Use of castalagin or its analogues for anti-cancer efficacy and to increase the response to immune checkpoint inhibitors.

Abstract

The object of the present invention is to provide a method for treating cancer in combination with an immune checkpoint inhibitor. [Solution] The present invention provides methods for enhancing or restoring an antitumor response, such as an antitumor immunity mediated by an immune checkpoint inhibitor, in a patient. These methods are particularly useful for treating tumors resistant to immunotherapy, such as immune checkpoint inhibitor therapy, based on the administration of castalagin or its analogues. Castalagin or its analogues may be administered in any suitable form, for example, as a crude plant or fruit extract such as Myrciaria dubia extract, or as a pharmaceutical composition.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 979,327, filed on February 20, 2020, which is incorporated herein by reference.

[0002] This invention generally relates to the field of cancer, and more particularly to the treatment of cancer in combination with immune checkpoint inhibitors. [Background technology]

[0003] The prevalence of cancer in human and animal populations, as well as its role in mortality, means there is a continuing need for new drugs that are effective against tumors. Removing tumors, reducing their size, or decreasing the number of cancer cells circulating in the blood or lymphatic system can be beneficial in various ways, such as reducing pain or discomfort, preventing metastasis, facilitating surgical intervention, and, more importantly, extending lifespan.

[0004] Various attempts have been made to help the immune system fight tumors. One early approach in the late 19th century involved general stimulation of the immune system, such as administering bacteria (live or dead) to induce a general immune response that could also be directed against tumors.

[0005] Recent approaches aimed at assisting the immune system in recognizing tumor-specific antigens (TSAs) (or tumor-associated antigens, TAAs) involve administering tumor-specific antigens (typically combined with adjuvants) to the target individual. However, a lack of a robust immune response to TAAs is frequently observed in cancer. One factor contributing to a weak response to TAAs is the induction of inhibitory pathways / signals (often referred to as "immune checkpoints") that suppress the immune response. While such inhibitory signals are important for maintaining self-tolerance and protecting tissues from damage when the immune system responds to pathogen infection, they can also reduce other potentially beneficial responses of the body to tumor development.

[0006] A new era of therapies using immune checkpoint inhibitors or blockers (ICBs) targeting inhibitory T cell receptors such as CTLA-4, PD-L1, and PD-1 has arrived (Marabelle, OncoImmunology 2016). This rapidly growing field even received the 2018 Nobel Prize in Medicine. These immunotherapies offer unprecedented clinical outcomes in several advanced cancers, including lung (Reck, NEJM 2016), melanoma (Robert, NEJM 2011), genitourinary tract (Motzer, NEJM 2018), and head and neck (Ferris, NEJM 2016). However, the primary resistance rate in patients with non-small cell lung cancer (NSCLC) ranges from 35 to 44%, while the secondary resistance rate approaches 100% (Reck, NEJM 2016).

[0007] Therefore, there is a need to develop novel approaches to enhance the response to ICB, more specifically in cancers resistant to ICB therapy.

[0008] This specification references several sources, and their contents are incorporated herein by reference in their entirety. [Overview of the project]

[0009] This application relates to the following items 1 to 55. 1. A method for treating a subject suffering from cancer resistant to immunotherapy such as immune checkpoint inhibitor therapy, comprising administering to the subject a therapeutically effective amount of castalagin or an analog thereof.

[0010] 2. The method of item 1, wherein the immune checkpoint inhibitor is a programmed cell death-1 (PD-1) inhibitor, a cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) inhibitor, or a programmed death-ligand 1 (PD-L1) inhibitor.

[0011] 3. The method of item 1 or 2, wherein the inhibitor is a blocking antibody.

[0012] 4. The method of item 2 or 3, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

[0013] 5. The method of any one of items 1 to 4, wherein castalagin or an analog thereof is present in a plant or fruit extract.

[0014] 6. The method of item 5, wherein the extract is a Myrciaria dubia (camu camu) extract.

[0015] 7. The method of any one of items 1 to 4, wherein the method comprises administering a pharmaceutical composition comprising castalagin or an analog thereof.

[0016] 8. The method of any one of items 5 to 7, wherein the extract or the pharmaceutical composition is formulated for delivering castalagin or an analog thereof to the intestine.

[0017] 9. The method of item 8, wherein the extract or the pharmaceutical composition is formulated as a capsule.

[0018] 10. The method of any one of items 1 to 9, wherein the cancer is lung cancer or breast cancer.

[0019] 11. The method of item 10, wherein the lung cancer is non-small cell lung cancer (NSCLC).

[0020] 12. How to determine if breast cancer is triple-negative breast cancer (TNBC) using item 10.

[0021] 13. Any one of items 1 to 12, further comprising administering an effective dose of an immune checkpoint inhibitor or castalagin alone.

[0022] 14. A method for enhancing an antitumor immune response in a subject suffering from cancer, the method comprising administering a therapeutically effective dose of castalagin or an analogue thereof to the subject.

[0023] 15. The method described in item 14, which states that the antitumor immune response is an antitumor T cell response.

[0024] 16. The method of item 14 or 15, further comprising administering a therapeutically effective dose of an immune checkpoint inhibitor to a subject.

[0025] 17. The method of item 16, wherein the immune checkpoint inhibitor is a programmed cell death-1 (PD-1) inhibitor, a cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) inhibitor, or a programmed cell death-ligand 1 (PD-L1) inhibitor.

[0026] 18. The method of item 16 or 17, wherein the inhibitor is a blocking antibody.

[0027] 19. The method of item 17 or 18, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

[0028] 20. Any one of items 14-19, wherein castalagin or an analogue thereof is present in a plant or fruit extract.

[0029] 21. The method of item 20, wherein the extract is Myrciaria dubia (camu camu) extract.

[0030] 22. Any one of items 14 to 21, wherein the method comprises administering a pharmaceutical composition comprising castalagin or an analogue thereof.

[0031] 23. Any one of items 20-22, wherein an extract or pharmaceutical composition is formulated for delivery of castalagin or its analogue into the intestine.

[0032] 24. The method of item 23, wherein the extract or pharmaceutical composition is formulated as a capsule.

[0033] 25. The subject has lung cancer or breast cancer, using one of the methods listed in items 14-24.

[0034] 26. The method for determining that lung cancer is non-small cell lung cancer (NSCLC) as described in item 25.

[0035] 27. How to determine if breast cancer is triple-negative breast cancer (TNBC) using item 25.

[0036] 28. Use of castalagin or its analogues to treat patients with cancer resistant to immunotherapy, such as immune checkpoint inhibitor therapy.

[0037] 29. Use of castalagin or its analogues to manufacture pharmaceuticals for the treatment of subjects with cancer resistant to immunotherapy such as immune checkpoint inhibitor therapy.

[0038] 30. Use of item 28 or 29, where the immune checkpoint inhibitor is a programmed cell death-1 (PD-1) inhibitor, a cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) inhibitor, or a programmed cell death-ligand 1 (PD-L1) inhibitor.

[0039] 31. Use of any one of items 28-30, where an immune checkpoint inhibitor is a blocking antibody.

[0040] 32. Use of any one of items 28-31, where the immune checkpoint inhibitor is a PD-1 inhibitor.

[0041] 33. Use of any one of items 28-32 in which castalagin or its analogues are present in a plant or fruit extract.

[0042] 34. Use of item 33, where the extract is Myrciaria dubia extract.

[0043] 35. Use of any one of items 28-32 in which castalagin or an analogue thereof is present in the pharmaceutical composition.

[0044] 36. Use of any one of items 33-35, in which an extract or pharmaceutical composition is formulated to deliver castalagin or its analogue into the intestines.

[0045] 37. Use of item 36, in which the extract or pharmaceutical composition is formulated as a capsule.

[0046] 38. Use of any one of items 28-37, where the cancer is lung cancer or breast cancer.

[0047] 39. Use of item 38 if the lung cancer is non-small cell lung cancer (NSCLC).

[0048] 40. Use of item 38 if the breast cancer is triple-negative breast cancer (TNBC).

[0049] 41. Use of castalagin or its analogues to enhance the antitumor immune response in subjects.

[0050] 42. Use of castalagin or its analogues to manufacture pharmaceuticals for enhancing the antitumor immune response in subjects.

[0051] 43. Use of item 41 or 42, where the antitumor immune response is an antitumor T cell response.

[0052] 44. Use of any one of items 41-43, in which castalagin or its analogues are intended for use in combination with an immune checkpoint inhibitor.

[0053] 45. Use of item 44, where the immune checkpoint inhibitor is a programmed cell death-1 (PD-1) inhibitor, a cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) inhibitor, or a programmed cell death-ligand 1 (PD-L1) inhibitor.

[0054] 46. ​​Use of item 44 or 45, where the immune checkpoint inhibitor is a blocking antibody.

[0055] 47. Use of any one of items 44-46, where the immune checkpoint inhibitor is a PD-1 inhibitor.

[0056] 48. Use of any one of items 41-47 in which castalagin or its analogues are present in a plant or fruit extract.

[0057] 49. Use of item 48, where the extract is Myrciaria dubia extract.

[0058] 50. Any use of any one of items 41-47, in which castalagin or an analogue thereof is present in the pharmaceutical composition.

[0059] 51. Use of any one of items 48-50, in which an extract or pharmaceutical composition is formulated to deliver castalagin or its analogue into the intestines.

[0060] 52. Use of item 51, in which the extract or pharmaceutical composition is formulated as a capsule.

[0061] 53. Use of any one of items 41 to 52 if the subject has skin cancer (e.g., melanoma, squamous cell carcinoma), lung cancer, kidney cancer (e.g., renal cell carcinoma), Hodgkin lymphoma, head and neck cancer, colon cancer, liver cancer, stomach cancer, or myeloma, preferably lung cancer or breast cancer.

[0062] 54. Use of item 53 if the subject has lung cancer, preferably non-small cell lung cancer (NSCLC).

[0063] 55. Use of item 53, where the subject has breast cancer, preferably triple-negative breast cancer (TNBC).

[0064] Other objects, advantages, and features of the present invention will become more apparent from reading the following non-limiting description of specific embodiments, which are given merely as examples with reference to the accompanying drawings. [Brief explanation of the drawing]

[0065] [Figure 1A] This is a schematic diagram of a protocol used to study the effects of camu camu extract (CC) alone and its additive effect when combined with anti-PD-1 therapy in a mouse tumor model sensitive to anti-PD-1 therapy. Syngeneic C57BL / 6 mice were subcutaneously transplanted with 0.8 × 10⁶ MCA-205 sarcomas. When the tumors reached a size of 20–35 mm², they were treated intraperitoneally (ip) with or without forced oral administration of 200 mg / kg of CC (SunFood camu camu powder) once daily, either with or without this administration, with or without anti-PD-1 mAb (250 μg / mouse, clone RMP1-14) or isotype control (clone 2A3). [Figure 1B] This graph shows the tumor size over time in mice transplanted with MCA-205 tumors, treated with or without once-daily forced oral administration of CC, either with or without. [Figure 1C] This graph shows the tumor size at euthanasia of mice transplanted with MCA-205 tumors, treated with or without once-daily forced oral administration of CC, or with or without. [Figure 2A]This is a schematic diagram of a protocol used to study the effects of camu camu extract (CC) alone and its additive effect when combined with anti-PD-1 therapy in a mouse tumor model resistant to anti-PD-1 therapy. Syngeneic C57BL / 6 mice were subcutaneously implanted with 0.5 × 10⁶ E0771 breast cancer tumor models. When the tumors reached a size of 20–35 mm², they were treated intraperitoneally (ip) with or without forced oral administration of 200 mg / kg of CC (SunFood) once daily, either with or without such administration, with an anti-PD-1 mAb (250 μg / mouse, clone RMP1-14) or an isotype control (clone 2A3). [Figure 2B] This graph shows the tumor size over time in mice transplanted with E0771 tumors after sequential injection of anti-PD-1 mAb (αPD-1) or isotype control (IsoPD-1) and once-daily forced oral administration of water or CC. [Figure 2C] This graph shows the tumor size at euthanasia of mice transplanted with E0771 tumors, treated with or without once-daily forced oral administration of CC, either with or without. [Figure 3A] This is a schematic diagram of the protocol used to study the effect of broad-spectrum antibiotics (ATBs) on the response to carcinomatous cell carcinoma (CC) in a mouse MCA-205 tumor model. Mice were treated with ATBs two weeks prior to tumor transplantation and antibiotics were continued until the end of the experiment. A mixture of ampicillin (1 mg / ml), streptomycin (5 mg / ml), and colistin (1 mg / ml) (Sigma-Aldrich) was added to sterile drinking water. The solution and bottles were changed three times a week. Antibiotic activity was confirmed by macroscopic changes observed at the cecal level at euthanasia (dilation) and by culturing fecal pellets resuspended in sterile NaCl on blood agar plates at 37°C for 48 hours under aerobic or anaerobic conditions. MCA-205 inoculation and CC treatment were performed as shown in Figure 1A. [Figure 3B]This graph shows the tumor size over time in mice transplanted with MCA-205 tumors, administered water (control, Figure 3B) (5 mice / group) with or without once-daily forced oral administration of CC. [Figure 3C] This graph shows the tumor size over time in mice transplanted with MCA-205 tumors, administered ATB (Figure 3C) (5 mice / group) with or without once-daily forced oral administration of CC. [Figure 4A] This is a schematic diagram of a protocol used to study the effect of fecal microbiota migration (FMT) from CC-treated mice on the response to anti-PD-1 in a mouse MCA-205 tumor model. Feces from CC-treated mice were frozen at -80°C in Eppendorf® tubes. MCA-205 was subcutaneously implanted, and when the tumor reached a size of 20–35 mm², it was treated intraperitoneally (ip) with or without once-daily forced oral administration of diluted feces in NaCl, either with or without. 100 μg of feces was resuspended in 1 mL of sterile NaCl. [Figure 4B] This graph shows the tumor size at euthanasia of mice transplanted with MCA-205 tumors, treated with or without once-daily forced oral administration of diluted feces in NaCl, or with or without such administration. [Figure 5A] This is a schematic diagram of the experimental design for the avatar mouse experiment. FMT was performed individually in SPF C57Bl6 mice after 3 days of ATB using fecal samples from non-responder (NR) and responder (R) non-small cell lung cancer (NSCLC) patients. Two weeks later, MCA-205 sarcoma cells were inoculated and administered once daily with water or CC, combined with sequential injections of αPD-1 or IsoPD-1 mAb. [Figure 5B]This graph shows the pooled mean tumor ± SEM values ​​for each CC group and water group, specifically for euthanasia (D+17) after FMT from two NR group and two R group patients. [Figure 5C] This graph shows the number of genera observed for alpha diversity in R and NR mice (n=10) with MCA-205 at baseline (before forced CC administration and 14 days after engraftment) in SPF-fed mice (n=10) treated with ATB and then receiving FMT from 4 NSCLC patients (n=2 NR, n=2 R). Mean ± SEM. [Figure 5D] This is the Bray-Curtis expression of beta diversity in 16s RNA sequencing of NR and R FMT at the genus level, 2 weeks after engraftment. *p<0.05, ***p<0.001. [Figure 5E] This is a Volcano plot representation of the differential abundance analysis results after 16s sequencing analysis of mouse feces 14 days after receiving NR or R FMT on day 0. [Figure 5F] This graph shows the alpha diversity, represented by genus, observed in the NR and R FMT groups at D+11. Values ​​are shown as mean ± SEM. *p<0.05, **p<0.01. [Figure 6A] This graph shows 16s rRNA fecal samples from mice in four groups in the MCA-205 experiment (Figure 1A), and represents the alpha diversity measured by the Shannon index in each group. [Figure 6B] This graph shows the results of a real-time PCR assay on DNA extracted from mouse feces after 6 days of forced administration of water or CC, using specific primers for 16s detection in the MCA-205 model (n=10 mice / group). [Figure 6C] This graph shows beta diversity, measured by the Bray-Curtis index, comparing baseline (pre-treatment) with the pooled CC or water (αPD1 and IsoPD-1) group. [Figure 6D]This graph shows 16s rRNA microbiome profiling of samples from the MCA-205 experiment (Figure 1A), and the representation of beta diversity as measured by the Bray-Curtis index, comparing all four groups 6 days after treatment. [Figure 6E] This is a Volcano plot representation of differential abundance analysis comparing pooled CC groups and water groups in MCA-205 tumors. The bacterial enrichment in each group is represented using adjusted p-values. **p<0.01.** [Figure 6F] This is a Volcano plot representation of the differential abundance analysis in the water / IsoPD-1 group compared to the CC / IsoPD-1 group in the MCA-205 tumor model. The bacterial enrichment in each group is represented using adjusted p-values. **p<0.01.** [Figure 6G] This is a Volcano plot representation of the differential abundance analysis in the water / IsoPD-1 group compared to the CC / αPD-1 group in the MCA-205 tumor model. The bacterial enrichment in each group is represented using adjusted p-values. **p<0.01.** [Figure 6H] This is a Volcano plot representation of differential abundance analysis comparing the water / αPD-1 group and the CC / αPD1 group in E0771 tumors. The bacterial enrichment in each group is represented using adjusted p-values ​​and p-values ​​(FDR: 0.1). *p<0.05, **p<0.01, ***p<0.001. [Figure 6I] This is a Volcano plot representation of the differential abundance analysis between the water / IsoPD-1 group and the CC / IsoPD-1 group in the E0771 model. [Figure 7A]These graphs show the results of immunocytological profiling by flow cytometry in mouse MCA-205 (Figures 7A-B) or E0771 (Figure 7C) tumor models treated with anti-PD-1 and / or CC. Tumors and spleens were harvested 9 or 19 days after the first injection of anti-PD-1 mAb into mice with MCA-205 or E0771 tumors, respectively. The excised tumors were cut into small pieces and digested at 37°C for 30 minutes in RPMI medium containing Liberase 25 μg / mL (Roche) and DNase 1 (Roche) at 150 μg / mL. The tissue was then pulverized using 100 and 70 μm cell strainers (Becton & Dickinson) and filtered twice. The spleens were pulverized in RPMI medium and then filtered through a 100 μm cell strainer. Prior to membrane staining (CD45, CD3, CD4, CD8, PD1, PDL1, ICOS, CXCR3, CCR9, CD45RB, CD62L, CD44), 2 million cells or splenocytes were pre-incubated at 4°C for 30 minutes with purified anti-mouse CD16 / CD32 (clone 93, eBioscience). For intracellular staining, the Foxp3 staining kit (eBioscience) was used. Dead cells were excluded using the Live / Dead Fixable Aquablue dead cell staining kit (Life Technologies). Figures 7A-B are graphs showing TCM CD8+ T cells (CD45RB-CD62L+CD8+ T cells) and relative CD8+ T cells / Foxp3+CD4+ T cells (Treg), respectively, in TILs from mice with MCA-205 tumors after treatment with or without once-daily forced oral administration of CC. [Figure 7B]These graphs show the results of immunocytological profiling by flow cytometry in mouse MCA-205 (Figures 7A-B) or E0771 (Figure 7C) tumor models treated with anti-PD-1 and / or CC. Tumors and spleens were harvested 9 or 19 days after the first injection of anti-PD-1 mAb into mice with MCA-205 or E0771 tumors, respectively. The excised tumors were cut into small pieces and digested at 37°C for 30 minutes in RPMI medium containing Liberase 25 μg / mL (Roche) and DNase 1 (Roche) at 150 μg / mL. The tissue was then pulverized using 100 and 70 μm cell strainers (Becton & Dickinson) and filtered twice. The spleens were pulverized in RPMI medium and then filtered through a 100 μm cell strainer. Prior to membrane staining (CD45, CD3, CD4, CD8, PD1, PDL1, ICOS, CXCR3, CCR9, CD45RB, CD62L, CD44), 2 million cells or splenocytes were pre-incubated at 4°C for 30 minutes with purified anti-mouse CD16 / CD32 (clone 93, eBioscience). For intracellular staining, the Foxp3 staining kit (eBioscience) was used. Dead cells were excluded using the Live / Dead Fixable Aquablue dead cell staining kit (Life Technologies). Figures 7A-B are graphs showing TCM CD8+ T cells (CD45RB-CD62L+CD8+ T cells) and relative CD8+ T cells / Foxp3+CD4+ T cells (Treg), respectively, in TILs from mice with MCA-205 tumors after treatment with or without once-daily forced oral administration of CC. [Figure 7C]These graphs show the results of immunocytological profiling by flow cytometry in mouse MCA-205 (Figures 7A-B) or E0771 (Figure 7C) tumor models treated with anti-PD-1 and / or CC. Tumors and spleens were harvested 9 or 19 days after the first injection of anti-PD-1 mAb into mice with MCA-205 or E0771 tumors, respectively. The excised tumors were cut into small pieces and digested at 37°C for 30 minutes in RPMI medium containing Liberase 25 μg / mL (Roche) and DNase 1 (Roche) at 150 μg / mL. The tissue was then pulverized using 100 and 70 μm cell strainers (Becton & Dickinson) and filtered twice. The spleens were pulverized in RPMI medium and then filtered through a 100 μm cell strainer. Prior to membrane staining (CD45, CD3, CD4, CD8, PD1, PDL1, ICOS, CXCR3, CCR9, CD45RB, CD62L, CD44), 2 million cells or splenocytes were pre-incubated at 4°C for 30 minutes with purified anti-mouse CD16 / CD32 (clone 93, eBioscience). For intracellular staining, the Foxp3 staining kit (eBioscience) was used. Dead cells were excluded using the Live / Dead Fixable Aquablue dead cell staining kit (Life Technologies). Figure 7C is a graph showing intratumoral CD8+ T cell activation (as assessed by MFI of ICOS+CD8+ T cells by flow cytometry) in TILs of the E0771 tumor model after treatment with CC+ / -αPD-1. [Figure 7D] This graph shows the effect of blocking CD8+ T cell activity on the antitumor effect of cytotoxicopropylglycation (CC) in a mouse MCA-205 tumor model. Syngeneic C57BL / 6 mice were subcutaneously transplanted with 0.8 × 10⁶ MCA-205 sarcomas. Three days after tumor inoculation, the mice were treated with 150 μg / mouse anti-CD8 (clones: 53-5.8, BioXCell) or an isotype control. Then, when the tumors reached a size of 20–35 mm², the mice were either or not subjected to forced oral administration of 200 mg / kg of CC once daily. [Figure 7E]This is a pairwise Spearman rank correlation heatmap of significantly different cells enriched in the CC / isoPD-1 group compared to the water / isoPD-1 group (n=1) in the MCA-205 experiment, using positively correlated TIL cytometry and adjusted for tumor size. The left panel shows cells within the TIL, and the right panel shows cells within the spleen. [Figure 7F] In experiment E0771, pairwise Spearman rank correlation heatmaps were obtained between significantly different fecal taxons enriched in the CC / αPD-1 group compared to the water / αPD-1 group, after matching flow cytometry and tumor size, as well as the frequencies of the indicated cell types. Unpaired t-tests were used. *p<0.05, **p<0.01, ***p<0.001. [Figure 8A] This diagram shows the fractionation workflow for CC extracts. [Figure 8B] This figure shows the retention times of complete camu camu extraction by high-performance liquid chromatography, followed by the retention times of the polar fraction and fraction P3, and the HPLD retention times of castalagin extracted from oak. [Figure 8C] Figure 7A shows graphs illustrating the effects of various fractions (P, NP, M, INS) in SPF-fed mice (n=5, mean ± SEM tumor size at euthanasia) with MCA-205, in or without the presence of anti-PD-1. Using the same experimental design as described earlier (Figure 1A), mice were either administered each fraction (polar fraction: P, nonpolar fraction: NP, moderately polar: M, and insoluble fraction: INS) at a concentration of 40.18 mg / kg, or received forced oral administration of CC once daily at a dose of 100 mg / kg, or not. Unpaired t-tests were used. *p<0.05, **p<0.01. [Figure 8D]This graph shows the effects of different fractions (P1, P2, P3, P4) from fraction P in Figure 7C in the presence or absence of anti-PD-1 in a mouse MCA-205 tumor model. Using the same experimental design as described earlier (Figure 1A), mice were administered either each fraction (P1, P2, P3, and P4) at a concentration of 0.85 mg / kg, or CC at a dose of 100 mg / kg once daily by forced oral administration, or not. Unpaired t-tests were used. *p<0.05, **p<0.01. [Figure 8E] This graph shows the effects of different doses of castalagin in the presence of anti-PD-1 in a mouse MCA-205 tumor model (mean MCA-205 tumor size represents the size at the time of euthanasia for Mic). Using the same experimental design as previously described (Figure 1A), mice were administered either increased doses of castalagin (from 0.11 mg / kg to 2.56 mg / kg), or forced oral administration of CC once daily at a dose of 100 mg / kg, or no administration at all. Notably, the dose present in CC corresponds to approximately 0.85 mg / kg. For a negative control at 0 mg / kg, mice were given water. [Figure 8F] This graph shows the effect of castalagin in the presence of anti-PD-1 in SPF-fed mice (n=5, tumor size at euthanasia) carrying E0771. Using the same experimental design as described above (Figure 2A), mice were administered castalagin (0.85 mg / kg per mouse) once daily by forced oral administration, or not, in the presence or absence of anti-PD-1. [Figure 9A] This graph shows the effect of administering castalagin at a standard concentration (0.85 mg / kg per mouse) under sterile conditions on tumor size in a mouse MCA-205 tumor model. [Figure 9B] This graph shows bacterial diversity (number of observed genera) 5 days (baseline) and 11 days after mandatory castalagin administration in the MCA-205 model. An independent t-test was used. *p<0.05, **p<0.01, ***p<0.001. [Figure 9C]This shows the Bray-Curtis beta diversity expression from 16s rRNA microbiome sequencing on castalagin or water-forced administration days in SPF-fed mice (n=5) carrying MCA-205. Each line corresponds to a mouse group, and each point corresponds to a single animal. Unpaired t-tests were used. *p<0.05, **p<0.01, ***p<0.001. [Figure 9D] This is a Volcano plot representation of the differential abundance analysis results after 16s sequencing analysis in the water / IsoPD-1 group compared to the castalagin / IsoPD-1 group in SPF-fed mice (n=5) carrying MCA-205. [Figure 9E] The relative abundance analyses of Ruminococcus, Alistepes, Christensenellaceae R7 group, Paraprevotella, and Lachnoclostridium after 16s sequencing analysis between the water group and the castalagin group in NR FMT experiments are shown. *p<0.05, **p<0.01, ***p<0.001. [Figure 9F] The relative abundance analyses of Ruminococcus, Alistepes, Christensenellaceae R7 group, Paraprevotella, and Lachnoclostridium after 16s sequencing analysis between the water group and the castalagin group in NR FMT experiments are shown. *p<0.05, **p<0.01, ***p<0.001. [Figure 9G] The relative abundance analyses of Ruminococcus, Alistepes, Christensenellaceae R7 group, Paraprevotella, and Lachnoclostridium after 16s sequencing analysis between the water group and the castalagin group in NR FMT experiments are shown. *p<0.05, **p<0.01, ***p<0.001. [Figure 9H]The relative abundance analyses of Ruminococcus, Alistepes, Christensenellaceae R7 group, Paraprevotella, and Lachnoclostridium after 16s sequencing analysis between the water group and the castalagin group in NR FMT experiments are shown. *p<0.05, **p<0.01, ***p<0.001. [Figure 9I] The relative abundance analyses of Ruminococcus, Alistepes, Christensenellaceae R7 group, Paraprevotella, and Lachnoclostridium after 16s sequencing analysis between the water group and the castalagin group in NR FMT experiments are shown. *p<0.05, **p<0.01, ***p<0.001. [Figure 9J] This graph shows the effect of castalagin treatment (0 mg / kg, 1 / 4 dose of 0.21 mg / kg, 1 dose of 0.85 mg / kg, and 3 doses of 2.55 mg / kg) on ​​the amount of Ruminococcaceae in feces. In SPF-bred mice carrying MCA-205 (n=5), DNA extracted from mouse feces was performed using real-time PCR after 6 days of oral forced administration of 0.21 mg / kg, 0.85 mg / kg, and 2.55 mg / kg with water or castalagin, using specific primers for Ruminococcaceae detection. *p<0.05, **p<0.01. [Figure 10A] This graph shows the effect of castalagin treatment on the immune cell profile in mouse MCA-205. Flow cytometry analysis of MCA-205 TILs in sterile and SPF experiments comparing CC to water at the time of euthanasia. [Figure 10B] Representative images of tumors stained with CD4, CD8, and Foxp3 immunofluorescence in both the water / IsoPD-1 group and the castalagin / IsoPD-1 group. [Figure 10C]Box plot of intratumoral ratio CD8+ / Foxp3+CD4+ obtained by immunofluorescence staining (n=8 / group). *p<0.05. [Figure 10D] This graph shows the effect of castalagin treatment on the immune cell profile in mouse E0771. Flow cytometry analysis of memory CD8+ T cells in tumors of E0771 at the time of euthanasia. [Figure 10E] This graph shows the effect of castalagin treatment on the immune cell profile in mouse E0771. Flow cytometry analysis of memory CD8+ T cells in the spleen of E0771 at the time of euthanasia. [Figure 11A] This graph shows the effect of castalagin (0.85 mg / kg) on ​​tumor growth dynamics in the presence or absence of anti-PD-1 in an ATB-avatar model after FMT from one NR NSCLC patient who received once-daily forced administration of castalagin or water in combination with αPD1 mAb or IsoPD-1. An unpaired t-test was used. Results are shown as mean ± SEM. *p<0.05. [Figure 11B] This graph shows the therapeutic effect of castalagin fecal microbiota transplantation (FMT) using fecal samples from NR NSCLC patients under ATB and sterile conditions. Fecal microbiota transplantation (FMT) of fecal samples from a non-responder (NR) non-small cell lung cancer (NSCLC) patient (n=1 NR) was performed in sterile C57BL / 6 mice (n=3). After 2 weeks, MCA-205 sarcoma cells were inoculated and administered once daily with water or castalagin. Each line corresponds to a mouse group, and each point corresponds to a single animal. An unpaired t-test was used. Results are shown as mean ± SEM. [Figure 12A] This is a schematic diagram of the hydrolysis of castalagin to ellagic acid and castalin, as well as the metabolism of ellagic acid to urolithin by the gut microbiota. [Figure 12B]This graph shows the effects of castalagin, vescalagin, ellagic acid, castalin, and urolithin A on tumor size at the time of euthanasia in a mouse MCA-205 tumor model, using the same experimental design as described earlier (Figure 1A). [Figure 12C] This figure shows the in vitro labeling of castalagin with fluorothane. [Figure 12D] This is a representation of one flow cytometry experimental representation of fluorescein-labeled castalagin in co-culture with Escherichia coli, Ruminococcus bromii, and Bacteroides thetaiotomicron. The upper panel shows the unstained state, and the lower panel shows staining with fluorescein-castalagin at 37°C. [Figure 12E] This graph shows the results of competitive assays of R. bromii and E. coli in the presence of fluorescein-bound castalagin at 37°C and 0°C, and in the presence of unbound castalagin at a 100-fold concentration. Each point represents one experiment. [Figure 12F] Images of R. bromii, E. coli, and B. thetaiotaomicron after fluorescein fluorescence inverted microscopy. [Figure 12G] This graph shows the results of diversity (16s) and ruminococcaceae qPCR assays in two non-cancer HIV patients treated with 1.5 mg of CC once daily. The graph shows the amount of diversity (16s RNA) before CC administration and 3 weeks later. [Figure 12H] This graph shows the diversity (16s) and qPCR assay results for Ruminococcaceae in two non-cancer HIV patients treated with 1.5 mg of CC once daily. The graph shows the amount of Ruminococcaceae DNA before CC administration and 3 weeks later. [Figure 13]This graph shows the results of a qPCR assay performed on DNA extracted from mouse feces after 6 days of oral forced administration of 0.85 mg / kg water or castalagin in SPF-bred mice (n=10) carrying MCA-205, using specific primers for Ruminococcaceae detection. *p<0.05. [Figure 14] This table shows a list of bacteria that increased after CC and / or castalagin administration compared to water. [Modes for carrying out the invention]

[0066] In the context describing the present invention (in particular in the context of the following claims), the use of the terms "a," "an," and "the," and similar references, should be interpreted to encompass both singular and plural forms, unless otherwise indicated herein or otherwise clearly contradicted by the context.

[0067] The terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., “including, but not limited to”) unless otherwise noted.

[0068] The enumeration of value ranges in this specification is intended to serve simply as a simplified method of individually referring to each individual value contained within the range, unless otherwise indicated herein, and each individual value is incorporated herein as if it were individually enumerated herein. All subsets of values ​​within the range are also incorporated herein as if they were individually enumerated herein.

[0069] All methods described herein may be carried out in any preferred order, unless otherwise indicated herein or unless the context clearly contradicts it.

[0070] Any and all examples or use of exemplary language (such as "e.g.", "etc.") provided herein are intended solely to better illustrate the invention and, unless otherwise claimed, do not limit the scope of the invention.

[0071] No language in this specification should be construed as indicating an element not claimed to be essential to the implementation of the invention.

[0072] In this specification, the term “approximately” has its usual meaning. The term “approximately” is used to indicate that a value includes inherent variability due to errors in the device or method used to determine that value, or that it includes values ​​close to the enumerated values, for example, values ​​within 10% of the enumerated values ​​(or range of values).

[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this invention pertains.

[0074] The scope of the claims should not be limited by the preferred embodiments described in the examples, but rather should be interpreted in the broadest way that is consistent with the specification as a whole.

[0075] Any and all combinations and partial combinations of the embodiments and features disclosed herein are incorporated into the present invention.

[0076] In the studies described herein, the inventors demonstrated that a crude extract from Myrciaria dubia (camu camu, CC) berries could induce an antitumor response and enhance the antitumor response to immune checkpoint inhibitors in two mouse tumor models, as well as restore the antitumor response to immune checkpoint inhibitors in resistant tumors. The inventors also provided compelling evidence that the effects of the camu camu extract are mediated, at least partially, by the modulation of the gut microbiota and involve a T cell-mediated immune response. Further characterization of the camu camu extract led to the identification of castalagin as the main active component responsible for the extract's effect on the antitumor response.

[0077] Accordingly, in a first aspect, the Disclosure provides a method for inducing or restoring a response to an immunotherapy, such as immune checkpoint inhibitor therapy, in a subject with an immunotherapy-resistant cancer, comprising administering a therapeutically effective dose of castalagin or an analogue thereof to the subject. The Disclosure also provides the use of castalagin or an analogue thereof to induce only antitumor activity and / or improve or restore a response to such an immunotherapy, such as immune checkpoint inhibitor (ICI) therapy, in a subject with an immunotherapy-resistant cancer. The Disclosure also provides the use of castalagin or an analogue thereof to manufacture a pharmaceutical product for inducing or restoring a response to an immunotherapy, such as immune checkpoint inhibitor therapy, in a subject with an immunotherapy-resistant cancer. This disclosure also provides castalagin or an analogue for use in inducing or restoring a response to immunotherapy, such as immune checkpoint inhibitor therapy, in subjects with cancer resistant to such immunotherapy (e.g., immune checkpoint inhibitor therapy).

[0078] In another aspect, the Disclosure provides a method for treating a subject with cancer resistant to immunotherapy, such as immune checkpoint inhibitor therapy, comprising administering to the subject a therapeutically effective dose of castalagin or an analogue in combination with immunotherapy (e.g., immune checkpoint inhibitor). The Disclosure also provides the use of castalagin or an analogue in combination with immunotherapy (e.g., immune checkpoint inhibitor) for treating a subject with cancer resistant to immunotherapy (e.g., immune checkpoint inhibitor therapy). The Disclosure also provides the use of castalagin or an analogue in combination with immunotherapy (e.g., immune checkpoint inhibitor) for manufacturing a pharmaceutical product for treating a subject with cancer resistant to immunotherapy (e.g., immune checkpoint inhibitor therapy). The Disclosure also provides a combination therapy comprising castalagin or an analogue in combination with immunotherapy (e.g., immune checkpoint inhibitor) for treating a subject with cancer resistant to immunotherapy (e.g., immune checkpoint inhibitor therapy).

[0079] In another aspect, the Disclosure provides a method for enhancing an immune response, such as an antitumor immune response, in a subject who requires such enhancement, the method comprising administering a therapeutically effective dose of castalagin or an analogue to the subject. The Disclosure also provides the use of castalagin or an analogue for enhancing an immune response, such as an antitumor immune response, in a subject. The Disclosure also provides the use of castalagin or an analogue for manufacturing a pharmaceutical product for enhancing an immune response, such as an antitumor immune response, in a subject. The Disclosure also provides castalagin or an analogue for use in enhancing an immune response, such as an antitumor immune response, in a subject.

[0080] In one embodiment, the above-described treatment increases the levels of immune cells such as T cells (e.g., tumor-infiltrating lymphocytes or TILs) in the tumor. In one embodiment, T cells are CD4 + and / or CD8 + T cells, for example, activated (ICOS +) and / or memory CD4 + and / or CD8 + T cells (central memory (T CM ) CD4 + and / or CD8 + cells). In another embodiment, the above-mentioned treatment increases the CD8 + T cell / Foxp3 + CD4 + T cell (Treg) ratio. In another embodiment, the above-mentioned treatment increases ICOS + Foxp3 - CD4 + CD4 T cells.

[0081] In another embodiment, the disclosure includes administering an effective amount of castalagin or an analogue to a subject to the subject's intestines to bacteria of the families or genera shown in Figure 14, e.g., Acetatifactor, Lachnospiraceae FCS020 group, Acetomaculum Lachnospiraceae GCA-900066225, Akkermansia, Lachnospiraceae GCA-900066575, Alistipes, Lachnospiraceae UCG-001, Anaeroplasma Lachnospiraceae UCG-004, Anaerosporobacter, Lachnospiraceae UCG-006, Anaerovorax, Lactobacillus, Angelakisella Monoglobus, Asaccharospora, Oscillibacter, Bifidobacteriaceae, Oscillospiraceae UCG-005. R-7 group, Roseburia, Clostridiales vadinBB60 group, Ruminiclostridium, Clostridium sensu stricto 1, Ruminiclostridium 5, Coprococcus 3, Ruminiclostridium 9, Eisenbergiella, Ruminococcaceae, Enterobacteriaceae, Ruminococcaceae UBA1819, Enterococccaceae, Ruminococcaceae UCG-005, Enterococcus, RuminococcaceaeThe present invention provides methods for increasing the levels of UCG-009, Erysipelotrichaceae, Ruminococcaceae UCG-014, Escherichia / Shigella, Ruminococcus, Family XIII, Sporacetigenium, Family XIII UCG-001, Staphylococcaceae, Flavonifractor, Staphylococcus, Herbinix, Tannerellaceae, Isobaculum, Turicibacter, Lachnospiraceae, Tyzzerella 3 and / or Lachnospiraceae A2, preferably Turicibacter, Bilophila, Ruminococcaceae (e.g., Ruminococcaceae UBA1819), Parasutterella, Clostridium sensu stricto 1, Chrinstensenellaceae, Alistipes, Ruminococcus, Akkermansia and / or Anaeroplasma. In another aspect, this disclosure relates to the following in the intestines of subjects: Acetatifactor, Lachnospiraceae FCS020 group, Acetimaculum Lachnospiraceae GCA-900066225, Akkermansia, Lachnospiraceae GCA-900066575, Alistipes, Lachnospiraceae UCG-001, Anaeroplasma Lachnospiraceae UCG-004, Anaerosporobacter, Lachnospiraceae UCG-006, Anaerovorax, Lactobacillus, Angelakisella Monoglobus, Asaccharospora, Oscillibacter, Bifidobacteriaceae, OscillospiraceaeUCG-005 Bifidobacterium Paraprevotella Bilophila Parasutterella Blautia Peptococcaceae Butyricicoccus Peptostreptococcaceae Candidatus soleaferrea, Porphyromonadaceae, Carnobacteriaceae, Rikenellaceae, Christensenellaceae, Rhomboutsia, Christensenellaceae R-7, Roseburia, Clostridiales vadinBB60グループ Ruminiclostridium Clostridium sensu stricto 1 Ruminiclostridium 5 Coprococcus 3 Ruminiclostridium 9 Eisenbergiella Ruminococcaceae Enterobacteriaceae Ruminococcaceae UBA1819、Enterococcaceae、Ruminococcaceae UCG-005、Enterococcus、Ruminococcaceae UCG-009、Erysipelotrichaceae、Ruminococcaceae UCG-014、Escherichia / Shigella、Ruminococcus、Family XIII, Sporacetigenium, Family XIII UCG-001, Staphylococcaceae, Flavonifractor, Staphylococcus, Herbinix, Tannerellaceae, Isobaculum, Turicibacter, Lachnospiraceae, Tyzzerella Group 3: Lachnospiraceae A2, Turicibacter, Bilophila, Ruminococcaceae (Ruminococcaceae UBA1819), Parasutterella, Clostridium sensu stricto1. To provide the use of castalagin or its analogues to increase the levels of bacteria of families or genera such as Chrinstensenellaceae, Alistipes, Ruminococcus, Akkermansia, and / or Anaeroplasma. In another aspect, this disclosure relates to the following intestines of subjects: Acetatifactor, Lachnospiraceae FCS020 group, Acetomaculum Lachnospiraceae GCA-900066225, Akkermansia, Lachnospiraceae GCA-900066575, Alistipes, Lachnospiraceae UCG-001, Anaeroplasma Lachnospiraceae UCG-004, Anaerosporobacter, Lachnospiraceae UCG-006, Anaerovorax, Lactobacillus, Angelakisella Monoglobus, Asaccharospora, Oscillibacter, Bifidobacteriaceae, Oscillospiraceae UCG-005. R-7 group, Roseburia, Clostridiales vadinBB60 group, Ruminiclostridium, Clostridium sensu stricto 1, Ruminiclostridium 5, Coprococcus 3, Ruminiclostridium 9, Eisenbergiella, Ruminococcaceae, Enterobacteriaceae, Ruminococcaceae UBA1819, Enterococccaceae, RuminococcaceaeUse of castalagin or an analog thereof for the manufacture of a medicament for increasing the level of bacteria of the family or genus selected from the group consisting of UCG-005, Enterococcus, Ruminococcaceae UCG-009, Erysipelotrichaceae, Ruminococcaceae UCG-014, Escherichia / Shigella, Ruminococcus, Family XIII, Sporacetigenium, Family XIII UCG-001, Staphylococcaceae, Flavonifractor, Staphylococcus, Herbinix, Tannerellaceae, Isobaculum, Turicibacter, Lachnospiraceae, Tyzzerella 3 and / or Lachnospiraceae A2, preferably Turicibacter, Bilophila, Ruminococcaceae (e.g., Ruminococcaceae UBA1819), Parasutterella, Clostridium sensu stricto 1, Chrinstensenellaceae, Alistipes, Ruminococcus, Akkermansia, and / or Anaeroplasma, is provided.

[0082] Acetatifactor Lachnospiraceae FCS020 Acetitomaculum Lachnospiraceae GCA-900066225、Akkermansia、Lachnospiraceae GCA-900066575、Alistipes、Lachnospiraceae UCG-001、Anaeroplasma Lachnospiraceae UCG-004、Anaerosporobacter、Lachnospiraceae UCG-006、Anaerovorax、Lactobacillus、Angelakicella Monoglobus、Asaccharospora、Oscillibacter、Bifidobacteriaceae、Oscillospiraceae UCG-005 Bifidobacterium Paraprevotella Bilophila Parasutterella Blautia Peptococcaceae Butyricicoccus Peptostreptococcaceae Candidatus soleaferrea, Porphyromonadaceae, Carnobacteriaceae, Rikenellaceae, Christensenellaceae, Rhomboutsia, Christensenellaceae R-7, Roseburia, Clostridiales vadinBB60グループ Ruminiclostridium Clostridium sensu stricto 1 Ruminiclostridium 5 Coprococcus 3 Ruminiclostridium 9 Eisenbergiella Ruminococcaceae Enterobacteriaceae Ruminococcaceae UBA1819、Enterococcaceae、Ruminococcaceae UCG-005、Enterococcus、Ruminococcaceae UCG-009、Erysipelotrichaceae、RuminococcaceaeThe present invention provides castalagin or analogues for use in increasing the levels of bacteria of families or genera such as UCG-014, Escherichia / Shigella, Ruminococcus, Family XIII, Sporacetigenium, Family XIII UCG-001, Staphylococcaceae, Flavonifractor, Staphylococcus, Herbinix, Tannerellaceae, Isobaculum, Turicibacter, Lachnospiraceae, Tyzzerella 3 and / or Lachnospiraceae A2, preferably Turicibacter, Bilophila, Ruminococcaceae (e.g., Ruminococcaceae UBA1819), Parasutterella, Clostridium sensu stricto 1, Chrinstensenellaceae, Alistipes, Ruminococcus, Akkermansia and / or Anaeroplasma.

[0083] In some embodiments, the above-described method or use increases the level of bacteria of the family or genus Turicibacter. In some embodiments, the above-described method or use increases the level of bacteria of the family or genus Bilophila. In some embodiments, the above-described method or use increases the level of bacteria of the family or genus Ruminococcaceae (e.g., Ruminococcaceae UBA1819). In some embodiments, the above-described method or use increases the level of bacteria of the family or genus Parasutterella. In some embodiments, the above-described method or use increases the level of bacteria of the family or genus Clostridium sensu stricto 1. In some embodiments, the above-described method or use increases the level of bacteria of the family or genus Akkermansia. In some embodiments, the above-described method or use increases the level of bacteria of the family or genus Anaeroplasma. In further embodiments, the bacteria of the family or genus Akkermansia is Akkermensia muciniphilia.

[0084] In another aspect, the Disclosure provides a method for reducing the levels of bacteria of the family or genus Lactobacillus and / or Pseudoflavonifractor in the intestines of a subject, comprising administering an effective amount of castalagin or an analogue to the subject. In another aspect, the Disclosure provides the use of castalagin or an analogue for reducing the levels of bacteria of the family or genus Lactobacillus and / or Pseudoflavonifractor in the intestines of a subject. In another aspect, the Disclosure provides the use of castalagin or an analogue for manufacturing a medicament for reducing the levels of bacteria of the family or genus Lactobacillus and / or Pseudoflavonifractor in the intestines of a subject. The Disclosure also provides castalagin or an analogue for use in reducing the levels of bacteria of the family or genus Lactobacillus and / or Pseudoflavonifractor in the intestines of a subject.

[0085] This disclosure also provides a combination therapy comprising castalagin or an analogue thereof and an immunotherapy such as an immune checkpoint inhibitor. This disclosure also provides the use of a combination therapy comprising castalagin or an analogue thereof and an immunotherapy such as an immune checkpoint inhibitor (e.g., an immunotherapy agent) for treating a subject with cancer (e.g., cancer resistant to immunotherapy such as immune checkpoint inhibitor monotherapy). This disclosure also provides the use of a combination therapy comprising castalagin or an analogue thereof and an immunotherapy such as an immune checkpoint inhibitor for manufacturing a pharmaceutical product for treating a subject with cancer (e.g., cancer resistant to immunotherapy such as immune checkpoint inhibitor monotherapy). This disclosure also provides a method for treating a subject with cancer (e.g., cancer resistant to immunotherapy such as immune checkpoint inhibitor monotherapy), comprising administering an effective dose of a combination therapy comprising castalagin or an analogue thereof and an immunotherapy such as an immune checkpoint inhibitor to the subject.

[0086] Castalagin (molecular weight 934.63, CAS number 24312-00-3) has the following structure. [ka]

[0087] It is the (33-beta) isomer of vescalagin (molecular weight 934.63, CAS number 36001-47-5) and has the following structure. [ka]

[0088] Castalagin and vescalagin belong to a specific group of ellagitannins, which are composed of a series of highly water-soluble C-glucoside variants.

[0089] Therefore, castalagin analogs include castalagin licosides such as grandinin (lyxose) and robulin E (xylose), casuarinin, and castalin. Castalagin analogs may also be ethoxylated castalagin, as described in WO2014 / 071438. Castalagin analogs retain or share the biological activity of castalagin, more specifically, the ability to improve the immune response (anti-tumor immune response) in subjects and restore the response to immunotherapy, such as immune checkpoint inhibitor therapy. In one embodiment, the castalagin analog is a castalagin salt, preferably a pharmaceutically acceptable salt. The term "salt," as used herein, refers to acidic salts formed with inorganic and / or organic acids, and basic salts formed with inorganic and / or organic bases. Salts for use in pharmaceutical compositions are pharmaceutically acceptable salts. As used herein, the term “pharmaceutically acceptable salt” refers to a salt of castalagin that retains the biological activity of castalagin and is biologically or otherwise undesirable.

[0090] For example, a salt of castalagin may be an acid addition salt of hydrochloric acid, hydrobromic acid, phosphoric acid, acetic acid, trifluoroacetic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, tartaric acid, maleic acid, citric acid, ascorbic acid, methanesulfonic acid or ethanesulfonic acid, or camphoric acid. A salt of castalagin may also be a base addition salt, such as sodium hydroxide or potassium hydroxide, triethylamine or tert-butylamine. Such salts can be formed fairly easily by those skilled in the art using standard techniques. In fact, chemically modifying pharmaceutical compounds (i.e., castalagin) into salts is a well-known technique among pharmaceutical chemists (e.g., H. Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995) at pp. 196 and 1456-1457; P. Stahl et al, Camille G. (eds.), Handbook of Pharmaceutical Salts. Properties, Selection and Use. (2002) Zurich: Wiley-VCH; S. Berge et al, Journal of Pharmaceutical Sciences (1977) 66(1) 1-19; P. Gould, International J. of Pharmaceutics (1986) 33 201-217; Anderson et al, The Practice of Medicinal Chemistry (1996), Academic Press, New York; and The Orange Book (Food & Drug). See Administration, Washington, DC (and their websites). Salts of castalagin can be formed, for example, by reacting castalagin with a certain amount (e.g., equivalent) of acid or base in an aqueous medium in which the salt precipitates, or in which it is subsequently freeze-dried.

[0091] Castalagin may be found or isolated from a variety of sources, including fruit and / or plant extracts, such as extracts from Myrciaria dubia (camu camu) berries, lythrum salicaria (see, e.g., WO / 2016 / 102874), oak (Quercus sp.), chestnut (Castanea sp.), trunk bark extracts of Anogeissus leiocarpus and Terminalia avicennoides (Shuaibu MN et al., Parasitology Research. 103(6):1333-8), and leaves of Syzygium samarangense (Blume) (Kamada et al., Fitoterapia, Volume 129, September 2018, Pages 94-101). Methods for isolating castalagin and / or other C-glucoside elagitannins are well known in the art and are described in some of the above references, as well as in particular in Araujo et al., Rsc Advances, 2015, 5, 96151-96157 and Stine et al., Methods in Molecular Biology (Clifton, NJ), 2011, 670, 13-32).

[0092] For the methods, uses, and therapies described herein, castalagin or its analogues may be used in the form of a suitable amount of castalagin or its analogues in the form of an extract (fruit and / or plant extract), including a crude extract or a concentrated, partially purified extract of castalagin or its analogues, or in a purified form (either isolated from a natural source or synthesized). Thus, in some embodiments, an extract containing castalagin or its analogues is used or administered. In other embodiments, purified or isolated castalagin or its analogues are used or administered. In some embodiments, purified or isolated castalagin or its analogues

[0093] Those skilled in the art will understand that an extract, or purified castalagin or its analogue, can be mixed with one or more carriers and / or excipients (pharmaceutically acceptable carriers and / or excipients) to obtain a composition suitable for administration to a subject.

[0094] When used herein, “excipient” has the ordinary meaning in the art and refers to any component other than the active ingredient (drug) itself. Examples of excipients include buffers, binders, lubricants, diluents, fillers, thickeners, disintegrants, plasticizers, coatings, barrier layer formulations, stabilizers, release retarders, and other components. As used herein, “pharmaceutically acceptable excipient” means any excipient that does not interfere with the efficacy of the biological activity of the active ingredient and is non-toxic to the subject, i.e., an excipient that is a type of excipient and / or intended for use in amounts that are non-toxic to the subject. Excipients are well known in the art, and this composition is not limited in these respects. Carriers / excipients may be suitable for, for example, intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, intraocular, intraventricular, intrasacral, intraspinal, subarachnoid, epidural, intracisional, intraperitoneal, intranasal, or pulmonary (e.g., aerosol) administration. The therapeutic composition is prepared using standard methods known in the art by mixing an active ingredient of desired purity with one or more optional pharmaceutically acceptable carriers, excipients, and / or stabilizers (Remington: The Science and Practice of Pharmacy, by Loyd V Allen, Jr, 2012, 22 nd edition,Pharmaceutical Press、Handbook of Pharmaceutical Excipients,by Rowe et al.,2012,7 th (See edition, Pharmaceutical Press.)

[0095] In some embodiments, castalagin or its analogues are formulated for oral administration. Formulations suitable for oral administration may include (a) an effective amount of the activator / composition suspended in a liquid solution, such as water, saline, or a diluent such as PEG400; (b) capsules, sachets, or tablets each containing a predetermined amount of the active ingredient as a liquid, solid, granule, or gelatin; (c) a suspension in a suitable liquid; and (d) a suitable emulsion. Tablet forms may contain lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and one or more other excipients, colorants, fillers, binders, diluents, buffers, humectants, preservatives, flavorings, dyes, disintegrants, and pharmaceutically suitable carriers. The lozenge form may include an active ingredient in the flavor, such as sucrose, and a pastille containing the active ingredient in the inert base, such as gelatin and glycerin, or a sucrose and acacia emulsion, gel, etc., which contain a carrier known in the art in addition to the active ingredient.

[0096] In some embodiments, castalagin or its analogues are formulated for parenteral administration (e.g., injection). Formulations for parenteral administration may contain, for example, excipients, sterile water or saline solution, polyalkylene glycols such as polyethylene glycol, plant-derived oils, or hydrogenated naphthalene. The release of the compound can be controlled using biocompatible, biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers. Other potentially useful parenteral delivery systems for castalagin or its analogues include ethylene vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients (e.g., lactose), or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or may be gels.

[0097] In some embodiments, castalagin or its analogues are formulated for intestinal delivery, i.e., delivery to the intestines. This can be achieved by methods well known in the art. For example, castalagin or its analogues may be coated or encapsulated with an enteric coating or material. The enteric coating allows, for example, release at a specific pH, or release in the presence of digestive enzymes or bacteria characteristically present at a specific location in the gastrointestinal tract where release is desired (e.g., the small intestine, large intestine, or a specific region thereof). In some embodiments, the enteric coating material is pH-sensitive and affected by changes in pH encountered in the gastrointestinal tract (pH-sensitive release). Typically, the enteric coating material remains insoluble at the pH of the stomach, and then, at the higher pH of the downstream gastrointestinal tract (e.g., often the duodenum, or sometimes the colon), release of the active ingredient becomes possible. In another embodiment, the enteric material comprises an enzymatically degradable polymer that is broken down by bacterial enzymes present in the lower gastrointestinal tract, particularly the colon (e.g., carbohydrate-processing enzymes such as glycosidases, polysaccharide lyases, and carbohydrate esterases).Examples of such enteric-coated materials include cellulose polymers such as hydroxypropylcellulose, hydroxyethylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate succinate, hydroxypropylmethylcellulose phthalate, methylcellulose, ethylcellulose, cellulose acetate, cellulose phthalate acetate, cellulose trimellitate acetate, and sodium carboxymethylcellulose; acrylic acid polymers and copolymers, preferably acrylic acid, methacrylic acid, methyl acrylate, acrylate Eudragit® (Rohm), which includes those formed from methyl methacrylate and / or ethyl methacrylate, and Acryl-EZE® (Colorcon, USA), Eudragit® L30D-55 and L100-55 (soluble at pH 5.5 or higher), Eudragit® L-IOO (soluble at pH 6.0 or higher), Eudragit® S (soluble at pH 7.0 or higher as a result of a greater degree of esterification), and Eudragits® NE, RL and RS (water-insoluble polymers with varying degrees of permeability and expansion). Other methacrylic acid resins marketed under trade names such as Pharma (Westerstadt, Germany); vinyl polymers and copolymers such as polyvinylpyrrolidone, vinyl acetate, vinyl acetate phthalate, vinyl acetate crotonic acid copolymer, and ethylene-vinyl acetate copolymer; enzymatically digestible polymers such as azopolymers, pectin, chitosan, amylose, and guar gum; and zein and shellac. Combinations of different enteric-coated materials may also be used.Approaches for colon-specific drug delivery are well known in the art (see, for example, Philip et al., Oman Med J. 2010 Apr;25(2):79-87 and Lee et al., Pharmaceutics. 2020 Jan;12(1):68), and include pH-dependent systems (e.g., using pH-dependent polymers), receptor-mediated systems, magnetically driven systems, delayed or time-dependent systems, microorganism-triggered drug delivery systems (e.g., including glycosyl polymers that can be degraded by enzymes produced by the colonic microbiota, such as glucuronidase, xylosidase, arabinosidase, and galactosidase), pressure-controlled colon delivery capsules (drug release induced by higher pressure encountered in the colon), osmotically controlled drug delivery, and any combination of these approaches (e.g., colon-targeted delivery systems (CODES™) using a combination of pH-dependent and microorganism-triggered drug delivery approaches).

[0098] In one embodiment, castalagin or its analogue is formulated in capsules made from enteric-coated material (enteric-coated capsules).

[0099] Any suitable amount of castalagin or its analogues may be administered to the subject. The dosage depends on many factors, including the mode of administration. Typically, the amount of castalagin or its analogue contained in a single dose is sufficient to effectively prevent, delay, or treat cancer without inducing significant toxicity.

[0100] For the prevention, treatment, or reduction of the severity of a given disease or condition (cancer), the appropriate dosage of castalagin or its analogues depends on the type of disease or condition being treated, the severity and course of the disease or condition, whether castalagin or its analogues are administered for preventive or therapeutic purposes, previous therapies, the patient's clinical history, and response to castalagin or its analogues, as well as the discretion of the attending physician. Castalagin or its analogues are preferably administered to the patient in a single dose or over a series of treatments. Preferably, it is desirable to determine the dose-response curve in vitro, and then in a useful animal model, before testing in humans. This disclosure provides dosages for castalagin or its analogues and compositions containing them. For example, depending on the type and severity of the disease, approximately 1 μg / kg to 1000 mg (mg / kg) per kilogram of body weight per day. Furthermore, the effective dose may be 0.5 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg / 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, or 200 mg / kg, and may increase in 25 mg / kg increments up to a maximum of 1000 mg / kg, or may be within a range between any two of the above values. A typical daily dose may be in the range of approximately 1 μg / kg to 100 mg / kg or greater, depending on the factors mentioned above. For repeated administration over several days or longer, treatment is maintained, depending on the condition, until the desired suppression of disease symptoms (e.g., reduction in tumor volume or tumor cell count) occurs. However, other dosage regimens may also be useful. The progress of this therapy is readily monitored by conventional techniques and assays. In one embodiment, the dosage for administration to human subjects corresponds to a dosage of at least 0.8 mg of castalagin / kg in mice.

[0101] As used herein, the term immunotherapy refers to antitumor treatments that enhance or elevate the immune response against tumor cells. Immunotherapy includes cell-based immunotherapy, such as the administration of tumor cells, including chimeric antigen receptor (CAR) T cells and NK cells, or T cells with tumor antigen-specific TCRs, or antigen-presenting cells (APCs such as dendritic cells) that can express tumor antigens on their surface. Immunotherapy also includes the administration of specific antibodies that recognize antigens expressed by tumor cells and target them for destruction by the immune system, or the administration of cytokines (interferons, interleukins) that stimulate the immune response. Another type of immunotherapy involves the administration of immune checkpoint inhibitors. Combinations of different types of immunotherapy, such as the administration of immune cells (CAR T or NK cells) in combination with immune checkpoint inhibitors, may also be used.

[0102] As used herein, the terms “immune checkpoint inhibitor” (ICI) or “immune checkpoint blocker” (ICB) refer to agents that block or inhibit the activity of negative regulators of the immune response. Examples of such negative regulators of the immune response (i.e., immune checkpoints) include adenosine A2A receptor (A2AR), B7-H3 (CD276), B7-H4 (VTCN1), B and T lymphocyte attenuators (BTLA or CD272), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin-like receptor (KIR), lymphocyte activator gene-3 (LAG3), nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2 (NOX2), programmed cell death 1 (PD-1) receptor, and PD-L1. PD-L2, T cell immunoglobulin domain and mucin domain 3 (TIM-3), V-domain Ig inhibitor of T cell activation (VISTA), and sialic acid-binding immunoglobulin lectin 7 (SIGLEC7 or CD328) and SIGLEC9 (CD329). In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-1, or PD-L1. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1, such as an anti-PD-1 antibody. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L1, such as an anti-PD-L1 antibody. In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4, such as an anti-CTLA-4 antibody.

[0103] Cancer may be any type of cancer, including primary (or original) cancer, recurrent cancer, or metastatic cancer. Examples of cancers include cardiac sarcoma, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), bronchogenic carcinoma (squamous cell, anaplastic small cell, anaplastic giant cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma (e.g., Ewing's sarcoma, Kaposi's sarcoma), lymphoma, chondrotoxic hamartoma, mesothelioma; cancers of the gastrointestinal system, e.g., esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (stomach) (carcinoma, lymphoma, leiomyosarcoma), gastric (gastric), pancreas (tubular adenocarcinoma, insulinocarcinoma) Cancers of the genitourinary tract, such as kidney cancer (adenocarcinoma, Wilms' tumor [nephroblastoma], lymphoma, carcinoid tumor, bipoma), small intestine (adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine (adenocarcinoma, tubular adenoma, chorioadenoma, hamartoma, leiomyoma), urogenital cancers, such as kidney cancer (adenocarcinoma, Wilms' tumor [nephroblastoma], lymphoma, leukemia), bladder and / or urethral cancer (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate cancer (adenocarcinoma, sarcoma), and testicular cancer (seminoma, teratoma, fetal carcinoma, malformation Cancers, choriocarcinomas, sarcomas, stromal cell carcinomas, fibromas, fibroadenomas, adenomatous tumors, lipomas; liver cancers, e.g., hepatoma (hepatocellular carcinoma, HCC), intrahepatic cholangiocarcinoma, hepatoblastoma, vascular sarcoma, hepatocellular adenoma, hemangioma, pancreatic endocrine tumors (pheochromocytoma, insulinoma, vasoactive intestinal peptide tumor, islet cell tumors and glucagonoma, etc.); bone cancers, e.g., osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, malignant lymphoma (reticular cell sarcoma), multiple myeloma, malignant giant cell tumor, chordoma, osteochondroma (osteo) Chronoblastoma (osteochondrosis), benign chordoma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumor; cancers of the nervous system, e.g., neoplasms of the central nervous system (CNS), primary CNS lymphoma, skull cancer (osteoma, hemangioma, granuloma, xanthomas, osteoosteitis), meninges (meningioma, meningiosarcoma, gliomatosis), brain cancer (astrocytoma, medulloblastoma, glioma, ependymoma, germ cell tumor [pineal glandoma], glioblastoma multiforme, oligodendroglioma, Schwann cell tumor, retinoblastoma, congenital tumor), spinal neurofibroma, meningioma, glioma, sarcoma);Cancers of the reproductive system, such as gynecological cancers, uterine cancer (endometrial carcinoma), cervical cancer (cervical carcinoma, preneoplastic cervical dysplasia), ovarian cancer (ovarian carcinoma [serous pancreatic cystadenoma, mucocystic carcinoma, undifferentiated carcinoma], granulosa follicular cell tumor, Sertoli-Leydegg cell tumor, undifferentiated germ cell tumor, malignant teratoma), vulvar cancer (squamous cell carcinoma, carcinoma in situ, adenocarcinoma, fibrosarcoma, melanoma), vaginal cancer (clear cell carcinoma, squamous cell carcinoma, staphyloid sarcoma (fetal rhabdomyosarcoma), fallopian tube cancer (carcinoma); placental cancer, penile cancer, prostate cancer , testicular cancer; blood cancers, e.g., hematological cancers (acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), myeloproliferative disorders, multiple myeloma, myelodysplastic syndromes), Hodgkin's disease, non-Hodgkin lymphoma [malignant lymphoma]; oral cancers, e.g., lip cancer, tongue cancer, gingival cancer, palate cancer, oropharyngeal cancer, nasopharyngeal cancer, maxillary sinus cancer; skin cancers, e.g., malignant melanoma, cutaneous melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, lentigo dysplastic nevi (moles) Examples include dysplastic neoplasms, lipomas, hemangiomas, dermatofibromas, and keloids; adrenal cancer; neuroblastoma; and cancers of other tissues including connective tissue and soft tissue, retroperitoneal space, and peritoneum; eye cancer, melanoma, and adnexal cancer; breast cancer (e.g., ductal carcinoma); head and / or neck cancer (head and neck squamous cell carcinoma); anal cancer; thyroid cancer; parathyroid cancer; secondary and unspecified malignant neoplasms of lymph nodes; secondary malignant neoplasms of the respiratory and digestive systems; and secondary malignant neoplasms of other sites.

[0104] Immune checkpoint inhibitors are used to treat lung cancer (e.g., non-small cell lung cancer (NSCLC) and small cell lung cancer, squamous cell lung carcinoma), head and neck cancer (e.g., head and neck squamous cell carcinoma, renal cell carcinoma, gastric adenocarcinoma, nasopharyngeal neoplasm, urothelial carcinoma, colorectal cancer, mesothelioma (e.g., pleural mesothelioma), breast cancer (e.g., triple-negative breast cancer, TNBC), esophageal neoplasm, multiple myeloma, gastric and gastroesophageal junction cancer, gastric adenocarcinoma, For several cancers, including melanoma, Merkel cell carcinoma (MCC), lymphoma (e.g., Hodgkin and non-Hodgkin lymphoma, diffuse large B-cell lymphoma), liver cancer (e.g., hepatocellular carcinoma), melanoma, ovarian cancer, fallopian tube cancer, peritoneal neoplasm, bladder cancer, transitional cell carcinoma, prostate neoplasm, and cholangiocarcinoma, the immune checkpoint inhibitor is either already approved or currently being tested in Phase III and Phase IV clinical trials (see, for example, Darvin et al., Experimental & Molecular Medicine volume 50, Article number: 165 (2018)). Therefore, in one embodiment, the cancer is one of the aforementioned cancers for which an immune checkpoint inhibitor is either already approved or currently being tested in Phase III and Phase IV clinical trials.

[0105] Currently approved immune checkpoint inhibitors include anti-CTLA-4 ipilimumab (melanoma and lung cancer), anti-PD-1 nivolumab (melanoma, lung cancer, renal cell carcinoma, Hodgkin lymphoma, head and neck cancer, colon cancer, and liver cancer), pembrolizumab (melanoma, lung cancer, head and neck cancer, Hodgkin lymphoma, renal cell carcinoma, and gastric cancer), and cemiplimab (squamous cell carcinoma, myeloma, and lung cancer), and anti-PD-L1 atezolizumab (NSCLC, small cell lung cancer, TNBC), avelumab (NSCLC, MCC), and durvalumab (urothelial carcinoma, lung cancer). Therefore, in one embodiment, the cancer is one of the aforementioned cancers for which an immune checkpoint inhibitor has already been approved. In further embodiments, the cancer is resistant to PD-1 inhibitor-based therapy (anti-PD-1 therapy) and is melanoma, lung cancer, renal cell carcinoma, Hodgkin lymphoma, head and neck cancer, colon cancer, liver cancer, gastric cancer, squamous cell carcinoma, or myeloma.

[0106] In some embodiments, the above-described treatment involves the use / administration of more than one (i.e., combination) of an active / therapeutic agent, castalagin, or analogues thereof, in combination with an immune checkpoint inhibitor (i.e., combination therapy). The combination of agents may be administered in any conventional dosage form or co-administered (e.g., consecutively, simultaneously, or at different times). Co-administration in the context of this disclosure refers to the administration of one or more therapeutic agents in the course of coordinated treatment to achieve improved clinical outcomes. Such co-administration may also occur coexistently, i.e., over overlapping periods. For example, the first agent may be administered to the patient before, simultaneously with, before and after, or after the administration of the second active agent. In some embodiments, the agents may be combined / formulated in a single composition and therefore administered simultaneously. Alternatively, they may be formulated in separate compositions and therefore administered separately (simultaneously or at different times).

[0107] In some embodiments, the dose of castalagin or its analogues and / or immune checkpoint inhibitors used / administered in the methods, uses, compositions and combination therapies of this disclosure is a suboptimal dose. As used herein, “suboptimal dose” is a dose of one compound (castalagin or its analogues and / or immune checkpoint inhibitors) in a combination described herein that, when used in the absence of the other compounds in the combination, produces a biological effect of 50% or less, in some embodiments 40% or less, in further embodiments 30% or less, in further embodiments 20% or less, and in further embodiments 10% or less. Thus, the use of the compound combinations described herein may achieve increased efficacy / biological effect at a comparable suboptimal dose compared to the use of the compound in the absence of the others, when one or more compounds in the combination are used at a suboptimal dose.

[0108] As used herein, a synergistic effect is achieved when the effect of a combined compound is greater than the theoretical sum of the effects of each agent in the absence of the other compound. One potential advantage of combination therapies with synergistic effects is that lower doses (e.g., below-optimal doses) of one or both of the drugs or therapies may be used to achieve high therapeutic activity with low toxicity. In one embodiment, the combination therapy (castalagin or an analogue thereof and an immune checkpoint inhibitor) increases the effect by at least 5% compared to the predicted theoretical additive effect of the drugs. In a further embodiment, the combination therapy increases the effect by at least 10% compared to the predicted theoretical additive effect of the drugs. In a further embodiment, the combination therapy increases the effect by at least 20% compared to the predicted theoretical additive effect of the drugs. In a further embodiment, the combination therapy increases the effect by at least 30% compared to the predicted theoretical additive effect of the drugs. In a further embodiment, the combination therapy increases the effect by at least 50% compared to the predicted theoretical additive effect of the drugs. A further advantage of using combination drugs is that efficacy can be achieved in situations where either drug alone would not be effective, for example, against cancer or tumors resistant to immune checkpoint inhibitors. Resistance means that monotherapy with an immune checkpoint inhibitor does not result in a significant therapeutic effect, such as a significant reduction in tumor volume or tumor cell count. Examples of cancers in which resistance to immune checkpoint inhibitors has been reported in patients and / or animal models include lung cancer (e.g., NSCLC), pancreatic cancer, prostate cancer, melanoma, ovarian cancer, urothelial carcinoma, and renal cell carcinoma (see, e.g., Fares et al., American Society of Clinical Oncology Educational Book 39, 147-164, 2019; Pandey et al., Cancer Drug Resist 2019; 2: 178-188).

[0109] Castalagin or its analogues and / or immune checkpoint inhibitors may be administered / used in combination with one or more additional active agents or therapies (chemotherapy, radiotherapy, surgery, vaccines, immunotherapy, etc.) for the treatment of a targeted disease / condition (cancer) or for the management of one or more symptoms of a targeted disease / condition (e.g., analgesics, anti-nausea agents, etc.). In some embodiments, castalagin or its analogues and / or immune checkpoint inhibitors may be used in combination with one or more chemotherapeutic agents, immunotherapies (e.g., using CAR T cells or CAR NK cells), antibodies, cell-based therapies, etc. Examples of chemotherapeutic agents suitable for use in combination with castalagin or its analogues and / or immune checkpoint inhibitors include, but are not limited to, vinca alkaloids, agents that inhibit microtubule formation (such as colchicine and its derivatives), anti-angiogenic agents, therapeutic antibodies, EGFR targeters, tyrosine kinase targeters (such as tyrosine kinase inhibitors), transition metal complexes, proteasome inhibitors, antimetabolites (such as nucleoside analogues), alkylating agents, platinum-based drugs, anthracycline antibiotics, topoisomerase inhibitors, macrolides, retinoids (such as all-trans retinoic acid or its derivatives), geldanamycin or its derivatives (such as 17-AAG), and other cancer therapies recognized in the art.In some embodiments, chemotherapeutic agents for use in combination with castalagin or its analogues and / or immune checkpoint inhibitors include adriamycin, colchicine, cyclophosphamide, actinomycin, bleomycin, duanorubicin, doxorubicin, epirubicin, mitomycin, methotrexate, mitoxantrone, fluorouracil, carboplatin, carmustine (BCNU), methyl-CCNU, cisplatin, etoposide, interferon, camptothecin and its derivatives, phenesterine, taxane and its derivatives (e.g., taxol, paclitaxel and its derivatives). This product contains one or more of the following: conductors, taxoteres and their derivatives, topetecan, vinblastine, vincristine, tamoxifen, piposulfan, nab-5404, nab-5800, nab-5801, irinotecan, HKP, ortataxel, gemcitabine, oxaliplatin, Herceptin®, vinorelbine, Doxil®, capecitabine, Alimta®, Avastin®, Velcade®, Tarceva®, Neulasta®, lapatinib, sorafenib, erlotinib, erbitux, and their derivatives.

[0110] The subject may be any animal, more specifically, a mammal such as a mouse, rat, dog, or human. In one embodiment, the subject is a human.

[0111] This disclosure is further illustrated by the following non-limiting embodiments.

[0112] Materials and Methods: Mouse studies. All animal studies were approved by the Institutional Animal Care Committee (CIPA) and conducted in accordance with Canadian Council on Animal Care guidelines. Mouse experiments were performed using 7-week-old female C57BL / 6 mice obtained from Charles River, Canada. Sterile female C57BL / 6 mice were purchased from the International Microbiome Centre Germ-Free Facility (University of Calgary, Canada) and maintained at the CR-CHUM Germ-Free Facility.

[0113] Cell culture, reagents, and tumor cell lines. MCA-205 fibrosarcoma cells and E0771 mammary gland adenocarcinoma cells, Class I MHC H-2 for C57BL / 6 mice. b Syngeneic cell lines were used in this study. MCA-205 cells were cultured at 37°C in Roswell Park Memorial Institute (RPMI) 1640 (Gibco-Invitrogen) containing 10% fetal bovine serum (FBS) (Wisent), 2 mM L-glutamine (Wisent), 100 IU / ml penicillin / streptomycin (Wisent), 1 mM sodium pyruvate (Wisent), and MEM non-essential amino acids (Gibco-Invitrogen) in the presence of 5% CO2. E0771 cells were cultured at 37°C in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco-Invitrogen) containing 10% FBS (Wisent), 2 mM L-glutamine, 100 IU / ml penicillin / streptomycin (Wisent), and 1 mM sodium pyruvate (Wisent) in the presence of 5% CO2.

[0114] Subcutaneous models of MCA-205 sarcoma and E0771 breast cancer. 0.8 × 10⁶ mice were used in syngeneic C57BL / 6 mice. 6 Individual MCA-205 or 0.5 × 10 6Several E0771 cells were subcutaneously transplanted. The tumors were 20-35 mm in size. 2 When the mice reached a certain size, they were treated intraperitoneally (ip) with anti-PD-1 monoclonal antibody (mAb) (250 μg / mouse, clone RMP1-14, BioXcell) or isotype control (clone 2A3, BioXcell) for four doses every three days (or twice for flow cytometry analysis; see section below). At the start of treatment, the mice received forced oral administration once daily with the following product: Myrciaria dubia, camu camu (CC) raw extract (Sunfood) (200 mg / kg per mouse), fractions from extraction round 1 (P, INT, NP, Insol) (40.18 mg / kg per mouse), fractions from extraction round 2 (P1, P2, P3 and P4) (equivalent to the dose of fraction P at 40.18 mg / kg per mouse), vescalagin (extracted from CC, see isolation process below) (0.85 mg / kg per mouse), ellagic acid (0.85 mg / kg per mouse) (Sigma-Aldrich), urolithin A (0 The mice were administered 0.85 mg / kg (Sigma-Aldrich), castalin (0.5 mg / kg per mouse) (PhytoProof(C), Sigma-Aldrich), and castalagin (2.55 mg / kg per mouse) at different concentrations: 1 / 8, 1 / 6, 1 / 4, 1 / 2 of the standard concentration, the standard concentration (0.85 mg / kg per mouse), 1.5 times (1.25 mg / kg per mouse), and 3 times increased concentrations (PhytoProof(C), Sigma-Aldrich, or isolated from food-grade oak; see isolation process below). In the control group, mice received forced oral administration once daily with water (100 μl), and tumor area was regularly monitored every 3 days using calipers. In depletion experiments, anti-CD8 mAb (150 μg / mouse, clone 53-6.7, BioXcell) or isotype control (clone 2A3, BioXcell) was used.

[0115] Antibiotic treatment. For the antibiotic (ATB) experiment, mice were treated with an ATB solution containing ampicillin (1 mg / ml), streptomycin (5 mg / ml), and colistin (1 mg / ml) (Sigma-Aldrich) added to sterile drinking water, as previously described (Routy et al, Science, 2018 Jan 5;359(6371):91-97. Epub 2017 Nov 2). Antibiotic activity was confirmed weekly under aerobic and anaerobic conditions by culturing fecal pellets resuspended at 0.1 g / ml in Brain Heart Infusion (BHI) medium + 15% glycerol at 37°C for 48 hours on COS (Columbia agar containing 5% sheep blood) plates. For fecal microbiota transplantation (FMT) experiments in SPF-fed mice, the mice received the same combination of ATB for 3 days prior to FMT.

[0116] Fecal microbiota transplantation (FMT) experiments. FMT was performed as previously published by Routy et al. by thawing fecal material from five different non-small cell lung cancer (NSCLC) patients suitable for immune checkpoint blockers (ICIs) after appropriate ethical approval at the Centre de recherche du Centre hospitalier de l'Universite de Montreal (CRCHUM) in Montreal. Patient records were retrospectively analyzed to identify their response status. Two weeks after FMT, tumor cells were subcutaneously injected, and mice were treated with anti-PD-1 mAb or isotype control + / -CC, castalagin, or water as described above.

[0117] Flow cytometry analysis. Tumors and spleens were harvested 9 days after the first injection of anti-PD-1 mAb into mice with MCA-205 tumors, and 11 days after the first injection of anti-PD-1 mAb into mice with E0771 tumors. The excised tumors were cut into small pieces and digested at 37°C for 30 minutes in RPMI medium containing 25 μg / mL of Liberase® (Roche) and 150 μg / mL of DNase I (Roche). The tissue was then pulverized using 100 and 70 μm cell strainers (Fisher Scientific) and filtered twice. The spleens were pulverized in RPMI medium and then filtered through a 100 μm cell strainer. Prior to membrane staining with anti-mouse antibodies for CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.7), CD44 (IM7), CD45 (30-F11), CD45RB (C363-16A), CD62L (MEL-14), Foxp3 (FJK-16s), CXCR3 (CXCR3-173), CCR9 (CW-1.2), PD-1 (29F.1A12), PD-L1 (MIH5), and ICOS (7E.17G9) (BD, BioLegend, R&D, and eBioscience), 2 million cells or splenocytes were pre-incubated at 4°C for 30 minutes with purified anti-mouse CD16 / CD32 (clone 93, eBioscience). For intracellular staining, the Foxp3 staining kit (eBioscience) was used. Dead cells were removed using the Live / Dead Fixable Aqua Dead Cell Staining Kit (Life Technologies). Samples were acquired using a BD Fortessa 16-color cytometer (BD), and analysis was performed using FlowJo software (BD).

[0118] Immunofluorescence staining. Mouse tumors stored in a compound at the optimal cutting temperature (OCT) were cut (5 μm thick sections), mounted on microscope slides, and stored at -80°C. At the start of the experiment, the slides were air-dried and washed with cold acetone. An Endogenous Avidin Biotin Blocking Kit (ThermoFisher) was used to prevent nonspecific binding to biotin. In addition, the tissues were incubated with 10% donkey serum to reduce background staining. The primary antibodies used were anti-CD4, anti-CD8, and anti-Foxp3. Donkey anti-goat and donkey anti-rat antibodies conjugated to AF-488 were used as secondary antibodies, and the slides were incubated with Cy3-streptavidin to detect biotinylated antibodies. Nuclei were visualized by counterstaining with DAPI (ThermoFisher). Images were generated using an Olympus BX61VS hall slide scanner (with a 20×0.75NA objective lens and a resolution of 0.3225mm). The images were analyzed using Visiomorph software (Visiopharm).

[0119] HPLC and LC-MS systems were used. A 1260 Infinity LC system connected to an Agilent Technologies 6120 Quadrupole LC / MS mass spectrometer was used for reversed-phase chromatography (C18) and mass spectrometry (MS), respectively. X-Select SCH and HSS columns (Waters) were used for HPLC. For chromatography, a two-component solvent system of MilliQ® water (solvent A) and acetonitrile (ACN) (solvent B) was used, each acidified with 0.1% formic acid (FA). This polarity was optimal for polyphenols in the acidified solution; therefore, only negative ionization data are reported.

[0120] Camu Camu (CC) Extraction. Polyphenols in CC were extracted according to a slightly modified procedure of Fracassetti et al. (Food Chem 2013 15;139(1-4):578-88). Lyophilized raw CC extract (SunFood) was extracted with 50% methanol (MeOH) aqueous solution in a ratio of 1:15 (g:mL) (analytical experiment) or 1:8 (preparative experiment). The suspension was vortex-stirred, sonicated, and incubated at room temperature for 60 minutes. The suspension was centrifuged, and the supernatant was collected. A second extraction was performed using 90% MeOH aqueous solution after sedimentation. Before analysis, the supernatants from both extracts were combined and filtered.

[0121] Analysis of camu camu (CC) extract and identification of LC-MS peaks. After extracting polyphenols from CC, the combined extract was injected into an LC-MS system for analysis. The solvent gradient used to separate the components in the sample was adapted from Fracassetti et al., 2013. The relative retention time at 254 nm and negative ion mass spectrum from the LC-MS analysis were compared with those from the characterization of CC polyphenols by Fracassetti et al., 2013. Peak identity was tentatively assigned based on the agreement between the data of this invention and the data reported by Fracassetti et al., 2013.

[0122] Identification of the active fraction P isolated from fractionation round 1 of camu camu (CC). To evaluate which components of CC are responsible for its activity, four fractions—polar (P), intermediate polar (M), nonpolar (NP), and insoluble (INS)—were generated by reverse-phase chromatography and serial extraction. Polyphenols were extracted from CC, concentrated to dry, and then redissolved in a mixture of 40% ACN:10% MeOH in water to solubilize most of the polyphenols. Insoluble material was separated and discarded by filtration. The same solvent gradient used for CC peak identification (described above) was used for preparative HPLC. Fractions were manually collected over a total of 60 minutes at 10-minute intervals. The fractions were then frozen at -80°C and lyophilized. The three fractions from 30–60 minutes were combined to generate fraction NP. Fraction P was generated using HPLC column breakthroughs from 0–10 minutes as the starting point. Briefly, four Strata C18-E solid-phase extraction (SPE) columns (Phenomenex) were set up in parallel and prepared with MeOH. HPLC breakthroughs from 0–10 minutes, lyophilized, were dissolved in Milli-Q water to a concentration of 10 mg / mL. 5 mL of sample (10 mg / mL) was added to each column, and the flow-through was collected. Then, 9 mL of 5% ACN was added to the column, and the flow-through was collected. The flow-throughs from each column were combined and lyophilized to produce fraction P. Fractions M and INS were produced by sequential extraction of the raw CC extract in water, removing highly polar compounds, 50% MeOH, and 90% MeOH. Fraction M consisted of the 50% MeOH extract, which was evaporated and then lyophilized. The INS fraction consisted of the dried precipitate after all extraction steps were completed.

[0123] Identification of the active fraction P3, fractionation round 2. To evaluate which components of fraction P are responsible for its activity, four fractions (P1, P2, P3, and P4) were generated. A novel solvent gradient was developed to focus on the polar polyphenols contained in fraction P. The gradient method was as follows: 0%B at 0 min, 16%B at 30 min, 95%B at 35 min, and 100%B from 36 to 46 min. For the generation of fraction P, the HPLC breakthrough from 0 to 10 minutes (fractionation round 1) dissolved in MilliQ water was used as the starting point for fractionation round 2. The fractions were manually collected every minute over 30 minutes. The fractions from each trial were analyzed by LC-MS, then lyophilized and combined to generate fractions P1, P2, P3, and P4 as follows. Fraction P1 was created by combining the 0-5 minute period, Fraction P2 by combining the 5-17 minute period, Fraction P3 by combining the 18-19 minute period, and Fraction P4 by combining the 20-30 minute period.

[0124] Characterization of fraction P3. The purity of the castalagin peak in fraction P3 was determined by peak integration of the analytical LC-MS chromatogram at 254 nm. Castalagin analytical standards dissolved in Milli-Q water were used for comparison of retention times and negative ion mass spectra at 254 nm. Both fraction P3 and the castalagin analytical standards were dissolved in D2O for NMR analysis. 1 H, 1 H- 1 H-correlation spectroscopy (COSY), and 1 H- 13 The 13C heteronuclide single quantum coherence (HSQC) NMR spectra were recorded using a Bruker AVIIIHD 500MHz NMR spectrometer. The peaks were compared with the castalagin structure reassignments by Matsuo et al., 2015 (Org Lett 2015 Jan 2;17(1):46-9. Epub 2014 Dec 12).

[0125] Isolation of castalagin and vescalagin from camu camu (CC) and food-grade oak. 20 grams of lyophilized CC powder was extracted as described above. The crude extract was pre-fractionated using a Strata C18-E SPE column. Briefly, 3 mL of the redissolved crude extract was loaded onto the SPE column, and 2 mL of MilliQ water was added to remove ascorbic acid. Then, 9 mL of 5% ACN was added to each column, and the flow-through was collected in 3 mL batches. Castalagin and vescalagin were then purified from the flow-through by HPLC. The isolates were analyzed by LC-MS to assess their purity.

[0126] Synthesis, purification, and characterization of fluorescein-castalagin. Castalagin was monofunctionalized with fluorescein via transesterification with 5 / 6-carboxyfluorescein succinimidyl ester (fluorescein-NHS). Briefly, castalagin was dissolved in DMF and then reacted with fluorescein-NHS (2 equivalents) in the presence of triethylamine (2 equivalents) and 4-dimethyaminopyridine. After workup using DOWEX 50WX8 resin, the crude mixture was analyzed by LC-MS. The peak corresponding to monofunctionalized fluorescein-castalagin was isolated by HPLC. Bacteria R. bromii, E. coli, and B. thetaoitomicron were stained at 37°C and 0°C in the presence of fluorescein-bound castalagin, and in the presence of unbound castalagin at a 100-fold concentration.

[0127] Inverted epifluorescence microscopy. Images of fluor-octalagine stained samples were acquired using an inverted optical microscope (Ti2, Nikon, Inc.) configured for epifluorescence and equipped with a high-sensitivity CCD camera (C14440-20UP, Hamamatsu, Inc.).

[0128] Genomic DNA extraction from mouse feces. Whole genomic DNA was extracted from fecal pellets using the ZymoBIOMICS DNA Miniprep Kit (Zymo Research Corporation) and immediately stored at -80°C. This protocol includes a bead disruption step to ensure complete recovery of bacterial DNA. DNA concentration and quality were measured using Nanodrop ND-1000 (ThermoFisher).

[0129] Quantitative real-time PCR (qRT-PCR) was performed to evaluate the relative levels of total bacterial DNA. The V6 region of the 16S rRNA gene was amplified using primer sets 891F (5'-TGGAGCATGTGGTTTAATTCGA-3', SEQ ID NO: 1) and 1033R (5'-TGCGGGACTTAACCCAACA-3', SEQ ID NO: 2) (Anhe et al. Diabetologia. 2018 Apr;61(4):919-931). The relative levels of Ruminococcaceae DNA were specifically evaluated using specific primers F (5'-ACTGAGAGGTTGAACGGCCA-3', SEQ ID NO: 3) and R (5'-CCTTTACACCCAGTAAWTCCGGA-3', SEQ ID NO: 4) (Garcia-Mazcorro JF et al FEMS Microbiol Ecol 2012;80(3):624-36). The extracted DNA (400 ng / well) was combined with the aforementioned 500 nM primer mix and 1× qPCRBIO SyGreen blue Mix Hi-ROX (PCRBIOSystems). The qPCR reaction was performed using an Applied Biosystems StepOnePlus Real-Time PCR System (ThermoFisher Scientific), denaturing the DNA at 95°C for 3 minutes, followed by amplification over 40 cycles of 5 seconds at 95°C and 30 seconds at 60°C, and completed at the melting curve stage. The raw threshold cycle (Ct) values ​​were compared to bacterial standard curves generated with Escherichia coli DNA for 16s analysis and bacterial standard curves generated with Ruminoccocus bicirculans for Rumniccocaceae analysis to approximate bacterial load.

[0130] Mouse fecal samples were processed and analyzed using 16S rRNA gene sequencing. Isolated DNA was analyzed using 16S ribosomal RNA (rRNA) gene sequencing to investigate the microbial composition of the fecal samples. The V3-V4 region of the 16S rDNA gene was amplified by PCR using a transposon-based Illumina Nextera adapter (Illumina) and primers Bakt_341F (5'-CCTACGGGNGGCWGCAG-3', SEQ ID NO: 5) and Bakt_805R (5'-GACTACHVGGGTATCTAATCC-3', SEQ ID NO: 6), which were adapted to incorporate a sample barcode enabling multiple paired-end sequencing. The PCR mixture contained 1×Q5 buffer (NEB), 1×Q5 enhancer (NEB), 200 μM dNTPs (VWR International), 0.2 μM forward and reverse primers (Integrated DNA Technologies), 1 unit of Q5 (NEB), and 1 μl of template DNA in 50 μl of reaction material. The PCR cycling conditions consisted of initial denaturation at 98°C for 30 seconds, followed by 15 cycles of the first set (10 seconds at 98°C, 30 seconds at 55°C, and 30 seconds at 72°C), then 15 cycles of the second step (10 seconds at 98°C, 30 seconds at 65°C, and 30 seconds at 72°C), followed by a final extension at 72°C for 2 minutes, and then cooling to 4°C without a set time. The PCR product was purified using 35 μl of magnetic beads per 50 μl of PCR reaction material (AxyPrep Mag PCR Cleanup Kit, Axygen Biosciences). Amplification was controlled using a Bioanalyzer 2100 with a DNA 7500 chip (Agilent Technologies). Samples were pooled in equimolar ratios, the pools were re-purified as described above, and quality was verified using a Bioanalyzer 2100 with a DNA high-sensitivity chip. The pools were quantified using PicoGreen (Life Technologies) and loaded into a MiSeq system (Illumina).High-throughput sequencing was performed at IBIS (Institut de Biologie Integrative et des Systemes-Universite Laval).

[0131] Gene sequencing and analysis were performed using R v4.0.0. The DADA2R package v1.16.0 (Callahan et al., Nat Methods. 2016 Jul;13(7):581-3) was used to generate accurate amplicon sequence variants (ASVs) for each sample from the raw amplicon sequences. The sequences were corrected for Illumina amplicon sequence errors, deduplified, chimeras removed, and paired-end reads were merged with 260 bases for forward reads and 190 bases for reverse reads. Taxonomy assignment was performed against the SILVA reference database v138 (Quast et al., Nucleic Acids Res. 2013 Jan;41 (database registration):D590-6). Residual sequences of Archea and Eukaryota were removed. Downstream analysis was performed at the genus level using the phyloseq R package v1.32.0 (McMurdie et al., PLoS One. 2013 Apr 22;8(4):e61217). Alpha diversity was estimated using the Shannon diversity index and the Inverse Simpson index. These indices were compared between groups using the Mann-Whitney U test.

[0132] Statistical analysis. Statistical analysis was performed using R v4.0.0. The Mann-Whitney U test was used to determine significant differences between different groups using alpha diversity, which represents the diversity in each individual sample measurement. Differential abundance analysis at the genus level was performed using DESeq2 (Love et al., Genome Biol. 2014;15(12):550). Spearman rank correlation tests were obtained using Graphpad Prism 8 and used to compare continuous variables between flow cytometry analysis parameters against significant bacteria identified using differential abundance analysis in the water vs. CC, water / αPD-1 vs. CC / αPD-1, water / αPD-1 vs. castalagin / αPD-1, and water / IsoPD-1 vs. castalagin / αPD-1 groups for the MCA-205 and E0771 tumor models. Unless otherwise noted, all p-values ​​are reported after Bonferroni correction when dealing with experimental conditions with more than two problems. The p-values ​​were two-sided with 95% confidence intervals: *p<0.05, **p<0.01, and ***p<0.001.

[0133] Example 1: Administration of camu camu (CC) extract enhances or restores anti-PD-1 antitumor activity in a mouse tumor model. To discover approaches to enhance or restore the antitumor activity of ICBs such as anti-PD-1 antibodies, syngeneic C57BL / 6 mice transplanted with MCA-205 sarcoma tumor cells (anti-PD-1 sensitive) were administered a crude extract from camu camu (Myrciaria dubia), an Amazonian fruit with a unique phytochemical profile, in combination with anti-PD-1 according to the protocol shown in Figure 1A. The camu camu extract used in the experiments described herein is raw camu camu powder, commercially available from Sunfood, obtained from camu camu berries from the South American rainforest that have been dried at low temperatures and ground into a fine powder.

[0134] The results shown in Figures 1B-1C indicate that once-daily forced oral administration of CC extract alone exhibits anticancer activity (similar to anti-PD-1 monotherapy) and enhances the anticancer activity of anti-PD-1 antibodies, as demonstrated by a reduction in tumor size.

[0135] Next, the antitumor effect of CC was tested in mice transplanted with anti-PD-1 resistant tumors (E0771 mammary carcinoma) according to the protocol shown in Figure 2A. As expected, administration of anti-PD-1 alone did not cause a significant reduction in tumor size in this model, confirming the resistance of E0771 mammary carcinoma cells to anti-PD-1 monotherapy (Figures 2B-2C). Similarly, administration of CC extract alone failed to significantly reduce E0771 tumor size. However, a significant reduction in E0771 tumor size was obtained after administration of both anti-PD-1 and CC extract (Figures 2B-C), providing evidence that CC extract has the ability to restore the anti-PD-1 antitumor response against anti-PD-1 resistant tumors.

[0136] Example 2: Camu camu extract acts by regulating the gut microbiota. To better understand the mechanism by which CC extract exerts its antitumor effect, experiments were conducted according to the protocol shown in Figure 3A. First, the results shown in Figures 3B-C demonstrate that administration of broad-spectrum antibiotics (ATBs) that affect the gut microbiota completely suppresses the antitumor effect of CC extract in a mouse MCA-205 tumor model. Second, fecal microbiota transfer (FMT) experiments were performed in specific pathogen-free (SPF) mice. More specifically, feces from mice previously treated with CC extract were transferred to mice transplanted with MCA-205 tumors, and the effect on tumor size was measured (Figure 4A). As shown in Figure 4B, the transfer of microbiota from mice previously treated with CC extract was sufficient to restore CC activity, either as monotherapy or in combination with anti-PD-1. To explore the potential of CC as a treatment, ATB-treated mice were recolonized by performing FMT from two responder (R) patients and two non-responder (NR) patients with non-small cell lung cancer (NSCLC). MCA-205 tumors were inoculated into these “avatar” mouse models, and the mice were treated with CC or water, with or without αPD-1 (Figure 5A). FMT from NR patients conferred resistance to αPD-1, while FMT from R patients restored the αPD-1 antitumor effect (Figures 5A and 5B). The baseline microbiome in FMT-treated mice was characterized at FMT engraftment (before CC+ / -αPD-1). FMT from R was associated with greater alpha diversity (Figure 5C). Analyzing the beta diversity of R compared to the NR group also revealed two objective clusters (Figure 5D). Interestingly, Bilophilia and Ruminococcaceae UBA1819 accounted for a large proportion of mice that received FMT from R and had tumors sensitive to water / αPD-1 (Figure 5E).

[0137] In FMT NR avatar mice, oral supplementation with CC / isoPD-1 restored the antitumor effect of CC (Figure 4B). Furthermore, concomitant use of CC / αPD-1 restored the efficacy of αPD-1 that had been impaired in FMT NR mice treated with water / αPD-1. Conversely, in FMT R avatar mice, CC alone or in combination with anti-PD-1 did not show any further enhancement of the antitumor response compared to water / αPD-1. At the microbiome level, CC / isoPD-1 increased alpha diversity in FMT NR avatars but did not affect the diversity of FMT R avatar mice treated with water / isoPD-1 (Figure 5F). At the genus level, the addition of CC / isoPD-1 to FMT NR was associated with an increase in the relative abundance of Ruminococcaceae (p=0.055).

[0138] Next, microbial profiling was performed on fecal samples from the experiments described in Figure 1A using 16S rRNA sequencing. The V3-V4 region of the 16S rDNA gene was amplified by PCR using primers Bakt_341F and Bakt_805R adapted to incorporate the transposon-based Illumina Nextera adapter (Illumina). High-throughput sequencing was performed at the Institut de biologie integrative et des systemses (IBIS). After dataset filtration (low-high readout), rRNA sequences that successfully passed the pretreatment step and had more than 97% nucleotide sequence identity were binned into Operational Taxonomic Units (OTUs) using USEARCH 61 (version 6.1.544). These experiments revealed that CC treatment was associated with increased alpha diversity compared to water / isoPD-1, independently of αPD-1 therapy (Figure 6A). Quantitative real-time (qRT-PCR) PCR using 16S rRNA gene-based specific primers confirmed an increase in bacterial abundance in the CC group compared to water (Figure 6B).

[0139] Beta diversity, as measured by Bray Curtis, revealed that forced oral administration with CC led to the development of distinct bacterial clusters compared to before CC supplementation (p<0.001) (Figure 6C), while forced oral administration with water / αPD-1 did not show a splitting of the microbiome into different clusters (p=0.16) (Figures 6C-D).

[0140] Differential abundance analysis showed that certain bacteria at the genus level were specifically enriched in the CC group compared to the water group. Ruminococcus (adjusted p<0.05) was the most distinctly abundant bacterium, followed by Turicibacter and Oscillospiraceae UCG 005 (unadjusted p<0.05) (Figure 6E). Furthermore, Ruminococcus was the only bacterium that consistently increased in both the CC / isoPD1 group and the CC / αPD-1 group compared to their respective water groups (Figures 6F-G).

[0141] Profiling of the gut microbiota using 16s rRNA sequencing in an αPD-1 resistant E0771 tumor model showed that Turicibacter, Bilophila, Ruminococcaceae UBA1819, Parasutterella, Clostridium sensu stricto 1, Ruminococcus, Akkermansia, and Anaeroplasma (adjusted p<0.05) were present in larger proportions in mice treated with CC / αPD-1 compared to water / αPD-1 (Figure 6H). Interestingly, Akkermansia and Ruminococcus were also present in larger proportions in mice treated with CC / isoPD-1 compared to water / isoPD-1 (Figure 6I). In summary, these results reveal a specific association between bacterial species and the anticancer effects of CC.

[0142] These results provide compelling evidence that the antitumor activity of the CC extract is at least partially dependent on the gut microbiota.

[0143] Example 3: Effect of CC administration on immune cells Immune replacement profiling was performed in a mouse tumor model to evaluate the effect of the treatment on immune cells. Administration of CC extract alone or in combination with anti-PD-1 resulted in central memory (T CM )CD8 + resulted in significant upregulation (Figure 7A), and the ratio of CD8 + / Foxp3 + CD4 + T (Treg) was significantly increased in three groups with antitumor efficacy, namely CC / isoPD-1, CC / αPD-1, or water / αPD-1, compared with water / isoPD-1 in the MCA-205 tumor model (Figure 7B). Furthermore, a significant increase in ICOS expression on CD8 + T cells was also observed in the E0771 tumor model administered CC extract alone or in combination with anti-PD-1 (Figure 7C).

[0144] To verify that the CC-associated antitumor activity was mediated by CD8 + T cells, mice bearing MCA-205 received CC and anti-CD8 + monoclonal antibody to deplete the CD8 + subpopulation, and showed increased tumor growth compared with CC / isoCD8 (control), indicating that the antitumor effect of CC was CD8 + T cell-dependent (Figure 7D).

[0145] Next, the effect of CC on gut microbiota, tumor size and immune profiling in both tumor and spleen in the MCA-205 mouse model was investigated. Pairwise comparisons using nonparametric Spearman correlation between bacteria enriched in CC / isoPD-1 and the water / isoPD-1 group were performed using intratumoral cytometry immune markers and tumor size. CD3 + T cell infiltration, CD8 + T cell PD-L1 expression, CD8 + proportion of TCM cells, and CD8+ An increase in the / Treg ratio was associated with CC-enriched bacteria such as Ruminoccocus, as well as downregulated Lactobacillus and Pseudoflavonifractor in MCA-205 (Figure 7E). Similarly, in the CC / isoPD-1 group, CD8 in spleen cells was associated with water / isoPD-1. + Cells and ratio CD8 + / Treg was correlated with Ruminoccocus and Oscillospiraceae UCG 005.

[0146] In parallel, TILs in E0771 tumors were analyzed after CC+ / -αPD-1. Combining CC with αPD-1 allowed for ICOS to be compared to water / αPD-1. + CD8 + As evidenced by the increase in MFI of T cells, intratumoral CD8 + T cell activation was induced (Figure 7C). Subsequently, ICOS was determined by the upregulated bacterial CD8 / Treg ratio in water / αPD-1 and CC / αPD-1, and the abundance of Rumincoccus, Bilophila, and Akkermansia that conferred a reduction in tumor size. + Foxp3 - CD4 + Spearman rank correlations (Figure 7F) were performed between immune markers and tumor size, further demonstrating a positive correlation with the upregulation of T cell infiltration.

[0147] Example 4: Isolation of castalagin polyphenol extract as a bioactive compound for the antitumor activity of CC To identify specific compounds in the CC extract that conferred antitumor activity observed in a mouse tumor model, HPLC separation of the CC extract was performed according to the fractionation workflow diagram shown in Figure 8A. Representative figures of the HPLC retention times for the complete camu camu extract, followed by the polar fraction and fraction P3, and the HPLD retention times for castalagin extracted from oak are shown in Figure 8B. Using this technique, the CC extract was first separated into four fractions (P - polar, M - intermediate / moderate polar, NP - nonpolar, INS - insoluble), and each fraction was tested in the MCA-205 tumor model. The results shown in Figure 8C indicate that only the polar fraction (P) was able to mimic the effect of the CC extract tested in parallel in this experiment at a dose of 200 mg / kg.

[0148] Next, the active polarity fraction P was further separated into different subfractions according to retention time (P1-P4) and analyzed by HPLC. Fraction P1 consisted almost entirely of ascorbic acid, P2 consisted of the polyphenol vescalagin and gallic acid, P3 consisted of the polyphenol castalagin, and P4 consisted of different impurities. When these four subfractions were tested using the MCA-205 model, P3, which was mainly composed of castalagin, was found to be the only fraction associated with an antitumor effect similar to that of CC (Figure 8D).

[0149] To confirm that the active ingredient in fraction D was indeed castalagin, additional tests were performed using purified castalagin obtained from a commercial source (Phytoproof® reference substance from Millipore Sigma) and castalagin extracted from CC and oak using HPLC. HPLC of various sources of castalagin confirmed that the retention times were similar to those of the P3 fraction extracted from CC. Similar tumor inhibition was obtained in MCA-205 and E0711 in combination with αPD-1 using castalagin from PhytoProof® or castalagin from oak at similar concentrations present in CC (0.85 mg / kg per mouse) (Figures 8E-F). To define whether the efficacy of castalagin is dose-dependent, oral forced administration of castalagin was performed in mice using six different concentrations ranging from 1 / 8 to 3 times the standard dose. Some anticancer activity was observed at half the dose (0.42 mg / kg), but the anticancer activity was only significant at the standard dose (0.85 mg / kg, Figure 8E). On the other hand, the 3-fold increase in concentration (2.55 mg / kg per mouse) was not equivalent to the standard dose. In summary, the results indicate that castalagin is a bioactive compound of CC and has a dose-dependent effect with a potential state of no change.

[0150] Example 5: Castalagin supplementation increases bacterial diversity in the gut microbiota and enhances the T cell-mediated ICI response. To define the microbiome-dependent effect of castalagin under sterile conditions, proof-of-principle experiments were performed. As shown in Figure 9A, the antitumor effect of castalagin was suppressed by conducting the experiment under sterile conditions. Next, the effect of castalagin on the microbiome in SPF mice treated with castalagin / isoPD-1 was evaluated. 16S microbiome profiling revealed an increase in alpha diversity after castalagin (Figure 9B) and significant clustering observed in beta diversity (Figure 9C). At the taxon level, castalagin caused enrichment of Akkermensia, Ruminococcaceae UBA1819, Ruminococcus, Staphylococcus, Escherichia / shigella, Blautia, and Alistipes, while feces from the water group enriched Lachnospiraceae UCG-001 (Figure 9D). Furthermore, compared to the water group, an increase in the relative abundance of Ruminococcus, Alistepes, Christensenellaceae R7 group, and Paraprevotella was observed in the castalagin group in NR FMT experiments after 16s sequencing analysis, while no difference in Lachnoclostridium was observed between the water group and the castalagin group in NR FMT experiments (Figures 9E-I). qRT-PCR using Ruminococcaceae-specific primers was performed on feces in dose-dependent castalagin experiments (Figure 9J). Ruminococcaceae did not increase with 1 / 4 concentration castalagin compared to the water control (no antitumor effect), while Ruminococcaceae abundance significantly increased after supplementation with standard castalagin dose or 3 times the standard dose (antitumor effect) (Figure 9J).

[0151] Two techniques were used to determine whether castalagin had the same effect on the systemic immune response as CC. First, flow cytometry analysis in the MCA-205 experiment was performed again. CM CD8 +revealed upregulation of T cells, while no effect of CC was observed under sterile conditions (Figure 10A). Second, immunofluorescence (IF) staining demonstrated that the ratio of CD8 + / Foxp3 + CD4 + was further increased in the castalagin / IsoPD-1 group compared with the water / IsoPD-1 group (Figures 10B-C). In E0771 cells, castalagin, regardless of the presence or absence of CC, was associated with an increased frequency of memory CD8 + T cells (CD44 高 CD62L - CD8 + T cells) in both the tumor microenvironment and splenocytes (Figures 10D and 10E).

[0152] As previously performed using avatar mice, the therapeutic effect of castalagin after FMT was tested in NR NSCLC patients under ATB and GF conditions. Addition of castalagin alone was able to restore antitumor activity with an additive effect when combined with αPD-1 (Figures 11A-B).

[0153] To elucidate the mechanism by which castalagin alters intestinal microbiota composition and modifies the microbiota, metabolites of castalagin and its isomer vescalagin were analyzed. Castalagin is hydrolyzed to ellagic acid and castalin, and ellagic acid is then further converted to urolithin by the intestinal microbiota (Figures 12A-12B). Therefore, the potential antitumor effects of some of these metabolites (ellagic acid and urolithin A), as well as the potential antitumor effect of vescalagin, were tested individually. In contrast to castalagin, no antitumor effect was observed for downstream metabolites or the isomer (Figure 12B).

[0154] Next, we evaluated whether castalagin conjugated with fluorescein (Figure 12C) could interact with Ruminococcus. The results shown in Figures 12D and 12E show that when fluorescein-labeled castalagin was co-cultured with Ruminococcus bromii, the majority of the bacteria (approximately 80%) were labeled, while less than 10% and 34% of Escherichia coli and bacteroides thetaiotaomicron, respectively, were labeled after co-culture under the same conditions. Incubation in the presence of an excess (100-fold) of unlabeled castalagin reduced the proportion of fluorescein-labeled Ruminococcus Bromii (Figure 12E).

[0155] Figures 12G and 12H show that in two non-cancer human patients, daily administration of 1.5 mg of camu camu for 3 weeks increased diversity (16s) and was consistent with the results obtained in mice in terms of Ruminococcaceae expression in fecal samples.

[0156] Figure 13 shows the results of duplicate qRT-PCR experiments using Ruminococcaceae-specific primers performed on feces in the castalagin experiment, confirming the increase in Ruminococcaceae in the castalagin treatment group (Figure 13).

[0157] While the present invention has been described in this specification by the specific embodiments described above, it may be modified without departing from the spirit and nature of the subject invention as defined in the appended claims. In the claims, the term “including” is used as an open-ended term which is substantially equivalent to the phrase “including but not limited to.” The singular forms “a,” “an,” and “the” include the corresponding plural subject matter unless the context clearly indicates otherwise.

[0158] [Sequence List] SEQUENCE LISTING <110> THE ROYAL INSTITUTION FOR THE ADVANCEMENT OF LEARNING / MCGILL UNIVERSITY VAL-CHUM, LIMITED PARTNERSHIP UNIVERSITE LAVAL <120> USE OF CASTALAGIN OR ANALOGS THEREOF FOR ANTI-CANCER EFFICACY AND TO INCREASE THE RESPONSE TO IMMUNE CHECKPOINT INHIBITORS <130> F89069A1 <140> JP2022-550877 <141> 2021-02-19 <150> US 62 / 979,327 <151> 2020-02-20 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 1 tggagcatgt ggtttaattc ga 22 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 2 tgcgggactt aacccaaca 19 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 3 actgagaggt tgaacggcca 20 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 4 cctttacacc cagtaawtcc gga 23 <210> 5 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <220> <221> misc_feature <222> (9)..(9) <223> n is a, c, g, or t <400> 5 cctacgggng gcwgcag 17 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 6 gactachvgg gtatctaatc c 21

Claims

1. A method for treating a subject suffering from immunotherapy-resistant cancer, comprising administering a therapeutically effective dose of castalagin or an analogue thereof to the subject.

2. The method according to claim 1, wherein the immunotherapy includes immune checkpoint inhibitor therapy.

3. The method according to claim 2, wherein the immune checkpoint inhibitor is a programmed cell death-1 (PD-1) inhibitor, a cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) inhibitor, or a programmed cell death-ligand 1 (PD-L1) inhibitor.

4. The method according to claim 2 or 3, wherein the inhibitor is a blocking antibody.

5. The method according to claim 3 or 4, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

6. The method according to any one of claims 1 to 5, wherein the castalagin or its analogue is present in a plant or fruit extract.

7. The method according to claim 6, wherein the extract is Myrciaria dubia (camu camu) extract.

8. The method according to any one of claims 1 to 5, wherein the method comprises administering a pharmaceutical composition containing castalagin or an analog thereof, preferably castalagin.

9. The method according to any one of claims 6 to 8, wherein the extract or pharmaceutical composition is formulated to deliver the castalagin or its analog into the intestine.

10. The method according to claim 9, wherein the extract or pharmaceutical composition is formulated as a capsule.

11. The method according to any one of claims 1 to 10, wherein the cancer is lung cancer or breast cancer.

12. The method according to claim 11, wherein the lung cancer is non-small cell lung cancer (NSCLC).

13. The method according to claim 12, wherein the breast cancer is triple-negative breast cancer (TNBC).

14. The method according to any one of claims 1 to 13, further comprising administering an effective amount of the immune checkpoint inhibitor or castalagin alone.

15. A method for enhancing an antitumor immune response in a subject suffering from cancer, the method comprising administering a therapeutically effective amount of castalagin or an analog thereof to the subject.

16. The method according to claim 15, wherein the antitumor immune response is an antitumor T cell response.

17. The method according to claim 15 or 16, further comprising administering a therapeutically effective amount of an immune checkpoint inhibitor to the subject.

18. The method according to claim 17, wherein the immune checkpoint inhibitor is a programmed cell death-1 (PD-1) inhibitor, a cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) inhibitor, or a programmed cell death-ligand 1 (PD-L1) inhibitor.

19. The method according to claim 17 or 18, wherein the inhibitor is a blocking antibody.

20. The method according to claim 18 or 19, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

21. The method according to any one of claims 15 to 20, wherein the castalagin or analog thereof is present in a plant or fruit extract.

22. The method according to claim 21, wherein the extract is Myrciaria dubia (camu camu) extract.

23. The method according to any one of claims 15 to 22, wherein the method comprises administering a pharmaceutical composition containing castalagin or an analog thereof, preferably castalagin.

24. The method according to any one of claims 21 to 23, wherein the extract or pharmaceutical composition is formulated to deliver the castalagin or its analog into the intestine.

25. The method according to claim 24, wherein the extract or pharmaceutical composition is formulated as a capsule.

26. The method according to any one of claims 15 to 25, wherein the subject is suffering from lung cancer or breast cancer.

27. The method according to claim 26, wherein the lung cancer is non-small cell lung cancer (NSCLC).

28. The method according to claim 26, wherein the breast cancer is triple-negative breast cancer (TNBC).

29. The use of castalagin or its analogues to treat patients with immunotherapy-resistant cancer.

30. Use of castalagin or its analogues to manufacture medicines for the treatment of subjects with immunotherapy-resistant cancer.

31. The use according to claim 29 or 30, wherein the immunotherapy includes immune checkpoint inhibitor therapy.

32. The use according to claim 31, wherein the immune checkpoint inhibitor is a programmed cell death-1 (PD-1) inhibitor, a cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) inhibitor, or a programmed cell death-ligand 1 (PD-L1) inhibitor.

33. The use according to claim 31 or 32, wherein the immune checkpoint inhibitor is a blocking antibody.

34. The use according to any one of claims 31 to 33, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

35. The use according to any one of claims 29 to 34, wherein the castalagin or analogue thereof is present in a plant or fruit extract.

36. The use according to claim 35, wherein the extract is Myrciaria dubia extract.

37. The use according to any one of claims 29 to 34, wherein the castalagin or an analog thereof, preferably castalagin, is present in the pharmaceutical composition.

38. The use according to any one of claims 35 to 37, wherein the extract or pharmaceutical composition is formulated to deliver the castalagin or its analogue into the intestine.

39. The use according to claim 38, wherein the extract or pharmaceutical composition is formulated as a capsule.

40. The use according to any one of claims 29 to 39, wherein the cancer is lung cancer or breast cancer.

41. The use according to claim 40, wherein the lung cancer is non-small cell lung cancer (NSCLC).

42. The use according to claim 40, wherein the breast cancer is triple-negative breast cancer (TNBC).

43. The use of castalagin or its analogues to enhance the antitumor immune response in subjects suffering from cancer.

44. Use of castalagin or its analogues to manufacture a pharmaceutical product for enhancing the antitumor immune response in subjects suffering from cancer.

45. The use according to claim 43 or 44, wherein the antitumor immune response is an antitumor T cell response.

46. The use according to any one of claims 43 to 45, wherein the castalagin or analogue thereof is intended for use in combination with an immune checkpoint inhibitor.

47. The use according to claim 46, wherein the immune checkpoint inhibitor is a programmed cell death-1 (PD-1) inhibitor, a cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) inhibitor, or a programmed cell death-ligand 1 (PD-L1) inhibitor.

48. The use according to claim 46 or 47, wherein the immune checkpoint inhibitor is a blocking antibody.

49. The use according to any one of claims 46 to 48, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

50. The use according to any one of claims 43 to 49, wherein the castalagin or its analogue is present in a plant or fruit extract.

51. The use according to claim 50, wherein the extract is Myrciaria dubia extract.

52. The use according to any one of claims 43 to 49, wherein the castalagin or an analog thereof, preferably castalagin, is present in the pharmaceutical composition.

53. The use according to any one of claims 50 to 52, wherein the extract or pharmaceutical composition is formulated to deliver the castalagin or its analogue into the intestine.

54. The use according to claim 53, wherein the extract or pharmaceutical composition is formulated as a capsule.

55. The use according to any one of claims 43 to 54, wherein the subject is suffering from lung cancer or breast cancer.

56. The use according to claim 55, wherein the lung cancer is non-small cell lung cancer (NSCLC).

57. The use according to claim 55, wherein the breast cancer is triple-negative breast cancer (TNBC).

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