become apparent from the detailed description below. However, it should be understood that the detailed description and specific examples are given only as examples, illustrating certain aspects of the disclosure, as various changes and modifications within the spirit and scope of this disclosure are expected to become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawing]

[0020] [Figure 1-1]Figures 1a, 1b, 1c, 1d, 1e, and 1f are a series of figures showing that intratumor Tregs increased the expression of CD36 and genes involved in lipid metabolism. Figure 1a shows a metabolic-focused pathway enrichment analysis of RNA expression in Tregs derived from breast cancer and PBMCs of breast cancer patients. It presents pathways (P<0.05) where expression differed significantly between intratumor Tregs and PBMC Tregs. Figure 1b shows enrichment plots of fatty acid metabolic pathways (top) and lipid binding pathways (bottom) in intratumor Tregs compared to PBMC Tregs, identified by gene set enrichment analysis (GSEA). The heatmap shows the expression levels of each signature gene. Columns represent individual samples, and rows represent each gene. High and low expression levels are shown. Figures 1c and 1d show representative histograms (left) and quantitative results of geometric mean (GeoMean) fluorescence intensity (right) for Bodipy FL C12 (Figure 1c) and Bodipy 493 / 503 (Figure 1d) in Treg cells derived from the indicated tissue of Yumm1.7 melanoma-carrying B6 mice. DLN: Inflowing lymph nodes (n≧5); LN: Non-inflowing lymph nodes (n=5); Spleen (n≧5); Thymus (n=5); Tumor (n≧5). Figures 1e and 1f show representative histograms (left) and quantitative results of geometric mean (GeoMean) fluorescence intensity of CD36 surface staining in Treg cells derived from PBMCs and TILs (n=12) (Figure 1e) from melanoma patients, and from displayed tissues (DLN, n=15; spleen, n=15; thymus, n=9; tumor, n=14) (Figure 1f) from Yumm1.7 melanoma B6 mice. TIL: Tumor-infiltrating lymphocytes. Data are representative results from three independent experiments (Figures 1c and 1d), or cumulative results from three independent experiments (Figures 1e and 1f). Each symbol represents one individual. Data are mean ± SD and analyzed by two-sided independent Student's t-test. **P<0.01, ***P<0.001. [Figure 1-2] This is a continuation of Figure 1-1. [Figure 2-1]Figures 2a, 2b, 2c, 2d, 2e, 2f, 2g, 2h, 2i, and 2j are a series of figures demonstrating that disruption of CD36 selectively impairs the accumulation and suppressive function of intratumoral Tregs. Figure 2a shows representative images of hematoxylin and eosin (H&E) staining of indicated tissues from 21-23 week old WT mice and TregCD36- / - mice. Scale bar, 200 μm. Figures 2b and 2c show representative histograms (left) and quantitative results of geometric mean (GeoMean) fluorescence intensity (right) of Bodipy FL C12 (Figure 2b) and Bodipy 493 / 503 (Figure 2c) in splenic Tregs and intratumoral Tregs from Yumm1.7 melanoma-bearing WT mice or TregCD36- / - mice (n=6 per group). Figures 2d and 2e show tumor growth (Figure 2d) and tumor weight (Figure 2e) of YUMM1.7 melanoma from wild-type (WT) mice or TregCD36- / - mice (WT, n>9; TregCD36- / -, n>13). Foxp3YFP-Cre mice were used as WT mice. Figure 2f shows representative plots (left) and percentages of FoxP3+ Tregs among CD4+ T cells in the indicated tissues of tumor-carrying WT mice and TregCD36- / - mice (spleen, n>12; LN, n>11; tumor, n>13). Figure 2g shows representative plots (left) and percentages (right) of indicated cytokine-producing CD8+ T cells among all tumor-infiltrating CD8+ T cells from the indicated mice (n=5 per group). Figures 2h and 2i show the ex vivo suppression of CFSE-labeled WT naive CD8+ T cell proliferation by WT Treg and TregCD36- / - Treg sorted from tumor (Figure 2h) or spleen (Figure 2i) at the annotated ratios. Figure 2j shows body mass measurements in Rag1- / - mice that received naive CD4+ T cells alone or in combination with either WT Treg or TregCD36- / - Treg (medium, n=4; naive CD4, n=7; WT Treg, n=6; TregCD36- / -, n=6).Data are representative results from at least two independent experiments (Figures 2b, 2c, and 2g) or cumulative results from three independent experiments (Figures 2d, 2e, 2f, 2h, 2i, and 2j). Each symbol represents one individual. Data are mean ± SD (Figures 2b, 2c, 2e, 2f, 2g, 2h, and i) or mean ± SEM (Figures 2d and 2j) and analyzed by two-tailed independent Student's t-test (Figures 2b, 2c, 2d, 2e, 2f, 2g, 2h, and 2i) or one-way ANOVA with Tukey's multiple comparison test (Figure 2j). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns, no significant difference. [Figure 2-2] This is a continuation of Figure 2-1. [Figure 3]Figures 3a, 3b, 3c, 3d, 3e, 3f, 3g, 3h, and 3i are a series of figures illustrating CD36 deficiency-induced apoptosis in intratumor Tregs. Figure 3a shows the expression of genes related to the apoptotic pathway in WT and TregCD36- / - intratumor Tregs, as evaluated by RNA-seq (n=3 per group). Genes expressed differentially with a P value < 0.05 are shown. Figure 3b shows representative histograms (left) and quantitative analysis (right) of cleavage caspase-3 levels in intratumor Tregs from tumor-carrying mice from WT (n=13) and TregCD36- / - (n=14). Figure 3c shows representative histograms (left) and quantitative analyses (right) of MitoTracker Deep Red (MDR) staining in Tregs from the spleen, non-absorbed lymph nodes (LN), absorbed lymph nodes (DLN), blood, thymus, and tumors of tumor-carrying WT mice and TregCD36- / - mice (n>8 per group). Figures 3d and 3e show representative electron microscopy images (left) and quantitative plots (right) of mitochondrial count (Figure 3d) and cristae density (Figure 3e) in spleen Tregs and intratumor Tregs from tumor-carrying WT mice and TregCD36- / - mice. Scale bars: 500 nm in (Figure 3d) and 200 nm in (Figure 3e). Figure 3f shows OCR of the indicated iTregs cultured for 48 hours in cancer cell-conditioned medium (n≧4 per group). Figure 3g shows the viability of either WT iTreg or TregCD36- / - iTreg cells cultured in cancer cell conditioned medium as described above, and then treated with the indicated concentration of lactate for a further 72 hours (n≧4 per group). Figure 3h shows the NAD / NADH ratio of the indicated iTreg cells cultured in cancer cell conditioned medium for 48 hours (WT, n=11; TregCD36- / -, n=13). Figure 3i shows the relative viability of either WT iTreg or TregCD36- / - iTreg cells treated for 72 hours in cancer cell conditioned medium supplemented with or without NR (400 μM) (n=11 per group). Results were normalized to the viable cells of the control treatment in the indicated groups. NR: Nicotinamide riboside.The data are representative results from three independent experiments (Figures 3f and 3g) or cumulative results from three independent experiments (Figures 3b, 3c, 3h, and 3i). Data are mean ± SD and analyzed by two-tailed independent Student t-tests. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns, no significant difference. [Figure 4]Figures 4a, 4b, 4c, 4d, 4e, 4f, 4g, 4h, and 4i are a series of figures demonstrating the need for PPARβ signaling for metabolic adaptation of intratumor Tregs. Figure 4a shows enrichment plots of PPAR signaling pathways in intratumor Tregs compared to PBMC Tregs, identified by gene set enrichment analysis (GSEA). Heatmaps show the expression levels of each signature gene. Figure 4b shows the percentage of FoxP3+ Tregs among CD4+ tumor-infiltrating T lymphocytes from tumor-carrying WT mice and TregPPARβ- / - mice. Figures 4c and 4d show tumor growth (Figure 4c) and tumor weight (Figure 4d) of YUMM1.7 melanoma from wild-type (WT) mice or TregPPARβ- / - mice (WT, n=15; TregCD36- / -, n=10). Foxp3YFP-Cre mice were used as WT mice. Figure 4e shows the quantitative results of geometric MFI by MDR staining in intratumoral Tregs from WT mice and TregPPARβ- / - mice (n=5 per group). Figures 4f and 4g show that YUMM1.7 melanoma cells were transplanted into WT mice and TregCD36- / - mice and then treated with either DMSO or a PPARβ agonist as described in the Methods. Tumor growth (Figure 4f) (WT+DMSO, n=6; TregCD36- / -+DMSO, n=11; WT+PPARβ agonist, n=5; TregCD36- / -+PPARβ agonist, n=9) and the proportion of FoxP3+ Tregs among CD4+ tumor-infiltrating T lymphocytes (Figure 4g) (n≧9 per group) were analyzed. Figures 4h and 4i show quantitative analysis of MitoTracker Deep Red (MDR) staining (Figure 4h) and cleavage caspase-3 expression (Figure 4i) in intratumoral Tregs of WT mice and TregCD36- / - mice treated with the indicated process (n≧9 per group). Data are representative results from three independent experiments (Figures 4b and 4e) or cumulative results from at least three independent experiments (Figures 4c, 4d, 4f, 4g, 4h, and 4i). Data are mean ± SD (Figures 4b, 4d, 4e, 4g, 4h, and 4i) or mean ± SEM (Figures 4c and 4f) and analyzed by two-sided independent Student's t-test.*P<0.05, **P<0.01, ***P<0.001, ns, no significant difference. [Figure 5]Figures 5a, 5b, 5c, 5d, 5e, 5f, 5g, and 5h are a series of figures showing that targeting CD36 impairs intratumoral Tregs and primes tumors to PD-1 blockade. Figures 5a, 5b, and 5c show tumor growth in YUMM1.7 melanoma-bearing B6 mice treated for the indicated study (Figure 5a) (control, n=4; α-CD36 Ab, n=6), the percentage of FoxP3+ Tregs among CD4+ T cells in the indicated tissue (Figure 5b) (control, n=4; α-CD36 Ab, n=4), and the expression of cleavage caspase-3 in Tregs isolated from the indicated tissue (Figure 5c) (control, n=4; α-CD36 Ab, n=5). Figure 5d shows tumor growth in YUMM1.7 melanoma-carrying WT mice and TregCD36- / - mice treated with either the control medium (control) or α-CD36 mAb (WT+ control, n=12; WT+ α-CD36 Ab, n=10; TregCD36- / -+ control, n=11; TregCD36- / -+ α-CD36 Ab, n=10). Figure 5e shows tumor growth in YUMM1.7 melanoma-carrying WT mice and TregCD36- / - mice treated as shown (WT+ control, n=12; WT+ α-CD36 Ab, n=10; TregCD36- / -+ control, n=11; TregCD36- / -+ α-CD36 Ab, n=10). Figures 5f and 5g show tumor growth (Figure 5f) and Kaplan-Meier survival curves (Figure 5g) of YUMM1.7 melanoma-carrying WT mice and TregCD36- / - mice treated with the indicated method (WT, n=5; TregCD36- / -, n=4; WT+α-PD1, n=4; TregCD36- / -+α-PD1, n=5). Differences in survival time were analyzed by the long-rank (Mantel-Cox) test. Figure 5h shows tumor growth of induced Braf / Pten melanoma-carrying mice treated with the indicated method (control, n=10; α-PD1, n=11; α-CD36, n=11; α-CD36+αPD-1, n=11). Arrows indicate the day of treatment. The data represent representative results from three independent experiments (Figures 5a, 5b, and 5c), or cumulative results from at least two independent experiments (Figures 5d, 5e, 5f, 5g, and 5h). Each symbol represents one individual.Data were presented as mean ± SD (Figures 5b and 5c) or mean ± SEM (Figures 5a, 5d, 5e, and 5f) and analyzed by two-tailed independent Student t-tests. *P<0.05, **P<0.01, ns, no significant difference. [Figure 6-1] Figures 6a, 6b, 6c, and 6d are a series of figures demonstrating that targeting CD36 deregulates the host's antitumor immunity. Figures 6a, 6b, 6c, and 6d show representative plots and percentages (n=10 per group) of the absolute number of FoxP3+ Tregs per gram of tumor (n=10 per group) (Figure 6a), the percentage of CD8+ T cells among tumor-infiltrating T cells (n=19 per group) (Figure 6b), and the indicated cytokine-producing CD8+ T cells among all tumor-infiltrating CD8+ T cells (Figure 6c) and indicated cytokine-producing CD4+ cells among all tumor-infiltrating CD4+ T cells (Figure 6d) from YUMM1.7 melanoma-bearing mice treated for display (Figure 6a). [Figure 6-2] This is a continuation of Figure 6-1. [Figure 7-1] Figures 7a, 7b, 7c, 7d, 7e, 7f, 7g, and 7h are a series of figures showing the synergistic effects of checkpoint blocker inhibitors. Figure 7a shows the percentage of FoxP3+ Tregs among CD4+ T cells in the displayed tissue (control, n=4; α-CTLA4 mAb, n=4; α-CD36 mAb, n=4). Each symbol represents one individual. Data are mean ± SD and analyzed by two-tailed independent Student's t-test. *P<0.05, **P<0.01. Figures 7b, 7c, 7d, 7e, 7f, 7g, and 7h show tumor growth and Kaplan-Meier survival curves for YUMM1.7 melanoma-carrying B6 mice treated as indicated (control, n=10; α-PD1, n=10; α-CTLA4, n=7; α-CD36, n=11; α-CTLA4+αPD-1, n=7; α-CD36+αPD-1, n=11). Arrows indicate the day of treatment. Each symbol represents one individual. [Figure 7-2] This is a continuation of Figure 7-1. [Figure 7-3] This is a continuation of Figure 7-2. [Modes for carrying out the invention]

[0021] Detailed description of the invention The disclosed methods for regulating intratumoral regulatory T (Treg) cells and inhibiting tumor growth in subjects are partly based on the unexpected discovery that Treg-specific removal of CD36 reduces intratumoral Treg accumulation and suppresses tumor growth. Advantageously, Treg-specific CD36 deficiency does not induce autoimmunity, and CD36-deficient Treg cells maintain their inhibitory activity, for example, in suppressing CD4 T cell-induced inflammatory bowel disease.

[0022] The induction of Treg cells is thought to be the fundamental reason why an immune response to tumor-associated antigens does not occur, by suppressing tumor-specific T cells, such as CD8+ cytotoxic T cells, from attacking tumor cells. Therefore, the aim is to inactivate Treg cells to avoid suppression of the immune system and enable the immune system to initiate a response to attack both primary and metastatic tumors.

[0023] This disclosure demonstrates that Treg cell inactivation can be induced by CD36 deficiency. CD36 is a surface glycoprotein also known as a fatty acid translocase (FAT). CD36 is involved in inflammatory responses and modulates several functions, including lipid uptake, lipid storage, and lipid utilization (Glatz and Luiken, JLR, 2018). Tumor-infiltrating T cells have abnormally high fatty acid uptake, high intracellular lipid content, and high levels of CD36 expression (Yin et al., J Immunol, 2016; Cui and Kaech, Cancer Immunol Res, 2016). CD36 expression supports the survival of intratumor Treg cells by fine-tuning mitochondrial adaptability via PPAR signaling. Thus, high levels of CD36 expression in intratumor Treg cells modulate the metabolic adaptation of intratumor Treg cells by intervening in metabolic regulation and further promote tumor growth by suppressing the anti-tumor immune response. Therefore, targeting CD36 or PPAR signaling pathways appears to be an attractive therapeutic approach for regulating intratumor Treg cells and inhibiting tumor growth.

[0024] I. Methods to decrease the number of intratumor Treg cells and increase the number of intratumor cytotoxic T cells This disclosure provides a method for reducing the number of intratumoral Treg cells in a subject. The method may include the step of administering an effective dose of a CD36 inhibitor or a PPARβ inhibitor to the subject.

[0025] This disclosure also provides a method for increasing the number of tumor-derived cytotoxic T cells in a subject. The method may include a step of administering an effective dose of a CD36 inhibitor to the subject. In some embodiments, the method may include a step of administering an effective dose of a PPARβ inhibitor to the subject.

[0026] As used herein, “subject” refers to human or non-human mammals. Non-human mammals include, for example, livestock and pets such as sheep, cattle, pigs, dogs, cats, and mouse mammals. In certain embodiments, the subject is human. A “tissue-specific” promoter is a nucleotide sequence that, when functionally linked to a polynucleotide encoding or specified by a gene, causes the gene product to be produced within a cell only if the cell is substantially the tissue type corresponding to its promoter.

[0027] As used herein, “intratumor” refers to T cells located within tumor cell islands (i.e., clearly proximal to malignant epithelial cells), whereas peritumor T cells are located in the stroma surrounding and infiltrating the tumor. Therefore, T cells may be located within a tumor but may not be considered intratumor T cells because they are closely associated with stroma other than actual malignant cells. Whether or not a T cell is intratumor can be determined using any method known in the art for detecting T cells that maintain tumor structure.

[0028] As used herein, the term “regulatory T cell” or “Treg cell” refers to a CD4+CD25+ FoxP3+ T cell with inhibitory properties. As used herein, the term “helper T cell” refers to a CD4 T cell. Helper T cells (e.g., Th1 and Th2) recognize antigens bound to MHC class II molecules and produce different cytokines. As used herein, the term “cytotoxic T cell” refers to a CD8+ T cell. Cytotoxic T cells recognize antigens bound to MHC class I molecules.

[0029] Treg cells may include T cells expressing CD4, CD25, and FOXP3, such as CD4+, CD4+CD25+, and CD4+Foxp3+ regulatory T cells. Cytotoxic T cells, also known as killer T cells, may include CD8+ T cells.

[0030] CD36 inhibitors may, in no particular order, include nucleic acid molecules (e.g., enzyme nucleic acid molecules, antisense nucleic acid molecules, triple-chain oligonucleotides, dsRNA, ssRNA, RNAi, siRNA, aptamers, 2,5-A chimeras), lipids, steroids, peptides, proteins, allozymes, antibodies, monoclonal antibodies, humanized monoclonal antibodies, and small molecules (e.g., antiviral compounds). Similarly, PPARβ inhibitors may, in no particular order, include nucleic acid molecules (e.g., enzyme nucleic acid molecules, antisense nucleic acid molecules, triple-chain oligonucleotides, dsRNA, ssRNA, RNAi, siRNA, aptamers, 2,5-A chimeras), lipids, steroids, peptides, proteins, allozymes, antibodies, monoclonal antibodies, humanized monoclonal antibodies, and small molecules (e.g., antiviral compounds). For example, an anti-CD36 antibody may be a human antibody, a humanized antibody, a chimeric antibody, or a bispecific antibody. Non-exclusive examples of small molecule CD36 inhibitors may include AP-5258, AP5055, EP-80317, MPE-002, CHEML1789142, CHEML1789302, CHEML1789297, CHEML1789141, CHEML1789270, and CHEML1789308.

[0031] Non-exclusive examples of small molecule PPARβ inhibitors may include FH535, GSK0660, GSK3787, PT-S58, PT-S77, and ST-247.

[0032] In some embodiments, a method for reducing the number of intratumoral Treg cells in a subject may include the step of administering a CD36 inhibitor or a PPARβ inhibitor to the subject in combination with an additional therapeutic agent, such as an anticancer agent.

[0033] Additional therapeutic agents may be immune checkpoint modifiers, such as antibodies specific to immune checkpoints. Examples of immune checkpoints may include, but are not limited to, CTLA-4, PD-1, PD-L1, PD-L2, killer immunoglobulin receptor (KIR), LAG3, B7-H3, B7-H4, TIM3, A2aR, CD40L, CD27, OX40, 4-IBB, TCR, BTLA, ICOS, CD28, CD80, CD86, ICOS-L, B7-H4, HVEM, 4-1BBL, OX40L, CD70, CD40, and GALS. Non-exclusive examples of immune checkpoint regulators include ipilimumab, tremelimumab, pembrolizumab, nivolumab, pizilizumab, MPDL3280A, MEDI4736, BMS-936559, MSB0010718C, and AMP-224.

[0034] CD36 inhibitors, PPARβ inhibitors, or additional therapeutic agents may be administered simultaneously or sequentially in any suitable carrier for oral, topical, or parenteral administration.

[0035] CD36 inhibitors, PPARβ inhibitors, or additional therapeutic agents may be administered intratumor, intravenously, subcutaneously, intraosseously, orally, percutaneously, by sustained release, controlled release, delayed release, as suppositories, or sublingually.

[0036] CD36 inhibitors, PPARβ inhibitors, or additional therapeutic agents can be prepared as pharmaceutical compositions. These pharmaceutical compositions can be prepared, packaged, or marketed in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, or ocular administration routes. Formulations may include protruding nanoparticles containing the active ingredient, liposome preparations, resealed red blood cells, and immunological formulations. For example, a pharmaceutical composition formulated for parenteral administration may contain an active ingredient (e.g., a CD36 inhibitor, a PPAR inhibitor) combined with a pharmaceutically acceptable carrier such as sterile water or sterile isotonic saline. Injectable formulations can be prepared, packaged, or marketed in unit dosage forms, such as in ampoules or multi-dose containers containing preservatives. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions, pastes, and embeddable sustained-release or biodegradable formulations in oily or aqueous media. Such formulations may further contain one or more additional components, such as suspending agents, stabilizers, or dispersants. In one example of a parenteral formulation, the active ingredient is provided in a dry (i.e., powder or granule) form for reconstitution with a suitable medium (e.g., sterile water free of pyrogens) before parenteral administration of the reconstituted composition.

[0037] II. Methods to inhibit tumor growth This disclosure also provides a method for inhibiting tumor growth in subjects having cancer. The method may include the step of administering a therapeutically effective dose of a CD36 inhibitor to the subject alone or in combination with an additional therapeutic agent (e.g., an anticancer agent). Or, in addition, the method may include the step of administering a therapeutically effective dose of a PPARβ inhibitor to the subject alone or in combination with an additional therapeutic agent (e.g., an anticancer agent).

[0038] CD36 inhibitors may, in no particular order, include nucleic acid molecules (e.g., enzymatic nucleic acid molecules, antisense nucleic acid molecules, triple-stranded oligonucleotides, dsRNA, ssRNA, RNAi, siRNA, aptamers, 2,5-A chimeras), lipids, steroids, peptides, proteins, allozymes, antibodies, monoclonal antibodies, humanized monoclonal antibodies, and small molecules (e.g., antiviral compounds). Similarly, PPARβ inhibitors may, in no particular order, include nucleic acid molecules (e.g., enzymatic nucleic acid molecules, antisense nucleic acid molecules, triple-stranded oligonucleotides, dsRNA, ssRNA, RNAi, siRNA, aptamers, 2,5-A chimeras), lipids, steroids, peptides, proteins, allozymes, antibodies, monoclonal antibodies, humanized monoclonal antibodies, and small molecules (e.g., antiviral compounds). Non-exclusive examples of small molecule PPARβ inhibitors may include FH535, GSK0660, GSK3787, PT-S58, PT-S77, and ST-247.

[0039] Additional therapeutic agents may be immune checkpoint modifiers, such as antibodies specific to immune checkpoints. Examples of immune checkpoints may include, but are not limited to, CTLA-4, PD-1, PD-L1, PD-L2, killer immunoglobulin receptor (KIR), LAG3, B7-H3, B7-H4, TIM3, A2aR, CD40L, CD27, OX40, 4-IBB, TCR, BTLA, ICOS, CD28, CD80, CD86, ICOS-L, B7-H4, HVEM, 4-1BBL, OX40L, CD70, CD40, and GALS. Non-exclusive examples of immune checkpoint regulators include ipilimumab, tremelimumab, pembrolizumab, nivolumab, pizilizumab, MPDL3280A, MEDI4736, BMS-936559, MSB0010718C, and AMP-224.

[0040] In some embodiments, the additional therapeutic agents may include one or more antitumor / anticancer agents, including chemotherapeutic agents and immunotherapeutic agents.

[0041] "Chemotherapy agents" are chemical compounds useful for treating cancer. Examples of chemotherapy agents include: alkylating agents, e.g., thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates, e.g., busulfan, improsulfan, and pigosulfan; aziridines, e.g., benzodopa, carbocone, methyldopa, and uredopa; and ethyleneimines and methylamelamines, e.g., altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine. Includes; acetogenins (especially bratacin and bratacinone); camptothecin (including its synthetic analog topotecan); bryostatin; callistatin; CC-1065 (including its synthetic analogs adzeresin, carzelsin, and bizeresin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dorastatin; duocalmycin (including its synthetic analogs KW-2189 and CBI-TMI); eryuterobin; pancrustine; sarcodictiin; spongistatin; nitrogen mustards, e.g., chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobuenvicin, fenesterine, prednimustine, trophosphamide, uracil mustard, etc.; nitrosourea (nitrosurea), e.g., carmustine, chlorozotosine, fotemustine, lomustine, nimustine, ranimustine, etc.; antibiotics, e.g., engine antibiotics (e.g., calicheamicin, see Agnew Chem. Intl. Ed. Engl. 33:183-186 (1994)); dynemicins, including dynemicin A; esperamicin;Furthermore, neocardinostatin chromophores and related pigment proteins (endiin antibiotic chromophores), aclasinomycin, actinomycin, anthramycin, azaserin, bleomycin, kakutinomycin, carbicin (Carabicin), caminocycline, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, sorbicin, etc.; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU) etc.; Folic acid analogs, e.g., denopterin, methotrexate, pteropterin, trimethrexate, etc.; Purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, etc.; Pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU, etc.; Androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone, etc.; Anti-adrenal substances, e.g., aminoglutethimide, mitotane, trilostane, etc.; Folic acid supplements, e.g., folic acid; Aceglutone; Aldophosphamide glycoside; Aminolevulinic acid; Amsacrin; Bestrabusil; Bisanthren; Edatrexate (edatraxate); defofamin; demecoltin; diaziquan; eflornithine; eriptinium acetate; epotilone; etogluside; gallium nitrate; hydroxyurea; lentinan; ronidamine; mytansinoids, e.g., mytansine and anthamitosine; mitogwazone; mitoxantrone; mopidamol;Nitracrine; Pentostatin; Fenamet; Pirarubicin; Podophyllinic acid; 2-Ethylhydrazide; Procarbazine; PSK®; Lazoxane; Rhizoxin; Sizofuran; Spirogermanium; Tenuazonic acid; Triadiquan; 2,2',2''-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, verracurin A, loridine A, and anguidin); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, e.g., Paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France), etc.; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, e.g., cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and any pharmaceutically acceptable salts, acids, or derivatives of any of the above. This definition also includes: antihormone agents that act to modulate or inhibit hormonal effects on tumors, such as antiestrogens, e.g., tamoxifen, raloxifene, aromatase inhibitors 4(5)-imidazole, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and toremifene (Fareston); as well as antiandrogens, e.g., flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;Furthermore, any of the above-mentioned pharmaceutically acceptable salts, acids, or derivatives.

[0042] "Immunotherapy agents" are biological drugs useful in treating cancer. Examples of immunotherapy agents include atezolizumab, avelumab, blinatumomab, daratumumab, semiprimab, durvalumab, elotuzumab, laharpalepbek, ipilimumab, nivolumab, obinutuzumab, ofatumumab, pembrolizumab, and tarimozine.

[0043] The method may include a step of administering a composition containing a CD36 inhibitor, a PPARβ inhibitor, an immune checkpoint modulator, or any combination thereof to a target. Additional therapeutic agents, CD36 inhibitors, and / or PPARβ inhibitors may be administered simultaneously or sequentially in any suitable carrier for oral, topical, or parenteral administration. CD36 inhibitors, PPARβ inhibitors, or immune checkpoint modulators It can be administered intratumorally, intravenously, subcutaneously, intraosseously, orally, percutaneously, by sustained release, controlled release, delayed release, as a suppository, or sublingually.

[0044] Cancer may include, but is not limited to, the following: cardiac cancer, e.g., sarcomas, e.g., angiosarcoma, fibrosarcoma, rhabdomyosarcoma, and liposarcoma; myxoma; rhabdomyomas; fibromas; lipomas, and teratomas; lung cancer, e.g., bronchogenic cancers, e.g., squamous cell carcinoma, anaplastic small cell carcinoma, anaplastic large cell carcinoma, and adenocarcinoma; alveolar and bronchiolar cancers; bronchial adenomas; sarcomas; lymphomas; chondromatoid hamartomas; and This includes mesothelioma; gastrointestinal cancers, e.g., esophageal cancers, e.g., squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, and lymphoma; stomach cancers, e.g., carcinoma, lymphoma, and leiomyosarcoma; pancreatic cancers, e.g., tubular adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, and bipoma; small intestine cancers, e.g., adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, and adipose tissue cancer. Cancers of the colon include, for example, adenocarcinoma, neurofibroma, and fibroma; cancers of the colon, such as adenocarcinoma, tubular adenoma, chorioadenoma, hamartoma, and leiomyoma; cancers of the genitourinary tract, such as cancers of the kidney, such as adenocarcinoma, Wilms' tumor (nephroblastoma), lymphoma, and leukemia; cancers of the bladder and urethra, such as squamous cell carcinoma, transitional cell carcinoma, and adenocarcinoma; cancers of the prostate, such as adenocarcinoma and sarcoma; cancers of the testes, such as seminoma and teratoma. This includes fetal cancers, teratomas, choriocarcinomas, sarcomas, stromal cell carcinomas, fibromas, fibroadenomas, adenomatous tumors, and lipomas; liver cancers, e.g., hepatomas, e.g., hepatocellular carcinomas; cholangiocarcinomas; germ cell carcinomas; angiosarcomas; hepatocellular adenomas; and hemangiomas; bone cancers, e.g., osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulosarcoma), multiple myeloma, and malignant giant cell tumors. This includes chordoma, osteochondroma (osteochondrial exostoma), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma, and giant cell tumor; cancers of the nervous system, e.g., cancers of the skull, e.g., osteoma, hemangioma, granuloma, xanthomas, and degenerative osteitis; cancers of the meninges, e.g., meningioma, angiosarcoma, and gliomas; cancers of the brain, e.g., astrocytoma, medulloblastoma, glioma, ependymoma, germ cell tumor (pineal glandoma), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, and congenital tumors; and cancers of the spinal cord, e.g., neurofibroma, meningioma, glioma, and sarcoma;Gynecological cancers, e.g., uterine cancers, e.g., endometrial cancer; cervical cancers, e.g., cervical cancer and preneoplastic cervical dysplasia; ovarian cancers, e.g., serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified cancer, granulosa-theca cell tumor, Sertoli-Leydig cell tumor, undifferentiated germ cell tumor, and malignant teratoma; vulvar cancers, e.g., squamous cell carcinoma, carcinoma in situ, adenocarcinoma, fibrosarcoma, and melanoma; vaginal cancers, e.g., clear cell carcinoma, squamous cell carcinoma, staphyloid sarcoma, and embryonal rhabdomyosarcoma; and fallopian tube cancers, e.g., carcinoma; hematological cancers, e.g., blood cancers, e.g., acute This includes myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, and myelodysplastic syndromes, Hodgkin lymphoma, non-Hodgkin lymphoma (malignant lymphoma), and Waldenström macroglobulinemia; skin cancers, such as malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, lentigo dysplastic nevi, lipoma, hemangioma, dermatofibroma, keloid, and psoriasis; breast cancers, such as ductal carcinoma, lobular carcinoma, inflammatory breast cancer, medullary carcinoma, mucinous (gelatinous) carcinoma, Paget's disease of the breast, tubular carcinoma, phyllodes tumor, metaplastic carcinoma, sarcoma, microcapillary carcinoma, and adenoid cystic carcinoma; and adrenal cancers, such as neuroblastoma.

[0045] Cancer can be a solid tumor, which may or may not be metastatic. Cancer can also originate as diffuse tissue, such as leukemia.

[0046] CD36 inhibitors, PPARβ inhibitors, or additional therapeutic agents may be administered to the patient either before or after the onset of symptoms associated with the disease or condition. Furthermore, several divided and staggered doses may be administered daily or sequentially, or the dose may be administered by continuous infusion or bolus injection. In addition, the dosage of the therapeutic formulation may be increased or decreased proportionally, as indicated by the urgency of the treatment or prevention situation.

[0047] Administration to the subject can be carried out using known procedures in a dosage and duration effective to treat the disease or condition in the patient. The effective amount of the therapeutic compound required to achieve a therapeutic effect may vary depending on factors such as the activity of the particular compound used; the time of administration; the elimination rate of the compound; the duration of treatment; other drugs, compounds, or materials used in combination with the compound; the state of the disease or disorder of the patient being treated, age, sex, weight, condition, overall health, and medical history, as well as similar factors well known in the medical field. The dosage plan can be adjusted to produce an optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the urgency of the treatment situation. A non-limiting example of the effective dose range of the therapeutic compound of the present invention is about 0.01 to 50 mg / kg body weight / day. Those skilled in the art will be able to consider the relevant factors and make a determination regarding the effective amount of the therapeutic compound without excessive experimentation.

[0048] Administration can be carried out several times a day, or less frequently, for example, once a day, once a week, once every two weeks, once a month, or even less frequently, for example, once every few months, or even once a year or less. The amount of the compound administered daily is understood to be daily, every other day, every two days, every three days, every four days, or every five days, in non-limiting examples. For example, in the case of every-other-day administration, a dose of 5 mg per day may be started on Monday, the first subsequent dose of 5 mg per day may be administered on Wednesday, and the second subsequent dose of 5 mg per day may be administered on Friday, and so on. The frequency of administration will depend on a number of factors, which will be readily apparent to those skilled in the art, including but not limited to the type and severity of the disease being treated, the type and age of the animal, etc.

[0049] Routes of administration may include inhalation, oral, nasal, rectal, parenteral, sublingual, percutaneous, transmucosal (e.g., sublingual, tongue, (trans) buccal, (trans) urethral, ​​vagina (e.g., transvaginal and perival), nasal (intra) cavity, and (trans) rectal), intravesical, intrapulmonary, intraduodenal, intragastric, subarachnoid, subcutaneous, intramuscular, intradermal, intracranial, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

[0050] As used herein, “parenteral administration” includes any route of administration characterized by the physical penetration of the target tissue and the administration of the pharmaceutical composition through an opening within the tissue. Therefore, parenteral administration includes, but is not limited to, the administration of the pharmaceutical composition by injection, application of the composition through surgical incision, application of the composition through a non-surgical wound penetrating the tissue, etc. In particular, parenteral administration may also include, but is not limited to, intracranial injection, subcutaneous injection, intravenous injection, intraperitoneal injection, intramuscular injection, intrasternal injection, and renal dialysis infusion techniques.

[0051] CD36 inhibitors, PPARβ inhibitors, or additional therapeutic agents may be provided in a variety of suitable compositions and dosage forms, including, for example, tablets, capsules, caplets, pills, gel capsules, lozenges, dispersants, suspensions, liquids, syrups, granules, beads, transdermal patches, gels, powders, pellets, magma preparations, licks, creams, pastes, ointments, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosol preparations for inhalation, intravesical compositions and formulations, etc. It should be understood that formulations and compositions that would be useful in the present invention are not limited to the specific formulations and compositions described herein. For oral administration, tablets, sugar-coated tablets, liquids, drops, suppositories, or capsules, caplets, and gel capsules are particularly suitable. Other formulations suitable for oral administration include, but are not limited to, powders or granules, aqueous or oily suspensions, aqueous or oily liquids, pastes, gels, toothpastes, mouthwashes, coatings, oral rinses, or emulsions.

[0052] III. definition For the purpose of aiding in the understanding of the detailed descriptions of compositions and methods provided herein, and to facilitate the explicit disclosure of various aspects of this disclosure, several clear definitions are provided. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs.

[0053] As used herein, "PPARβ" and "PPARδ" are interchangeable terms and refer to the same PPAR receptor.

[0054] As used herein, “subject” refers to mammals, including humans. Non-human animal subjects receiving diagnosis or treatment may include, for example, primates, cattle, goats, sheep, horses, dogs, cats, mice, rats, and the like.

[0055] As used herein, the term “treatment” refers to an intervention intended to prevent the onset of a disorder or to alter the pathology or symptoms of a disorder. Thus, “treatment” includes both therapeutic treatments and preventive or protective measures. Those requiring treatment include those who already have a disorder and those whose disorder should be prevented. In the treatment of tumors (e.g., cancer), the therapeutic agent may directly reduce the pathology of the tumor cells or make the tumor cells more sensitive to other therapeutic agents, such as radiation therapy and / or chemotherapy.

[0056] Therefore, “treating” may include suppressing, inhibiting, preventing, treating, or a combination thereof. “Treatment” particularly refers to extending the time to persistent progression, promoting remission, inducing remission, enhancing remission, accelerating recovery, increasing the effectiveness of alternative treatments or reducing resistance to alternative treatments, or a combination thereof. “Suppressing” or “inhibiting” particularly refers to delaying the onset of symptoms, preventing disease relapses, reducing the number or frequency of relapsed episodes, extending the incubation period between symptomatic episodes, reducing the severity of symptoms, reducing the severity of acute episodes, reducing the number of symptoms, reducing the incidence of disease-related symptoms, shortening the incubation period of symptoms, improving symptoms, reducing secondary symptoms, reducing secondary infections, extending patient survival, or a combination thereof.

[0057] As used herein, the terms “modulate” or “to modulate” mean to increase, enhance, boost, increase, stimulate (acting as an agonist), promote, decrease, reduce, inhibit, block, or weaken (acting as an antagonist) any of the activities mentioned. Modulation can increase an activity to more than 1, 2, 3, 5, 10, 100 times, etc., compared to the baseline value. Modulation can also reduce an activity to below the baseline value.

[0058] Terms such as "prevent," "prevention," "prevention," and "preventive measures" refer to reducing the likelihood of developing a disability or condition in individuals who do not currently have the disability or condition but are at risk of developing it or are prone to developing it.

[0059] As used herein, the term “disease” is intended to be generally synonymous with and interchangeable with the terms “disorder” and “condition” (as in a medical condition). This is because all these terms reflect an abnormal condition of one of the bodies or parts of a human or animal that impairs normal function, typically manifests as characteristic signs and symptoms, and reduces the lifespan or quality of life of the human or animal.

[0060] The terms “decrease,” “decrease,” “decrease,” or “inhibit” are all used herein to generally mean a statistically significant decrease. However, to avoid misunderstanding, “decrease,” “decrease,” or “inhibit” means a decrease of at least 10% compared to a reference level, e.g., a decrease of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to a reference level, or a decrease of up to 100% including 100% (e.g., no level compared to a reference sample), or any decrease between 10% and 100%.

[0061] The terms “increased,” “increased,” “enhanced,” or “activated” are all used herein to generally mean an increase of a statistically significant amount; to avoid any misunderstanding, the terms “increased,” “increased,” or “enhanced,” or “activated” mean an increase of at least 10% compared to a reference level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or an increase of up to 100%, including 100%, or any increase between 10% and 100%, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or any increase between 2 times and 10 times or more compared to a reference level.

[0062] The terms “effective dose,” “effective dosage,” or “effective dosage” are defined as the amount sufficient to achieve, or at least partially achieve, the desired effect. The “therapeutic effective dose” or “therapeutic effective dosage” of a drug or therapeutic agent is any amount of the drug, when used alone or in combination with another therapeutic agent, that promotes disease regression, as demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of functional or physical impairment due to the disease. The “preventive effective dose” or “preventive effective dosage” of a drug is the amount of the drug, when administered alone or in combination with another therapeutic agent, that inhibits the onset or recurrence of the disease in a subject at risk of developing or relapsing the disease. The ability of a therapeutic or preventive agent to promote disease regression or inhibit the onset or recurrence of the disease can be evaluated using various methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems to predict human effects, or by assaying the activity of the drug in in vitro assays.

[0063] Dosage is often expressed in relation to body weight. Therefore, a dose expressed as [g, mg, or other units] / kg (or g, mg, etc.) usually refers to [g, mg, or other units] "per 1 kg (or g, mg, etc.) of body weight," even if the term "body weight" is not explicitly mentioned.

[0064] The term “drug” is used to refer to chemical compounds, mixtures of chemical compounds, biomacromolecules (nucleic acids, antibodies, proteins, or parts thereof, such as peptides), or extracts made from biomaterials such as cells or tissues of bacteria, plants, fungi, or animals (especially mammals). Such drugs may, by their activity, be suitable as “therapeutic agents”—substances that are biologically, physiologically, or pharmacologically active in a target, either locally or systemically.

[0065] The terms “therapeutic agent,” “therapeutic drug,” or “treatment agent” are used interchangeably and refer to molecules or compounds that, when administered to a subject, impart some beneficial effect. Beneficial effects include enabling a diagnostic determination; improving a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder, or condition; and generally counteracting a disease, symptom, disorder, or pathological condition.

[0066] As used herein, “therapeutic dose” or “effective dose” refers to a non-toxic but sufficient amount of a drug to produce a desired biological outcome. This outcome may be a reduction and / or mitigation of signs, symptoms, or causes of a disease or disorder, or any other desired change in the biological system. The appropriate therapeutic dose in any individual case can be determined by those skilled in the art using routine experiments.

[0067] Where used herein, “combination” therapy means, unless otherwise evident from the context, the administration of two or more therapeutic agents in a coordinated manner, including but not limited to concurrent administration. Specifically, combination therapy includes both concurrent administration (e.g., administration of a combination or concurrent administration of separate therapeutic compositions) and sequential or sequential administration, insofar as the administration of one therapeutic agent is in some way conditioned on the administration of the other therapeutic agent. For example, one therapeutic agent may be administered only after a different therapeutic agent has been administered and acted for a specified period of time. See, for example, Kohrt et al. (2011) Blood 117:2423.

[0068] As used herein, the term “depletion” means reducing or eliminating the function of a given cell type, inactivating a cell, partially or completely eliminating cell proliferation, and / or killing a cell.

[0069] As used herein, the term “antibody” (Ab) includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies and multireactive antibodies), and antibody fragments. Therefore, as used in any context herein, the term “antibody” means including, but not limited to, any specific binding member, a class and / or isotype of immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM); and biologically relevant fragments or their specific binding members, including but not limited to Fab, F(ab')2, Fv, and scFv (single-chain or related entities). It is understood in the art that an antibody is a glycoprotein or its antigen-binding moiety having at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH1, CH2, and CH3). The light chain consists of a light chain variable region (VL) and a light chain constant region (CL). Both the heavy and light chain variable regions contain a framework region (FWR) and a complementarity-determining region (CDR). While the four FWR regions are relatively conserved, the CDR regions (CDR1, CDR2, and CDR3) represent hypervariable regions, arranged from the NH2 terminus to the COOH terminus as follows: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, and FWR4. The heavy and light chain variable regions contain binding domains that interact with antigens, but depending on the isotype, the constant region may mediate the binding of immunoglobulins to host tissues or factors. As used herein, the definition of “antibody” also includes chimeric antibodies, humanized antibodies, and recombinant antibodies, human antibodies produced from transgenic non-human animals, and antibodies selected from libraries using enrichment techniques available to those skilled in the art.

[0070] As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, or in a cell culture, rather than within a multicellular organism.

[0071] As used herein, the term "in vivo" refers to events occurring within multicellular organisms, such as non-human animals.

[0072] It should be noted herein that, as used herein and in the appended claims, the singular forms “one (a),” “one (an),” or “it” include multiple subjects unless the context clearly indicates otherwise.

[0073] The terms “including,” “comprising,” “containing,” and “having,” and their variations, unless otherwise specified, mean to include the items listed thereafter, their equivalents, and any additional matters.

[0074] Phrases such as "in one aspect," "in various aspects," and "in several aspects" are used repeatedly. While such phrases do not necessarily refer to the same aspect, they may refer to the same aspect unless the context indicates otherwise.

[0075] The terms "and / or" or " / " mean any one of the items related to this term, any combination of items, or all of the items.

[0076] The word "substantially" does not exclude "completely"; for example, a composition that "substantially does not contain" Y does not have to contain Y completely. If necessary, the word "substantially" may be omitted from the definition of this invention.

[0077] As used herein, the term “each” is intended to identify individual items within a collection when used in relation to an item collection, but does not necessarily refer to every item within the collection. Exceptions may also be made where explicit disclosure or context clearly indicates otherwise.

[0078] The use of any examples or illustrative language provided herein (e.g., "etc.") is intended solely to better illustrate the invention and, unless otherwise claimed, does not imply any limitation to the scope of the invention. No language herein should be construed as indicating any unclaimed element as essential to the practice of the invention.

[0079] All methods described herein are to be performed in any appropriate order, unless otherwise indicated herein or unless it is clearly inconsistent with the context. With respect to any of the methods provided, the steps of the method may be performed simultaneously or sequentially. If the steps of the method are performed sequentially, the steps may be performed in any order, unless otherwise specified.

[0080] If a method involves a combination of steps, any combination or subcombination of steps is included within the scope of this disclosure unless otherwise specified herein.

[0081] Each publication, patent application, patent, and other reference cited herein is incorporated by reference in whole to the extent that it does not contradict this disclosure. Publications disclosed herein are provided solely because they were disclosed prior to the filing date of the present invention. Nothing herein should be construed as admitting that the present invention is not entitled to precede such publications for the purposes of prior art. Furthermore, the dates of the publications provided may differ from the actual publication dates and may need to be verified individually.

[0082] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes taking them into consideration should be suggested to those skilled in the art and should be included in the spirit and scope of this application and the appended claims. [Examples]

[0083] IV. Examples Example 1 This embodiment describes the materials and methods used in subsequent embodiments.

[0084] mouse C57BL / 6 / J, FoxP3 YFP-Cre Rag1 - / - (B6.129S7-Rag1 tm1Mom The mice were purchased from Jackson Laboratories. CD36fl / fl mice were prepared as previously described (Son, NH et al. J Clin Invest 128, 4329-4342 (2018)). PPARγ fl / fl and PPARβ fl / fl The mice were prepared as described in Dammone, G. et al. (International Journal of Molecular Sciences 19 (2018)). BRafCA; Tyr::CreER; Ptenlox4-5 (Braf / Pten) is described in Dankort et al. (Nature Genetics volume 41, pages 544-552 (2009)). K-ras of NSCLC LSL-G12D / + / p53 fl / fl The conditional mouse model is described by DuPage et al. (DuPage et al. Nature Protocols volume 4, pages 1064-1072 (2009)). The animals were housed in a pathogen-free facility at the University of Lausanne, and all experimental studies were approved and conducted in accordance with the guidelines and regulations enforced by the Swiss Animal Welfare Ordinance.

[0085] Cell lines and in vitro cultures The YUMM1.7 melanoma cell line is described in Meeth, K., et al. (Meeth, K., et al. Pigment cell & melanoma research 29, 590-597, (2016)). The YUMM1.7 and B16-ova melanoma cell lines were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin and used in experiments when in the logarithmic growth phase. The MC38 colon adenocarcinoma cell line is described in Hoves et al. (Hoves et al. Journal of Experimental Medicine, 215 (3) 859-876 (2018)). The MC38 cell line was maintained in IMDM containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0086] Culture of cancer cell-conditioned medium and iTregs iTregs were generated by activating naive CD4 + T cells for 3 days using anti-CD3 mAb and anti-CD28 mAb-conjugated Dynabeads (ThermoFisher) in RPMI medium supplemented with 10% FBS, 10 ng / ml TGFβ, and 50 U / ml IL-2. Then, the activated CD4 +T cells were maintained for an additional 2 days in RPMI medium + 10% FBS and 50 U / ml IL-2. Differentiated iTregs were first sorted using a FACS cell sorter and then incubated for 48 hours under the indicated culture conditions. Subsequently, the viability and NAD / NADH levels of the iTregs were determined by live / dead staining and ELISA kits, respectively. For NAD / NADH measurement of CD36-KO iTregs treated with a PPARβ agonist, sorted iTregs were cultured for 48 hours in cancer cell conditioned medium in the presence of DMSO or GW50156. Control RPMI for iTreg in vitro treatment was prepared by supplementing RPMI1640 medium (Biological Industries) with 2 mM glucose, 10 mM glutamine, 10% dialyzed FBS, 0.1% β-ME, and indicated levels of lactate. YUMM1.7 cancer cell conditioned medium was collected by incubating YUMM1.7 cells (70-80% density) with the control RPMI described above for 18 hours. The culture medium was then collected and centrifuged at 2000 rpm for 15 minutes to remove debris and cancer cells, and used as cancer cell conditioned medium. The YUMM1.7 cancer cell conditioned medium collected as described above was treated with Cleanascite® reagent (Biotech Support Group) in a 1:5 volume ratio according to the manufacturer's instructions before Treg culture.

[0087] Ex vivo suppression assay CD8 T cells derived from the spleen of Ly5.1 mice were enriched using a negative selection kit (MojoSort mouse CD8 T cell isolation kit, Biolegend) and stained with CellTrace® CFSE cell proliferation kit (ThermoFisher) at 37°C for 15 minutes. 1 × 10⁻⁶ 4 Individual CD8 cells were seeded in RPMI medium containing 50 U / ml IL-2 in a 96-well round plate. FoxP3 YFP-Cre Mouse or Treg CD36- / - CD44 isolated from mouse splenocytes or TILs + / YFP + Treg (CD45.2 +) was added according to the Treg:Teff ratio. Then, after removing the negative control group, anti-CD3 / CD28-conjugated Dynabeads (ThermoFisher) were added to the culture. The cells were incubated at 37°C and 5% CO2 for 72 hours, and then CD8 + T cell proliferation was determined by CFSE dilution using flow cytometry analysis.

[0088] Tumor transplantation and treatment of tumor-carrying mice For tumor induction, the skin surface of 3-week-old Braf / Pten mice was treated with 4-hydroxytamoxifen as previously described to induce tumor formation (Ho, PC et al. Cancer Res 74, 3205-3217 (2014)). For tumor transplantation, 5 × 10 4 Individual YUMM1.7 tumor cells, B16-OVA tumor cells, or 1 million MC38 tumor cells were subcutaneously injected in 50 μl PBS. Tumors were measured and calculated every 2-3 days after tumor transplantation or processing. For transplanted tumors, volume = (length × width) 2 ) / 2, or in the case of induced tumors, volume was calculated as (length × width × height). For in vivo treatment, Yumm1.7-carrying mice were administered either DMSO or a PPARβ agonist (GW 501516) (1 mg per kg of body weight, Cayman Chemical) by intraperitoneal injection every 3 days. For antibody-based treatment, tumor-bearing mice were treated by intraperitoneal injection with anti-PD-1 antibody (200 μg per injection, BioXcell, clone 29F.1A12) and anti-CD36 antibody (200 μg per injection, clone CRF D-2712 (Driscoll, WS, et al. Circulation research 113, 52-61 (2013)) according to the indicated combination. For antibody treatment in the Braf / Pten mouse model, tumor-bearing Braf / Pten mice were treated with anti-CD36 antibody and / or anti-PD-1 antibody for 10 days four weeks after tumor induction, as described above. All experiments were conducted in accordance with Swiss Federal Regulations.

[0089] Tumor digestion and cell isolation The tumor was fragmented into small pieces in RPMI containing 2% FBS, 1% penicillin-streptomycin (p / s), DNase I (1 μg / ml, Sigma-Aldrich), and collagenase (0.5 mg / ml, Sigma-Aldrich), maintained at 37°C for 40 minutes for digestion, and then filtered through a 70 μm cell strainer. The filtered cells were incubated with ACK lysis buffer (Invitrogen) to lyse erythrocytes, and then washed with fluorescence-activated cell sorter (FACS) buffer (phosphate-buffered saline containing 2% fetal bovine serum and 2 mM EDTA). Tumor-infiltrating leukocytes were further concentrated by Percoll density gradient centrifugation (800 × g, 30 min) at room temperature, as previously described (Cheng, WC et al. Nat Immunol 20, 206-217 (2019)).

[0090] Flow cytometry, cell sorting, and antibodies Single-cell suspensions were incubated on ice for 10 minutes with Fc receptor-blocking anti-CD16 / 32 (93) antibody and anti-CD351 (TX61) antibody (Biolegend) before staining. The cell suspensions were first stained at 37°C for 10 minutes using the LIVE / DEAD® fixable violet dead cell staining kit (ThermoFisher). After washing, surface proteins were stained at 4°C for 30 minutes. To detect cytokine production upon ex vivo restimulation, the cell suspensions were resuspended in RPMI 1640 containing 10% FBS and then added to plates coated with 1 μg / ml anti-CD3 antibody (clone 145-2C11, Biolegend) and anti-CD28 antibody (clone 37.51, Biolegend), and further incubated at 37°C for 5 hours in the presence of 2.5 μg / ml brefelzin A solution (BFA) (Biolegend). Cells were processed and stained with surface markers as described above, followed by intracellular cytokine staining. Samples were analyzed using an LSRII flow cytometer (BD Biosciences), and data were analyzed using FlowJo. Cells were sorted using either a FACSAria® III sorter (BD Biosciences) or an SH800S cell sorter (Sony). The following antibodies against mouse proteins were used: anti-CD45 (30-F11), anti-CD3ε (17A2), anti-CD4 (RM4-5), anti-CD8a (53.6.7), anti-CD44 (IM7), anti-62L (Mel-14), anti-PD1 (RMP1-30), anti-CD134 (OX40) (OX-86), anti-CD357 (GITR). (DTA-1), anti-CD36 (CRF D-2712), anti-IgA (mA-6E1), anti-FoxP3 (MF-14), anti-IFN-γ (XMG1.2), anti-TNF-α (MP6-XT22), anti-IL17A (TC11-18H10.1), anti-Ki67 (16A8), anti-CD278 (ICOS) (15F9), anti-CD152 (CTLA4) (UC10-4B9), cleavage caspase-3 (Asp175). These antibodies were purchased from Biolegend, eBiosciences, and Cell Signaling.

[0091] Mitochondrial assays, fatty acid uptake, and lipid content To measure mitochondrial membrane potential, cells were washed and incubated for 15 minutes with pre-warmed (37°C) staining solutions (RPMI containing 2% FBS) containing MitoTracker® Deep Red FM (ThermoFisher) and MitoTracker® Green FM (ThermoFisher) at working concentrations of 10 nm and 100 nM, respectively. After staining, cells were washed, resuspended in fresh FACS buffer, and surface marker staining was performed as described above. To measure fatty acid uptake, cells were incubated for 15 minutes at 37°C in RPMI medium (or human T cell culture medium) containing C1-BODIPY® 500 / 510 C12 (Life Technologies) at a final concentration of 0.5 μM. After incubation, cells were washed with FACS buffer and surface staining was performed. For the detection of lipid content, cells were permeabilized and immobilized, and then stained together with other intracellular proteins using BODIPY® 493 / 503 (Life Technologies) at a final concentration of 500 ng / ml.

[0092] RNA sequencing and bioinformatics analysis FoxP3 YFP-Cre Mouse or Treg CD36- / - 500-600 viable CD4 cells from mice + / CD44 + / YFP +Intratumor Tregs were directly isolated by FACS cell sorting into 4 μl lysis buffer consisting of 0.2% (vol / vol) Triton X-100 solution (MgBCH-Axon Lab) and an RNase inhibitor (Clontech) (to at least 99% purity). Plates containing the samples were sealed, flash-frozen, and maintained at -80°C, after which they were further processed according to a previously described version of the Smart-Seq2 protocol (Picelli, S. et al. Nature protocols 9, 171-181 (2014)). RNA sequencing raw data were processed using a standard RNA-seq analysis pipeline. Briefly, read alignment was examined using tophat2 v2.1.0 and then compared to the mouse (Mus musculus) GRCm38.p4 genome version. After alignment, reads mapped to each gene were annotated using HTseq counts. Differential expression analysis was performed using the DESeq2 R library. Subsequently, differential expression testing and visualization were investigated using START Web-based RNA-seq analysis resources (Nelson, JW, et al. Bioinformatics 33, 447-449 (2017)). Gene set enrichment analysis (GSEA) was performed using GSEA software.

[0093] Electron microscopy analysis and histological analysis For electron microscopy analysis, selected cells were fixed overnight at 4°C in 2.5% glutaraldehyde (EMS) and 1% osmium tetroxide (EMS), washed several times with water and acetone (Sigma) the following day, and embedded in Epon (Sigma) resin. Prior to imaging, 50 nm slides were prepared using a Leica UltraCut microtome and contrasted with uranyl acetate (Sigma) and Reynolds lead citrate (Sigma). Electron microscopy images were acquired at 4800x and 11000x magnification using a TVIPS TemCam-F416 digital camera with 80 kV acceleration using a Philips CM100 transmission electron microscope. Image analysis and quantification were performed using EMMENU, 3dmod (University of Colorado), and Fifi (ImageJ) software. To quantify the number of mitochondria per selected cell, a grid was applied, defining each intersection as part of the nucleus, cytoplasm, or mitochondria. Cristae density was determined by dividing the length of each cristae by the mitochondrial area. For histological analysis, organs were excised, placed in labeled cassettes, fixed in formalin for 24 hours, and then embedded in molten paraffin wax. Paraffin sections 3–5 μm thick were stained with hematoxylin and eosin according to standard procedures. Images were captured and exported using a Nikon Eclipse Ti-S inverted microscope.

[0094] Evaluation of human patients This study was conducted under approved protocols and in accordance with ethical guidelines for human samples. Human samples were analyzed in accordance with safety regulations and stained with the following antibodies for FACS analysis: anti-CD45 (2D1), anti-CD3 (SK7), anti-CD4 (SK3), anti-CD25 (BC96), anti-CD8 (RPA-TP), anti-CD36 (TR9), anti-PD1 (E12.1), and anti-FoxP3 (150D).

[0095] T-cell transplantation model for colitis FoxP3 YFP-Cre Mouse or TregCD36- / - WT Treg and CD36-KO Treg were sorted from either mouse spleen, and naive CD4 T cells were sorted using a combination of negative magnetic selection (MojoSort mouse CD4 T cell isolation kit, Biolegend) and FACS sorting. + T cells were collected (purity >98%). Naive CD4 cells were used to induce colitis. + cells (5×10 5 (individual cells) Rag1 - / - The transplant was administered intravenously to the recipient. In some recipients, FoxP3 YFP-Cre Mouse or Treg CD36- / - 4 × 10¹⁶ cells isolated from mouse splenocytes 5 individual CD44 + / YFP + Treg naive CD4 + T cells were transplanted simultaneously. Recipient mice were monitored every 2 or 3 days post-transplant for signs of disease, such as weight loss, and their weight was measured. Disease onset typically occurs 4-5 weeks post-transplant. Endpoints in this study included weight loss, duration of colitis, and assessment of diarrhea. In addition, the colon and small intestine were harvested, processed, and further evaluated by hematoxylin and eosin staining.

[0096] Measurement of NAD and NADH The ratio of nicotinamide adenine dinucleotide (NAD) to nicotinamide adenine dinucleotide hydrate (NADH) was measured using a commercially available NAD / NADH quantification kit (Sigma-Aldrich MAK037). To prevent enzymatic consumption of NAD and NADH, the cells were first deproteinized. After washing with cold PBS, the cell pellet suspended in NAD / NADH extraction buffer (200 μl) was treated with two repeated freeze-thaw cycles, and then centrifuged at 13000 × g at 4°C for 5 minutes. The supernatant was then divided into two equal volumes, one of which was NAD 総量 One sample was used for detection, and the other was heated at 60°C for 30 minutes for NAD degradation. The sample was then transferred to a 96-well plate and the absorbance at 450 nm was measured. Oxidized NAD (NAD + The amount of NAD総量 The NAD / NADH ratio in the sample was calculated by subtracting NADH from the NAD ratio. The NAD / NADH ratio in the sample could be determined by the following formula: Ratio = (NAD 総量 -NADH) / NADH.

[0097] Seahorse extracellular flux analysis Extracellular flux analysis was performed using the XF96 Seahorse extracellular flux analyzer, with minor modifications, as previously described (Liu, PS et al. Nat Immunol 18, 985-994 (2017)). Cells were treated with oligomycin (0.5 μM, Sigma-Aldrich), FCCP (2 μM, Sigma-Aldrich), rotenone (0.5 μM, Sigma-Aldrich), antimycin A (0.5 μM, Sigma-Aldrich), glucose (10 mM, Sigma-Aldrich), and 2-DG (50 mM, Sigma-Aldrich). Each condition was repeated 3–6 times in a single experiment.

[0098] statistical analysis Statistical analysis was performed using a two-tailed independent Student's t-test. The log-rank (Mantel-Cox) test was used for survival curve analysis. Each point represents a biological replica, and all data are presented as mean ± SD or mean ± SEM as indicated. P-values ​​are shown as follows: *** P<0.001, ** P<0.01, and * P<0.05. P<0.05 was considered statistically significant.

[0099] Data availability RNA-seq data for intratumor Tregs are available in the Gene Expression Omnibus database.

[0100] Example 2 Intratumor Tregs increased lipid metabolism and CD36 expression. To determine whether intratumor Tregs preferentially participate in specific metabolic pathways, we first analyzed RNA sequencing results from intratumor and circulating Tregs obtained from breast cancer patients in previously published studies (Plitas, G. et al. Immunity 45, 1122-1134). Gene pathway analysis, particularly focusing on metabolic pathways, revealed that intratumor Tregs highly expressed metabolic genes involved in lipid metabolism compared to circulating Tregs (Figures 1a and 1b), suggesting that intratumor Tregs may enhance lipid metabolism. Indeed, a comparison of peripheral blood mononuclear cells (PBMCs) from non-small cell lung cancer (NSCLC) patients with intratumor Tregs showed that intratumor Tregs took up more Bodipy FL C12, a green fluorescent fatty acid, and contained more neutral lipids based on Bodipy staining. To further explore these phenotypes, we evaluated the lipid metabolism of Tregs present in tumors and other peripheral tissues using a melanoma cell transplantation model. The results showed that intratumor Tregs exhibited a higher capacity for fatty acid uptake (Figure 1c) and higher lipid content (Figure 1c) compared to Tregs from other tissues of YUMM1.7 melanoma-carrying mice. Similarly, intratumor Tregs from B16 melanoma-carrying mice also showed enhanced fatty acid uptake. These findings suggest that the increased lipid metabolism by intratumor Tregs is a conserved phenotype in both human and mouse models. Notably, among the genes that regulate lipid uptake, CD36, a scavenger receptor involved in the uptake of long-chain fatty acids and oxidized low-density lipoproteins, was significantly upregulated in intratumor Tregs compared to circulating Tregs from breast cancer patients (Plitas, G. et al. Immunity 45, 1122-1134). Examination of Tregs in PBMCs and tumor-infiltrating lymph nodes (TILNs) from melanoma patients confirmed that the majority of patient-derived intratumor Tregs expressed high levels of CD36 (Figure 1e). In addition, intratumoral Tregs from Yumm1.7 melanoma-carrying mice expressed high levels of CD36, while Tregs present in other peripheral tissues or secondary lymphoid organs did not (Figure 1f).Notably, increased CD36 expression observed in intratumor Tregs was also seen in B16 melanoma models, genetically engineered Braf / PTEN melanoma mouse models, and K-ras in NSCLCs. LSL-G12D / + / p53 fl / fl This was also observed in a conditional mouse model. Furthermore, when inducible Tregs (iTregs) were cultured in a conditioned medium derived from cancer cell cultures, CD36 expression dramatically increased, whereas hypoxia and lactate could not induce CD36 expression in Tregs. Notably, the effect of the cancer cell conditioned medium on stimulating CD36 expression in Tregs disappeared when lipids were removed. In summary, these results suggest that TME can stimulate CD36 expression in Tregs, which may support the metabolic adaptation requirements of intratumor Tregs.

[0101] Example 3 CD36 controls the accumulation and suppression of intratumor Treg cells. To investigate whether CD36 expression modulates the behavior of Tregs in tumors, we investigated CD36 fl / fl Mouse and Foxp3 YFP-Cre By crossbreeding mice, Treg-specific CD36-deficient mice (Treg CD36- / - We created (named [Treg]). Considering that genetic removal of important regulatory factors in Tregs can lead to impaired Treg suppression function, resulting in systemic activation of T lymphocytes and autoimmunity, we first investigated whether CD36 deficiency in Tregs affects immune homeostasis. Aged Tregs CD36- / - Mice (21-23 weeks old) are all Foxp3, both male and female. YFP-Cre It was found that the mice (referred to as wild-type mice throughout this study) exhibited a body weight comparable to that of mice. CD36- / - The mouse also has a CD4 compared to the WT mouse. + and CD8 + Similar proportions of effectors or memory populations (CD44) in both T cell compartments hi CD62L lo ) included. Furthermore, Treg CD36- / -The mice did not show abnormal infiltration of lymphocytes and myeloid cells in various organs, nor severe systemic inflammatory damage (Figure 2a), suggesting that CD36 is not necessary for Tregs to maintain immune homeostasis.

[0102] Next, YUMM1.7 melanoma cells were transferred to WT mice and Treg CD36- / - The cells were transplanted into mice. Genetic removal of CD36 in Tregs dramatically reduced lipid uptake and content in intratumoral Tregs, but not in splenic Tregs (Figures 2b and 2c), indicating that intratumoral Tregs support enhanced lipid uptake in a CD36 expression-dependent manner. CD36- / - Slowing of growth of transplanted YUMM1.7 melanoma (Figures 2d and 2e), B16 melanoma, and MC38 colon cancer was also observed in mice. Furthermore, Treg CD36- / - In mice, intratumor Tregs were significantly reduced at the end of the analysis, but Tregs in the spleen and inflow lymph nodes were not reduced in the same way (Figure 2f). This is a favorable parameter associated with a strong antitumor response, CD8 + TIL frequency and CD8 + This was accompanied by a significant increase in the ratio of TIL to Treg TIL. In addition, Treg CD36- / - High frequency CD8 in mice + TIL and CD4 + / FoxP3 - TILs produce antitumor effector cytokines including interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα) (Figure 2g), and therefore, Treg CD36- / - The results suggest that TME in mice causes less immunosuppression.

[0103] To further investigate the dependence on CD36 in supporting intratumor Treg accumulation, heterozygous FoxP3 possesses both a WT Treg population and a CD36 knockout Treg population driven by FoxP3 expression, mediated by X chromosome inactivation. YFP-Cre / + / CD36 fl / flFemale mice were generated. WT Tregs and CD36-deficient Tregs can be detected based on the expression of yellow fluorescent protein (YFP). To eliminate potential toxicity induced by Cre recombinase, heterozygous Foxp3 mice were used as control mice. YFP-Cre / + Female mice were also created. Foxp3 mice carrying tumors YFP-Cre / + / CD36 fl / fl Female mice and Foxp3 YFP-Cre / + FoxP3 in both female mice + YFP within Treg + When comparing populations, CD36-deficient Tregs (Foxp3) were found to be present in tumors. YFP-Cre / + / CD36 fl / fl Cre in mice + Only a decrease in the frequency of the population was detected, while Cre in both the spleen and the lymph nodes in the drainage region was detected. + Treg and Cre - The frequency ratio with Treg is Foxp3 YFP-Cre / + / CD36 fl / fl Mouse and Foxp3 YFP-Cre / + The results were comparable to those observed in mice. These findings suggest that the absence of CD36 expression selectively inhibits the accumulation of tumor Tregs through endogenous regulation. In summary, these results highlight the important role of CD36 in selectively conferring the ability of Tregs to accumulate in TMEs.

[0104] Example 4 CD36 was not required in Tregs to maintain peripheral homeostasis. Interestingly, in a mouse melanoma model, the effector Treg (CD44) within the tumor... hi / CD62L lo ) is CD44 in the tumor lo It was also found that they expressed higher levels of CD36 compared to Tregs (dormant Tregs). Similarly, tumor-invasive GITRs, which are the most suppressive subset of effector Tregs derived from TILs in melanoma patients. + / CD25 + Effector Treg is used in GITR in PBMCs derived from melanoma patients and healthy donors.+ / CD25 + Compared with effector Tregs, a higher percentage expressed CD36. The expression of immune regulatory receptors in intratumoral Tregs was also examined. CD36-KO Tregs showed a decreased expression of glucocorticoid-induced TNFR-related protein (GITR) and OX40 compared with WT Tregs, but did not show a decrease in the expression of programmed cell death protein 1 (PD-1), CD25, cytotoxic T lymphocyte-associated protein 4 (CTLA-4), or inducible T cell co-stimulator (ICOS). These results suggest that CD36 expression contributes to the suppressive function of effector Tregs. To support this idea, Tregs CD36- / - Intratumoral Tregs derived from mice showed a reduced suppressive ability compared with WT intratumoral Tregs in an ex vivo suppression assay (Figure 2h). However, WT and CD36-deficient splenic Tregs showed comparable suppressive abilities (Figure 2i), suggesting that CD36 is only required to support the suppressive activity of intratumoral Tregs and not for supporting the suppressive activity of splenic Tregs.

[0105] To further investigate whether CD36 is required for Tregs to suppress peripheral inflammation, the ability of CD36-deficient Tregs to suppress T cell transplantation-induced colitis was analyzed. Naive CD4 + At 2 weeks after naive CD4 T cell transplantation, disease onset such as weight loss was detected. However, co-transplantation of CD36-KO Tregs was able to improve the weight loss of recipient mice as well as WT Tregs (Figure 2j). Furthermore, several organs were removed and processed at the end of the study for further evaluation by histological examination. Naive CD4 +Compared to T cell transplantation, co-transplantation of either WT Tregs or CD36-KO Tregs inhibited lymphocyte and myeloid cell infiltration, as well as morphological changes in the colon and small intestine, colonic shortening, and splenic hypertrophy in recipient mice. Furthermore, genetic removal of CD36 was found to have no effect on the expression of activation markers, including CD44, CD103, and KLRG1, or FoxP3 expression (indicated by the fluorescence intensity of YFP) in intratumor Tregs. However, CD36-deficient intratumor Tregs showed a slight enhancement in the production of the inflammatory cytokines IFNγ and TNF, suggesting that CD36 suppresses the ability of intratumor Tregs to produce inflammatory cytokines. In summary, these results suggest that CD36 expression specifically supports the suppressive function of intratumor Tregs.

[0106] Example 5 CD36 deficiency stimulated apoptosis in intratumoral Tregs. To investigate the underlying basis of the decreased cell solidity in CD36-deficient Tregs in TMEs, we first examined their proliferative capacity by staining for Ki67. We found that CD36 deficiency did not alter the proliferation of intratumor Tregs. Comparison of transcriptomes between wild-type intratumor Tregs and CD36-deficient intratumor Tregs showed that CD36-deficient Tregs exhibited increased expression of genes regulating apoptosis (Figure 3a). Indeed, higher levels of cleavage caspase-3 (Figure 3b) and annexin V staining (Figure 4h) were observed in CD36-deficient intratumor Tregs. Notably, except for a slight increase in aspiration lymph nodes, CD36 deficiency did not enhance cleavage caspase-3 levels in Tregs derived from the thymus and other secondary lymphoid organs, suggesting that intratumor Tregs require CD36-mediated regulation to prevent apoptosis.

[0107] Since mitochondrial metabolism and adaptability are suggested to regulate the repressive function and survival of Tregs (Yang, K. et al. Nature 548, 602-606 (2017); Weinberg, SE et al. Nature 565, 495-499 (2019); He, N. et al. PNAS 114, 12542-12547 (2017); Beier, UH et al. FASEB J 29, 2315-2326 (2015)), we next investigated whether CD36-deficient intratumoral Tregs could not maintain mitochondrial adaptability. Surprisingly, compared to WT Tregs, CD36-deficient intratumoral Tregs showed a decrease in mitochondrial membrane potential as measured by MitoTracker DeepRed staining, whereas Tregs in other tissues did not show such a decrease (Figure 3c). This finding suggests that Tregs CD36- / - This was further supported by electron microscopy analysis showing that mouse-derived tumor Tregs had a lower number of mitochondria (Figure 3d) and a lower number of cristae within each mitochondria (Figure 3e). Nevertheless, WT and CD36-deficient splenic Tregs showed comparable mitochondrial numbers and cristae densities. To further elucidate the effect of CD36 on mitochondrial metabolism in Tregs, we investigated CD4 in WT or CD36-deficient Tregs. +iTreg cells, generated from either type of T cell, were treated with cancer cell-conditioned medium to induce CD36 expression, as previously described. The Seahorse extracellular flux assay was then performed. As shown in Figure 3f, CD36-deficient Treg cells showed a decrease in oxygen consumption rate (OCR) while showing an increase in glycolysis rate. These results suggest that removal of CD36 impairs oxidative phosphorylation (OXPHOS), potentially shifting the metabolic priority of Treg cells towards aerobic glycolysis. These findings suggest that enhanced CD36 expression in intratumor Tregs may support the metabolic flexibility of Tregs by regulating mitochondrial adaptability in response to metabolic stress imposed by TMEs (Li, X. et al. Nat Rev Clin Oncol (2019); Ho, PC et al. Cell 162, 1217-1228 (2015); Siska, PJ & Rathmell, JC Trends Immunol 36, 257-264 (2015)).

[0108] We also investigated whether CD36-deficient Tregs exhibit impaired survival in response to metabolic load. In contrast to normal culture conditions (RMPI + 10% FBS; named RMPI), CD36-deficient Tregs exposed to cancer cell-conditioned medium showed reduced survival rates. Since lactate levels can worsen in cancer cell-conditioned medium, and lactate accumulation is a common feature of TMEs, it was hypothesized that CD36-deficient Tregs may not be able to maintain survival under these conditions due to high levels of lactate. Supporting this hypothesis, CD36-deficient Tregs were found to exhibit severe impaired survival in response to escalating lactate doses (Figure 3g). Consistent with these results, recent studies have suggested that increased electron transport chain activity leads to an increased NAD / NADH ratio in Tregs, supporting the conversion of lactate to pyruvate (Angelin, A. et al. Cell Metab 25, 1282-1293 e1287 (2017)), which may support Treg survival under lactate-enriched conditions. Therefore, it was hypothesized that CD36-deficient Tregs may have a lower NAD / NADH ratio compared to WT Tregs due to reduced mitochondrial adaptability and OXPHOS. Indeed, as shown in Figure 3h, CD36-deficient Tregs had a lower NAD / NADH ratio compared to WT Tregs, and supplementation with nicotinamide riboside (NR) to replenish NAD partially restored the survival rate of CD36-deficient Tregs exposed to cancer cell-conditioned medium (Figure 3i). Thus, the in vivo inability of CD36-deficient intratumoral Tregs may be due to reduced mitochondrial adaptability and OXPHOS, which allow Tregs to survive in lactate-enriched conditions via NAD-regulated metabolic processes.

[0109] Example 6 CD36-PPARβ signaling modulated metabolic adaptation in intratumor Tregs. To understand how CD36 stimulates mitochondrial fitness in intratumoral Tregs, we evaluated transcriptomic changes in intratumoral and circulating Tregs from breast cancer patients. As predicted, intratumoral Tregs upregulated genes that control mitochondrial function and biogenesis. Furthermore, intratumoral Tregs were found to show increased expression of genes involved in the PPAR signaling pathway (Figure 4a). Since CD36 has been suggested to support metabolic flexibility in metabolic tissues by enhancing PPARβ (also known as PPARβ) - dependent and PPARγ - dependent regulation of mitochondrial activity and biogenesis, CD36 - induced metabolic reprogramming may promote mitochondrial fitness in intratumoral Tregs by providing lipid signals and modulating PPAR transcriptional regulation. To test this idea, we crossed PPARβ fl / fl mice and PPARγ fl / fl mice with Foxp3 YFP-Cre mice to obtain Treg - specific PPARβ - deficient mice (named Treg PPARβ- / - for short) and PPARγ - deficient mice (named Treg PPARγ- / - for short), respectively. Genetic ablation of PPARγ in Tregs was observed not to impair the accumulation of intratumoral Tregs or the growth of YUMM1.7 melanoma. In contrast, Treg PPARβ- / - mice recapitulated the characteristic phenotypes of Treg + mice, including decreased accumulation of intratumoral Tregs (Figure 4b), decelerated growth of transplanted YUMM1.7 melanoma (Figure 4c and 4d), and increased CD8 CD36- / - TILs. Similar to CD36 - deficient intratumoral Tregs, PPARβ - deficient intratumoral Tregs showed a decrease in mitochondrial membrane potential compared to WT intratumoral Tregs (Figure 4e). Notably, PPARβ - deficient intratumoral Tregs expressed less CD36 compared to WT intratumoral Tregs. Considering that lipid removal abolished the ability of cancer - conditioned medium to induce CD36 expression in Tregs, these results further suggest that lipid - induced PPARβ signaling may contribute to CD36 induction in intratumoral Tregs.

[0110] To clarify the relationship between CD36 and PPARβ activation and their roles in supporting intratumoral Treg accumulation, we used WT mice transplanted with YUMM1.7 melanoma and Tregs. CD36- / - Mice were treated for two weeks with either a PPARβ-selective agonist (GW501516) or a control medium. As shown in Figures 4f and 4g, treatment with GW501516 resulted in Treg CD36- / - In mice, tumor growth and the abundance of intratumoral Tregs were restored (Figures 4f and 4g). In addition, Tregs treated with GW501516 were also restored. CD36- / - In mouse-derived intratumor Tregs, mitochondrial membrane potential was elevated and levels of cleaved caspase-3 were low (Figures 4h and 4i). In parallel, GW501516 treatment in CD36-deficient Tregs increased the NAD / NADH ratio, suggesting that CD36-regulated lipid uptake activates the PPARβ pathway, supporting enhanced mitochondrial adaptability and NAD / NADH ratio in intratumor Tregs. Furthermore, activation of the PPARβ pathway may further amplify CD36-mediated metabolic adaptation in intratumor Tregs by enhancing CD36 expression. In summary, these results indicate that CD36-PPARβ signaling modulates metabolic programs to support Treg persistence in TMEs.

[0111] Example 7 Targeting CD36 enhanced anti-tumor immunity by damaging intratumor Tregs. Next, we investigated whether it was possible to specifically inhibit intratumoral Tregs without systemic Treg elimination or overall impairment of Treg suppression function by blocking CD36-mediated metabolic adaptation. Mice transplanted with Yumm1.7 melanoma were treated with an anti-CD36 monoclonal antibody (mAb) that inhibited CD36-mediated uptake of fatty acids and oxidized low-density lipoproteins. As shown in Figure 5a, treatment with the anti-CD36 mAb reduced tumor growth and decreased accumulation of intratumoral Tregs, while the Treg population was maintained in the spleen and aspiration lymph nodes (Figures 5b and 6a). Similar to genetic removal of CD36 in Tregs, anti-CD36 mAb treatment promoted apoptosis in intratumoral Tregs (Figure 5c) and CD8 + This resulted in a significant increase in T cell tumor infiltration (Figure 6b). In addition, treatment of mice with an anti-CD36 mAb resulted in CD8 + TIL and CD4 + Production of antitumor effector cytokines in TILs was improved (Figures 6c and 6d). Since CD36 expression may support metabolic flexibility and metastasis in cancer cells and other immune cells, the antitumor response induced by anti-CD36 mAbs may be Treg-independent. To test this idea, Treg CD36- / - The same treatment was performed using mice as recipients. From the results, it was found that anti-CD36 mAb treatment was effective against Treg CD36- / - The study showed that tumor progression could not be suppressed in mice (Figure 5d), suggesting that the antitumor response induced by anti-CD36 mAb treatment may be primarily due to targeting CD36 expressed in Treg cells rather than other CD36-expressing cells.

[0112] T cell exhaustion may limit the therapeutic outcomes of Treg-targeted interventions; therefore, reactivating exhausted T cells by PD-1 blockade may enhance the antitumor effect of CD36 blockade and suppress tumor progression. In fact, anti-PD-1 mAbs reduced tumor-carrying Tregs compared to WT mice. CD36- / -In mice, tumor progression was more effectively limited and survival was extended (Figures 5e and 5f). In addition to genetic removal of CD36 in Tregs, anti-PD-1 mAbs also enhanced the antitumor response to anti-CD36 mAbs in both genetically engineered Braf / PTEN melanoma mouse models (Figure 5g) and YUMM1.7 transplantation models (Figure 5h). These results demonstrate that targeting CD36 in Tregs can reprogram TMEs to a more immunostimulatory state, which may therapeutically complement the effects of PD-1 blockade to prevent T cell exhaustion. This suggests that CD36 blockade is a novel potential immunotherapeutic intervention with reduced side effects caused by systemic dysfunction of Tregs.

[0113] The synergistic effects of checkpoint blocker inhibitors (e.g., PD1 inhibitors and CTLA4 inhibitors) are further demonstrated in Figures 7a-f. For tumor transplantation, 5 × 10 4 Individual YUMM1.7 tumor cells were subcutaneously injected in 50 μl PBS. Tumors were measured and calculated every 2-3 days after tumor transplantation or processing. Tumor volume was calculated as: Volume = (Length × Width) 2 The formula was calculated as ) / 2. For antibody-based treatment, tumor-bearing mice were treated by intraperitoneal injection with anti-PD-1 antibody (200 μg per injection, BioXcell, clone 29F.1A12), anti-CTLA4 antibody (200 μg per injection, BioXcell, clone 9D9), and anti-CD36 antibody (200 μg per injection, clone CRF D-2712) according to the combinations shown.

[0114] As shown in Figures 7a, 7c, 7d, and 7e, mice transplanted with Yumm1.7 melanoma were treated with anti-CD36 monoclonal antibody (mAb) and anti-CTLA4 mAb. Treatment with anti-CD36 mAb reduced tumor growth and decreased intratumoral Treg frequency, while the Treg population was maintained in the spleen and aspiration lymph nodes. This could not be achieved with anti-CTLA4 mAb treatment. In addition, as shown in Figure 5h, anti-PD-1 mAb also enhanced the antitumor response to anti-CD36 mAb in the YUMM1.7 transplantation model, but tumor growth was also reduced with combination treatment of anti-PD-1 mAb and anti-CTLA4 mAb. These results suggest that targeting CD36 in Tregs can reprogram TMEs to a more immunostimulatory state, which may therapeutically compensate for the effect of PD-1 blockade in preventing tumor progression.

[0115] The results presented herein indicate that intratumor Tregs upregulate CD36 expression to promote fatty acid uptake. The uptaken fatty acids further support mitochondrial adaptability by activating a PPARβ-mediated transcriptional program that regulates mitochondrial nascentization and function. Enhanced mitochondrial adaptability in CD36-expressing intratumor Tregs leads to NAD regeneration via electron transport chain complex I, which then maintains lactate-to-pyruvate conversion. As a result of the continued support of lactate-to-pyruvate conversion via NAD regeneration, intratumor Tregs can survive in the acidic tumor microenvironment and potentially utilize lactate-derived pyruvate to support immunosuppressive activity.

[0116] Utilizing regulatory circuits in which metabolic processes modulate immune responses in immune cells is an intriguing strategy for fine-tuning host immunity in disease. This disclosure demonstrates that CD36-PPARβ signaling maintains survival and functional adaptability in intratumoral Tregs by modulating mitochondrial adaptability and NAD levels. Due to the uniqueness of metabolic stress generated in TMEs and the selectivity of CD36-PPARβ signaling to intratumoral Tregs, targeting CD36 offers broad therapeutic possibilities in cancer patients with minimal adverse effects on immune and peripheral tissue homeostasis. Furthermore, the additive antitumor effects induced by combined PD-1 blockade and CD36 targeting further warrant the development of CD36 inhibitory approaches as a potential cancer treatment.

[0117] All methods and apparatus disclosed and claimed herein can be fabricated and performed without excessive experimentation in the context of this disclosure. While the present invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications can be made to the apparatus, methods, and the order of the steps of the methods without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that the same or similar results can be achieved by adding, combining with, or substituting certain components for the components described herein. All such similar substitutions and modifiers, which are apparent to those skilled in the art, are considered to be within the defined spirit, scope, and concept of the invention.

Claims

1. A method for reducing the number of intratumoral regulatory T cells in a target in need, comprising the step of administering an effective dose of a CD36 inhibitor to the target.

2. A method for reducing the number of intratumoral regulatory T cells in a target in need, comprising the step of administering an effective dose of a PPARβ inhibitor to the target.

3. The method according to claim 1 or 2, wherein the intratumor regulatory T cells are CD4+ cells.

4. A method for increasing the number of intratumor cytotoxic T cells in a target in need, comprising the step of administering an effective dose of a CD36 inhibitor to the target.

5. A method for increasing the number of intratumor cytotoxic T cells in a target in need, comprising the step of administering an effective dose of a PPARβ inhibitor to the target.

6. The method according to claim 4 or 5, wherein the intratumor cytotoxic T cells are CD8+ cells.

7. The method according to claim 1 or 4, wherein the CD36 inhibitor is an anti-CD36 antibody or a small molecule CD36 inhibitor.

8. The method according to claim 7, wherein the anti-CD36 antibody is a human antibody, a humanized antibody, a chimeric antibody, or a bispecific antibody.

9. The method according to claim 7, wherein the small molecule CD36 inhibitor is selected from the group consisting of AP-5258, AP5055, EP-80317, MPE-002, CHEML1789142, CHEML1789302, CHEML1789297, CHEML1789141, CHEML1789270, and CHEML1789308.

10. The method according to any one of claims 1, 4, and 7-9, wherein the CD36 inhibitor is administered intratumor, intravenously, subcutaneously, intraosseously, orally, percutaneously, by sustained release, controlled release, delayed release, as a suppository, or sublingually.

11. The method according to claim 2 or 5, wherein the PPARβ inhibitor is an anti-PPARβ antibody or a low-molecular-weight PPARβ inhibitor.

12. The method according to claim 11, wherein the anti-PPARβ antibody is a human antibody, a humanized antibody, a chimeric antibody, or a bispecific antibody.

13. The method according to claim 11, wherein the small molecule PPARβ inhibitor is selected from the group consisting of FH535, GSK0660, GSK3787, PT-S58, PT-S77, and ST-247.

14. The method according to any one of claims 2, 5, and 11-13, wherein the PPARβ inhibitor is administered intratumor, intravenously, subcutaneously, intraosseously, orally, percutaneously, by sustained release, controlled release, delayed release, as a suppository, or sublingually.

15. The method according to any one of the claims, further comprising the step of administering an additional therapeutic agent to the target.

16. The method according to claim 15, wherein the additional therapeutic agent comprises an immune checkpoint modifier.

17. The method according to claim 16, wherein the immune checkpoint regulator comprises antibodies specific to CTLA-4, PD-1, PD-L1, PD-L2, killer immunoglobulin receptor (KIR), LAG3, B7-H3, B7-H4, TIM3, A2aR, CD40L, CD27, OX40, 4-IBB, TCR, BTLA, ICOS, CD28, CD80, CD86, ICOS-L, B7-H4, HVEM, 4-1BBL, OX40L, CD70, CD40, and GALS.

18. The method according to claim 15, wherein the additional therapeutic agent comprises an anti-PD-1 mAb, an anti-CTLA4 mAb, or a combination thereof.

19. The method according to any one of the claims, wherein the subject has cancer.

20. The method according to claim 19, wherein the cancer is selected from the group consisting of oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, digestive cancer, cancer of the central or peripheral nervous system, endocrine or neuroendocrine cancer or hematopoietic cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, islet cell carcinoma, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma, multiple neuroendocrine tumors type I and II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, and skin cancer.

21. A method for inhibiting tumor growth in a subject with cancer, comprising the step of administering a therapeutically effective dose of a CD36 inhibitor to the subject alone or in combination with an additional therapeutic agent.

22. A method for inhibiting tumor growth in a subject with cancer, comprising the step of administering a therapeutically effective dose of a PPARβ inhibitor to the subject, either alone or in combination with an additional therapeutic agent.

23. The method according to claim 21, wherein the CD36 inhibitor is an anti-CD36 antibody or a small molecule CD36 inhibitor.

24. The method according to claim 23, wherein the anti-CD36 antibody is a human antibody, a humanized antibody, a chimeric antibody, or a bispecific antibody.

25. The method according to claim 23, wherein the small molecule CD36 inhibitor is selected from the group consisting of AP-5258, AP5055, EP-80317, MPE-002, CHEML1789142, CHEML1789302, CHEML1789297, CHEML1789141, CHEML1789270, and CHEML1789308.

26. The method according to any one of claims 21 and 23-25, wherein the CD36 inhibitor is administered intratumor, intravenously, subcutaneously, intraosseously, orally, percutaneously, by sustained release, controlled release, delayed release, as a suppository, or sublingually.

27. The method according to claim 22, wherein the PPARβ inhibitor is an anti-PPARβ antibody or a small molecule PPARβ inhibitor.

28. The method according to claim 27, wherein the anti-PPARβ antibody is a human antibody, a humanized antibody, a chimeric antibody, or a bispecific antibody.

29. The method according to claim 27, wherein the low molecular weight PPARβ inhibitor is selected from the group consisting of FH535, GSK0660, GSK3787, PT-S58, PT-S77, and ST-247.

30. The method according to any one of claims 22 and 27-30, wherein the CD3 inhibitor is administered intratumor, intravenously, subcutaneously, intraosseously, orally, percutaneously, by sustained release, controlled release, delayed release, as a suppository, or sublingually.

31. The method according to any one of claims 21 to 30, wherein the additional therapeutic agent comprises an immune checkpoint modulator.

32. The method according to claim 31, wherein the immune checkpoint regulator comprises antibodies specific to CTLA-4, PD-1, PD-L1, PD-L2, killer immunoglobulin receptor (KIR), LAG3, B7-H3, B7-H4, TIM3, A2aR, CD40L, CD27, OX40, 4-IBB, TCR, BTLA, ICOS, CD28, CD80, CD86, ICOS-L, B7-H4, HVEM, 4-1BBL, OX40L, CD70, CD40, and antibodies specific to GALS.

33. The method according to any one of claims 21 to 32, wherein the cancer is selected from the group consisting of oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, digestive cancer, cancer of the central or peripheral nervous system, endocrine or neuroendocrine cancer or hematopoietic cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, islet cell carcinoma, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma, multiple neuroendocrine tumors type I and II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, and skin cancer.

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