Use of castanea ellagitannin or its analogs for anticancer efficacy and increased response to immune checkpoint inhibitors

Abstract

Methods and uses for enhancing or restoring anti-tumor responses in cancer patients, such as anti-tumor immunity mediated by immune checkpoint inhibitors, are described. These methods are based on the administration of chestnut ellagitannins or their analogs, and are particularly suitable for treating tumors that are resistant to immunotherapy, such as immune checkpoint inhibitors. Chestnut ellagitannins or their analogs can be administered in any suitable form, such as in a crude plant or fruit extract such as a Myrtles extract, or in a pharmaceutical composition.

Description

[0001] Cross-references

[0002] 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. Technical Field

[0003] The present invention relates generally to the field of cancer, and more particularly to treating cancer in combination with immune checkpoint inhibitors. Background Art

[0004] The prevalence of cancer in both human and animal populations and its role in mortality means there is a continuing need for new drugs that are effective against tumors. Eliminating a tumor or reducing its size or decreasing the number of cancer cells circulating in the blood or lymph node system can be beneficial in a variety of ways; reducing pain or discomfort, preventing metastasis, facilitating surgical intervention and, most importantly, prolonging life.

[0005] Various attempts have been made to help the immune system fight tumors. One early approach in the late 1800s involved general stimulation of the immune system, for example by administering bacteria (live or killed) to elicit a general immune response that was also directed against the tumor.

[0006] Recent approaches aimed at helping the immune system specifically recognize tumor-specific antigens (TSAs) (or tumor associated antigens (TAAs)) involve administering tumor-specific antigens to subjects, usually in combination with an adjuvant. However, a lack of a robust immune response to TAAs is often observed in cancer. One of the factors that contribute to a weak response to TAAs is the induction of inhibitory pathways / signals (commonly referred to as "immune checkpoints") that suppress immune responses. Although such inhibitory signals are important for maintaining self-tolerance and protecting tissues from damage when the immune system responds to pathogenic infections, they may also reduce the body's beneficial responses to tumor development.

[0007] A new era of treatment has matured with immune checkpoint inhibitors or blockers (ICBs) targeting inhibitory T cell receptors such as CTLA-4, PD-L1, and PD-1 (Marabelle, OncoImmunology 2016). This emerging field even won the 2018 Nobel Prize in Medicine. These immunotherapeutics have provided unparalleled clinical results in a variety of advanced cancers including lung cancer (Reck, NEJM 2016), melanoma (Robert, NEJM 2011), genitourinary system (Motzer, NEJM 2018), and head and neck tumors (Ferris, NEJM 2016). However, the primary resistance rate in patients with non-small cell lung cancer (NSCLC) is 35-44%, while the secondary resistance rate is close to 100% (Reck, NEJM 2016).

[0008] Therefore, there is a need to develop new approaches to enhance responses to ICBs, especially in cancers that are resistant to ICB treatment.

[0009] This specification refers to a number of documents, the contents of which are all incorporated herein by reference. Summary of the invention

[0010] This application relates to the following items 1 to 55:

[0011] 1. A method for treating a subject having cancer that is resistant to immunotherapy such as immune checkpoint inhibitor therapy, the method comprising administering to the subject an effective therapeutic amount of castalagin or an analog thereof.

[0012] 2. The method according to 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.

[0013] 3. The method according to item 1 or 2, wherein the inhibitor is a blocking antibody.

[0014] 4. The method according to item 2 or 3, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

[0015] 5. The method according to any one of items 1 to 4, wherein the chestnut ellagitannin or its analogue is present in a plant or fruit extract.

[0016] 6. The method according to item 5, wherein the extract is a Myrciaria dubia (Camu-Camu) extract.

[0017] 7. The method according to any one of items 1 to 4, wherein the method comprises administering a pharmaceutical composition comprising castanea ellagitannin or an analog thereof.

[0018] 8. The method according to any one of items 5 to 7, wherein the extract or pharmaceutical composition is formulated for delivering the castanea ellagitannin or its analogue to the intestine.

[0019] 9. The method according to item 8, wherein the extract or pharmaceutical composition is formulated into capsules.

[0020] 10. The method according to any one of items 1 to 9, wherein the cancer is lung cancer or breast cancer.

[0021] 11. The method according to item 10, wherein the lung cancer is non-small cell lung cancer (NSCLC).

[0022] 12. The method according to item 10, wherein the breast cancer is triple-negative breast cancer (TNBC).

[0023] 13. The method according to any one of items 1 to 12 further comprises administering alone an effective amount of an immune checkpoint inhibitor or chestnut ellagitannin.

[0024] 14. A method for enhancing an anti-tumor immune response in a subject suffering from cancer, the method comprising administering to the subject an effective therapeutic amount of castanea glabramin or an analog thereof.

[0025] 15. The method according to item 14, wherein the anti-tumor immune response is an anti-tumor T cell response.

[0026] 16. The method according to item 14 or 15, wherein the method further comprises administering to the subject an effective therapeutic amount of an immune checkpoint inhibitor.

[0027] 17. The method according to 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 death ligand 1 (PD-L1) inhibitor.

[0028] 18. The method according to item 16 or 17, wherein the inhibitor is a blocking antibody.

[0029] 19. The method according to item 17 or 18, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

[0030] 20. The method according to any one of items 14 to 19, wherein the castanea ellagitannin or its analogue is present in a plant or fruit extract.

[0031] 21. The method according to item 20, wherein the extract is a Myrtles camu extract.

[0032] 22. A method according to any one of items 14 to 21, wherein the method comprises administering a pharmaceutical composition comprising castanea ellagitannin or an analog thereof.

[0033] 23. The method according to any one of items 20 to 22, wherein the extract or pharmaceutical composition is formulated for delivering the castanea ellagitannin or an analog thereof to the intestine.

[0034] 24. The method according to item 23, wherein the extract or pharmaceutical composition is formulated into capsules.

[0035] 25. A method according to any one of items 14 to 24, wherein the subject has lung cancer or breast cancer.

[0036] 26. The method according to item 25, wherein the lung cancer is non-small cell lung cancer (NSCLC).

[0037] 27. The method according to item 25, wherein the breast cancer is triple-negative breast cancer (TNBC).

[0038] 28. Use of castanea ellagitannin or an analog thereof for treating a subject suffering from cancer that is resistant to immunotherapy, such as immune checkpoint inhibitor therapy.

[0039] 29. Use of castanea ellagitannin or an analog thereof in the preparation of a medicament for treating a subject with cancer that is resistant to immunotherapy, such as immune checkpoint inhibitor therapy.

[0040] 30. The use according to item 28 or 29, 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.

[0041] 31. The use according to any one of items 28 to 30, wherein the immune checkpoint inhibitor is a blocking antibody.

[0042] 32. The use according to any one of items 28 to 31, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

[0043] 33. The use according to any one of items 28 to 32, wherein the castanea ellagitannin or its analogue is present in a plant or fruit extract.

[0044] 34. The use according to item 33, wherein the extract is a Myrtlesia scabra extract.

[0045] 35. The use according to any one of items 28 to 32, wherein the castanea ellagitannin or its analogue is present in a pharmaceutical composition.

[0046] 36. The use according to any one of items 33 to 35, wherein the extract or pharmaceutical composition is formulated for delivering the castanea ellagitannin or its analogue to the intestine.

[0047] 37. The use according to item 36, wherein the extract or pharmaceutical composition is formulated into capsules.

[0048] 38. The use according to any one of items 28 to 37, wherein the cancer is lung cancer or breast cancer.

[0049] 39. The use according to item 38, wherein the lung cancer is non-small cell lung cancer (NSCLC).

[0050] 40. The use according to item 38, wherein the breast cancer is triple-negative breast cancer (TNBC).

[0051] 41. Use of chestnut ellagitannin or its analogs for enhancing the anti-tumor immune response of a subject.

[0052] 42. Use of chestnut ellagitannin or its analogs in the preparation of a drug for enhancing the anti-tumor immune response of a subject.

[0053] 43. The use according to item 41 or 42, wherein the anti-tumor immune response is an anti-tumor T cell response.

[0054] 44. The use according to any one of items 41 to 43, wherein the chestnut ellagitannin or its analogue is used in combination with an immune checkpoint inhibitor.

[0055] 45. The use according to item 44, 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.

[0056] 46. ​​The use according to item 44 or 45, wherein the immune checkpoint inhibitor is a blocking antibody.

[0057] 47. The use according to any one of items 44 to 46, wherein the immune checkpoint inhibitor is a PD-1 inhibitor.

[0058] 48. The use according to any one of items 41 to 47, wherein the castanea ellagitannin or its analogue is present in a plant or fruit extract.

[0059] 49. The use according to item 48, wherein the extract is a Myrtlesia scabra extract.

[0060] 50. The use according to any one of items 41 to 47, wherein the castanea ellagitannin or its analogue is present in a pharmaceutical composition.

[0061] 51. The use according to any one of items 48 to 50, wherein the extract or pharmaceutical composition is formulated for delivering the castanea ellagitannin or its analogue to the intestine.

[0062] 52. The use according to item 51, wherein the extract or pharmaceutical composition is formulated into capsules.

[0063] 53. The use according to any one of items 41 to 52, wherein the subject suffers from skin cancer (e.g., melanoma, squamous cell skin cancer), lung cancer, kidney cancer (e.g., renal cell carcinoma), Hodgkin's lymphoma, head and neck cancer, colon cancer, liver cancer, gastric cancer or myeloma, preferably lung cancer or breast cancer.

[0064] 54. The use according to item 53, wherein the subject suffers from lung cancer, preferably non-small cell lung cancer (NSCLC).

[0065] 55. The use according to item 53, wherein the subject suffers from breast cancer, preferably triple-negative breast cancer (TNBC).

[0066] Other objects, advantages and features of the present invention will become more apparent upon reading the following non-restrictive description of specific embodiments of the invention, given by way of example only with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] In the attached picture:

[0068] Figure 1A Schematic diagram of the protocol used to study the effects of camu camu extract (CC) alone and when combined with anti-PD-2 therapy in a mouse tumor model sensitive to anti-PD-1 therapy. Syngeneic C57BL / 6 mice were implanted subcutaneously with 0.8×10 6 MCA-205 sarcoma, and when the tumor size reaches 20 to 35 mm 2 Mice were treated intraperitoneally (ip) with anti-PD-1 mAb (250 μg / mouse; clone RMP1-14) or isotype control (clone 2A3) with or without daily oral administration of 200 mg / kg CC (Camu camu powder from SunFood).

[0069] Figure 1B is a graph showing the change in tumor size over time in mice implanted with MCA-205 tumors, treated with anti-PD-1 monoclonal antibody (mAb) or isotype control, with or without daily oral gavage of CC.

[0070] Figure 1C is a graph showing tumor size at sacrifice of mice implanted with MCA-205 tumors, treated with anti-PD-1 mAb or isotype control, with or without daily oral gavage of CC.

[0071] Figure 2A Schematic representation of the protocol used to study the effects of camu camu extract (CC) alone and its additive effects when combined with anti-PD-2 therapy in a mouse tumor model treated with anti-PD-1. Syngeneic C57BL / 6 mice were implanted subcutaneously with 0.5×10 6 E0771 breast cancer tumor model, and when the tumor size reaches 20 to 35 mm 2 At 4 hr, mice were treated intraperitoneally (ip) with anti-PD-1 mAb (250 μg / mouse; clone RMP1-14) or isotype control (clone 2A3), with or without daily gavage of 200 mg / kg CC (SunFood).

[0072] Figure 2B is a graph showing the changes in tumor size over time in mice implanted with E0771 tumors after continuous injections of anti-PD-1 mAb (αPD-1) or isotype control (IsoPD-1) and daily oral gavage with water or CC.

[0073] Figure 2C is a graph showing tumor size at sacrifice of mice implanted with E0771 tumors, treated with anti-PD-1 mAb or isotype control, with or without daily oral gavage of CC.

[0074] Figure 3ASchematic representation of the protocol used to study the effects of broad-spectrum antibiotics (ATB) on CC responses in the mouse MCA-205 tumor model. Mice received ATB treatment 2 weeks prior to tumor implantation and continued with antibiotics 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. Solutions and bottles were changed 3 times a week. Antibiotic activity was confirmed by macroscopic changes observed at the cecal level at the time of sacrifice (dilatation) and by incubation of fecal pellets resuspended in sterile NaCl on blood agar plates for 48 h at 37°C under aerobic or anaerobic conditions. Figure 1A MCA-205 inoculation and CC treatment were performed as indicated.

[0075] Figure 3B -C is a graph showing the change in tumor size over time in mice implanted with MCA-205 tumors with or without daily oral gavage of CC, water (control, Figure 3B ) or ATB( Figure 3C )(5 mice / group).

[0076] Figure 4A Schematic diagram of the protocol used to study the effects of fecal microbiota transplantation (FMT) of CC-treated mice on anti-PD-1 responses in the mouse MCA-205 tumor model. Feces from mice treated with CC were frozen at -80°C. MCA-205 was implanted subcutaneously when the tumor size reached 20 to 35 mm. 2 At 4 hr, mice were treated intraperitoneally (ip) with anti-PD-1 mAb (250 μg / mouse; clone RMP1-14) or isotype control (clone 2A3), with or without daily oral administration of fecal NaCl dilution. 100 μg of feces was resuspended in 1 mL of sterile NaCl.

[0077] Figure 4B is a graph showing tumor size at sacrifice in mice implanted with MCA-205 tumors, treated with anti-PD-1 monoclonal antibody or isotype control, with or without daily oral gavage with diluted feces in NaCl.

[0078] Figure 5A Schematic diagram of the experimental design of the avatar mouse experiment. After 3 days of ATB in SPF C57Bl6 mice, fecal samples from non-responders (NR) and responders (R) of non-small cell lung cancer (NSCLC) patients were subjected to FMT. Two weeks later, MCA-205 sarcoma cells were inoculated, and water or CC was gavaged daily, while αPD-1 or IsoPD-1 monoclonal antibodies were continuously injected.

[0079] Figure 5B Figure 2 shows the combined mean tumor ± SEM of 2 NR and 2 R groups for each CC and water group at sacrifice (D+17) after FMT.

[0080] Figure 5C is a graph showing the number of observed genus of R and NRα diversity at baseline (before CC gavage and 14 days after implantation in SPF-bred mice (n=10) carrying MCA-205) treated with ATB and then receiving FMT from 4 NSCLC patients (n=2NR, n=2R). Means ± SEM are shown.

[0081] Figure 5D is a Bray-Curtis plot of beta diversity of 16s RNA sequenced at the genus level for NR and R FMT 2 weeks after implantation. *p<0.05, ***p<0.001.

[0082] Figure 5E Figure 1 is a volcano plot image of the differential abundance analysis results after 16S sequencing analysis of feces of mice that received NR or R FMT on day 0 for 14 days.

[0083] Fig. 5F is a graph showing the alpha diversity represented by observed genera in the D+11NR and R FMT groups. Mean ± SEM is shown. *p<0.05, **p<0.01.

[0084] Fig. 6A is a graph showing the results from the MCA-205 experiment ( Figure 1A ) of four groups of mouse 16s rRNA fecal samples, and an image of alpha diversity measured by the Shannon index in each group.

[0085] Figure 6B is a graph showing the results of real-time PCR assay of DNA extracted from mouse feces 6 days after oral gavage with water or CC in the MCA-205 model (n=10 mice / group) using specific primers for 16s detection.

[0086] Figure 6C is a graph showing beta diversity as measured by the Bray-Curtis index, comparing baseline (before treatment) to CC or combined water (αPD1 and IsoPD-1) groups.

[0087] Fig.6D is a graph showing 16s rRNA microbiome analysis of samples from the MCA-205 experiment ( Figure 1A ) and compared the beta diversity images measured by the Bray-Curtis index for all four groups after 6 days of treatment.

[0088] Fig. 6E The volcano plot image of the differential abundance analysis comparing the combined CC group and the water group in MCA-205 tumors. The bacteria enriched in each group are indicated using adjusted p-values ​​and p-values ​​**p<0.01.

[0089] Fig. 6F Figure 1 is a volcano plot image of differential abundance analysis between water / IsoPD-1 and CC / IsoPD-2 groups in the MCA-205 tumor model. The bacteria enriched in each group are indicated using adjusted p-values ​​and p-values ​​**p<0.01.

[0090] Figure 6G Volcano plot of differential abundance analysis of water / IsoPD-1 and CC / αPD-1 groups in the MCA-205 tumor model. The bacteria enriched in each group are indicated using adjusted p-values ​​and p-values ​​**p<0.01.

[0091] Figure 6H Volcano plot comparing differential abundance analysis of water / αPD-1 and CC / αPD1 groups in E0771 tumors. Bacteria enriched in each group are indicated using adjusted p-values ​​and p-values ​​(FDR: 0.1) *p<0.05, **p<0.01, ***p<0.001.

[0092] Fig.6I It is a volcano plot image of the differential abundance analysis between the water / IsoPD-1 and CC / IsoPD-2 groups in the E0771 model.

[0093] Figure 7A-7C Figure 2 shows the effect of anti-PD-1 and / or CC-treated mice MCA-205 ( Figure 7A-7B ) or E0771( Figure 7C) Figure 1. Results of immune cell profiling by flow cytometry in a tumor model. Tumors and spleens were collected 9 or 19 days after the first injection of anti-PD-1 monoclonal antibodies into mice bearing MCA-205 or E0771 tumors, respectively. The excised tumors were cut into small pieces, digested in RPMI medium containing 25 μg / ml liberase (Roche) and 150 UI / mL DNase1 (Roche) at 37°C for 30 minutes, and then crushed and filtered twice using 100 and 70 μm cell strainers (Becton & Dickinson). The spleen was crushed in RPMI medium and filtered through a 100 μm cell strainer. Two million tumor cells or splenocytes were preincubated with purified anti-mouse CD16 / CD32 (clone 93; eBioscience) for 30 min prior to membrane staining (CD45, CD3, CD4, CD8, PD1, PDL1, ICOS, CXCR3, CCR9, CD45RB, CD62L, CD44). For intracellular staining, the Foxp3 staining kit (eBioscience) was used. Dead cells were excluded using the Live / Dead Fixable Aqua Blue Dead Cell Staining Kit (Life Technologies). Figure 7A-7B Figure 2 shows the TCM CD8 in TILs of MCA-205 tumor-bearing mice after treatment with anti-PD-1 mAb or isotype control (with or without oral CC gavage). + T cells (CD45RB - CD62L + CD8 + T cells) and CD8 + Graph of the ratio of B cells / Foxp3+CD4+T cells (Tregs). Figure 7C Figure 2 shows intratumoral CD8 TILs in the E0771 tumor model after CC+ / -αPD-1 treatment. + T cell activation (by flow cytometry with ICOS + CD8 + MFI evaluation of T cells).

[0094] Fig.7D Blockade of CD8 in the mouse MCA-205 tumor model was shown + Figure 2. Effect of T cell activity on the antitumor effect of CC. Syngeneic C57BL / 6 mice were subcutaneously implanted with 0.8×10 6 MCA-205 sarcoma mice were treated with 150 μg / mouse of anti-CD8 (clone: ​​53-5.8, BioXCell) or isotype control 3 days after tumor inoculation. Then, when tumor size reached 20 to 35 mm 2At the same time, mice were intragastrically administered with 200 mg / kg CC or no CC administration every day.

[0095] Fig. 7E Figure 2 is a pairwise Spearman rank correlation heatmap between significantly different bacteria enriched in CC / isoPD-1 and water / isoPD-2 groups (n=1) and positively correlated TIL cell counts and matched tumor size in the MCA-205 experiment. Left image is in TILs, right image is in splenocytes.

[0096] Figure 7F Paired Spearman rank correlation heatmap of significantly different fecal taxa enriched in CC / αPD-1 vs. water / αPD-1 groups with the frequencies of the indicated cell types detected by flow cytometry and matched tumor sizes in the E0771 tumor model. Unpaired t-test was used to compare *p < 0.05, **p < 0.01, ***p < 0.001.

[0097] Fig. 8A A workflow diagram for fractionation of CC extracts is shown.

[0098] Figure 8B is a plot of the HPLC retention times of the intact camu camu extract, followed by the polar fractions and fraction P3 and the HPLD retention times of castanea ellagitannins extracted from oak.

[0099] Figure 8C It shows Fig. 7A Figure 2. Effect of various fractions (P, NP, M, INS) shown in the presence or absence of anti-PD-1 on SPF-bred mice bearing MCA-205 (n=5, mean + / - SEM tumor size at sacrifice). The same experimental design as previously described was used ( Figure 1A ), mice were given daily oral administration of 40.18 mg / kg or 100 mg / kg of CC. Unpaired t-test was used to compare *p<0.05, **p<0.01.

[0100] Fig.8D is to show that in the presence or absence of anti-PD-1 in the murine MCA-205 tumor model, Figure 7C The same experimental design as described previously was used ( Figure 1A ), mice were given oral administration of 0.85 mg / kg or 100 mg / kg daily (P1, P2, P3 and P4). Unpaired t-test was used for *p<0.05, **p<0.01.

[0101] Fig. 8Eis a graph showing the effect of different doses of castanea ellagitannin in the presence of anti-PD-1 in the mouse MCA-205 tumor model (mean MCA-205 tumor size expressed at the time of mouse sacrifice). The same experimental design as previously described ( Figure 1A ), mice were gavaged daily or not, increasing doses of castanea ellagitannin (from 0.11 mg / kg to 2.56 mg / kg) or CC at a dose of 100 mg / kg. It is worth noting that the dose in CC is equivalent to about 0.85 mg / kg. For the negative control of 0 mg / kg, mice received water.

[0102] Fig.8F is a graph showing the effect of castanea ellagitannin in the presence of anti-PD-1 in SPF-fed mice carrying E0771 (n=5, tumor size at sacrifice). The same experimental design as described previously was used ( Figure 2A ), mice were given orally daily with or without chestnut ellagitannin (0.85 mg / kg per mouse) in the absence of anti-PD-1.

[0103] Fig.9A is a graph showing the effect of Castanopsis ellagitannin administered at a standard concentration (0.85 mg / kg per mouse) under sterile conditions on tumor size in the mouse MCA-205 tumor model.

[0104] Fig. 9B Graph showing bacterial diversity (# observed genera) on day 5 (baseline) and day 11 after intragastric administration of castanea ellagitanni in the MCA-205 model. Unpaired t-test was used to compare *p<0.05, **p<0.01, ***p<0.001.

[0105] Fig. 9C Bray-Curtis β-diversity images of 16srRNA microbiota in SPF-housed mice (n=5) carrying MCA-205 and given daily oral administration of chestnut ellagitannins or water. Each row corresponds to a mouse group and each dot corresponds to a single animal. Unpaired t-test was used to compare *p<0.05, **p<0.01, ***p<0.001.

[0106] Fig.9D It is a volcano plot image of the differential abundance analysis results of water / IsoPD-1 and chestnut ellagitannin / IsoPD-2 groups after 16s sequencing analysis in SPF-fed mice carrying MCA-205 (n=5).

[0107] Fig.9E-I shows the relative abundance analysis results of Ruminococcus, Alistipes, Christensenellaceae R7 group, Paraprevotella and Lachnoclostridium after 16S sequencing analysis between water and chestnut ellagitannin groups in the NR FMT experiment *p<0.05, **p<0.01, ***p<0.001.

[0108] Figure 9J Graph showing the effect of chestnut ellagitanni treatment (0 mg / kg, 1 / 4X, 0.21 mg / kg, 1X, 0.85 mg / kg and 3X, 2.55 mg / kg) on ​​the number of Ruminococci in feces. Real-time PCR was performed on DNA extracted from mouse feces 6 days after intragastric administration of water or chestnut ellagitanni at 0.21 mg / kg, 0.85 mg / kg and 2.55 mg / kg, using Ruminococcaceae specific primers to detect SPF mice (n=5) carrying MCA-205, *p<0.05, **p<0.01.

[0109] Fig. 10A Graph showing the effect of castanea ellagitannin treatment on immune cell distribution in mice MCA-205. In sterile and SPF experiments, CD8 + T central memory (TCM) cells (CD45RB-CD62L + ) of MCA-205TILs.

[0110] Fig. 10B Representative images of CD4, CD8, and Foxp3 immunofluorescence staining of tumors in the water / ISoPD-1 and chestnut ellagitannin / ISoPD-2 groups.

[0111] Fig. 10C CD8+ / Foxp3 in tumors obtained by immunofluorescence staining + CD4 + Box plot of ratios (n=8 / group) *p<0.05.

[0112] Figure 10D-10E : is a graph showing the results of the effect of castanea ellagitannin treatment on the distribution of immune cells in E0771 mice. Fig. 10D ) and spleen ( Fig.10E )Memory CD8 + Flow cytometric analysis of E0771 T cells.

[0113] Fig.11AGraph showing the effect of ellagic acid (0.85 mg / kg) on ​​tumor growth kinetics in the ATB-avatar model (daily gavage of ellagic acid or water in combination with αPD1 mAb or IsoPD-1) after FMT from 1 NR NSCLC patient in the presence or absence of anti-PD-1. Unpaired t-test was used. Mean ± SEM is shown. *p<0.05.

[0114] Fig. 11B is a graph showing the therapeutic effect of chestnut ellagitannin after FMT using fecal samples from NRNSCLC patients under ATB and sterile conditions. Fecal microbiota transplantation (FMT) of fecal samples from non-responder (NR) non-small cell lung cancer (NSCLC) patients (n=1NR) was performed in sterile C57BL / 6 mice (n=3). Two weeks later, MCA-205 sarcoma cells were inoculated and gavage with water or chestnut ellagitannin was given daily. Each row corresponds to a mouse group and each point corresponds to an animal. An unpaired t-test was used. Mean ± SEM is shown.

[0115] Fig. 12A Schematic diagram of the hydrolysis of castanea ellagitannin into ellagic acid and castalin, and the metabolism of ellagic acid into urolithins by the intestinal microbiota.

[0116] Fig. 12B Figure 2 shows the expression of 100% leukemia cells in the mouse MCA-205 tumor model using the same experimental design as previously described ( Figure 1A ), a graph showing the effect of castanein, vescalagin, ellagic acid, castanein, and urolithin A on tumor size at the time of sacrifice.

[0117] Fig. 12C is a diagram depicting in vitro labeling of chestnut ellagitannin with fluorescein.

[0118] Fig.12D Flow cytometry images of fluorescein-labeled chestnut ellagitannin in co-culture with Escherichia Coli, Ruminococcusbromii and Bacteroides thetaiotaomicron. The upper panel shows unstained conditions, and the lower panel shows staining with fluorescein-chestnut ellagitannin at 37°C.

[0119] Fig.12E is a graph showing the results of a competition assay of Ruminococcus braunii and E. coli in the presence of fluorescein-conjugated chestnut ellagitannin and unconjugated chestnut ellagitannin at 100-fold concentration at 37° C. and 0° C. Each point represents one experiment.

[0120] Fig.12F Shown are epifluorescence inverted microscope images of Ruminococcus brunneri, Escherichia coli, and Bacteroides thetaiotaomicron after treatment with flavonoids.

[0121] Figure 12G -H is a graph depicting the results of qPCR analysis of diversity (16s) and Ruminococcaceae in two non-cancer HIV patients treated with 1.5 mg CC daily. The graph depicts the diversity (16s RNA) before and 3 weeks after CC administration ( Figure 12G ) and the amount of Ruminococcaceae DNA ( Fig.12H ).

[0122] Fig.13 is a graph depicting the results of qPCR analysis of DNA extracted from mouse feces, using specific primers to detect Ruminococcaceae in SPF-fed mice (n=10) carrying MCA-205 after oral gavage with 0.85 mg / kg of water or chestnut ellagitannin for 6 days. *p<0.05.

[0123] Fig.14 is a table showing the list of bacteria that were increased after administration of CC and / or chestnut ellagitannin compared to water. DETAILED DESCRIPTION

[0124] The use of the terms "a" and "an" and "the" and similar references in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0125] The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (ie, "including, but not limited to,") unless otherwise noted.

[0126] Unless otherwise specified herein, the description of numerical ranges herein is intended only as a shorthand method of individually referencing each individual value falling within the range, and each individual value is incorporated into this specification as if it were individually described herein. All value subsets within these ranges are also incorporated into this specification as if they were individually described herein.

[0127] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0128] The use of any and all examples, or exemplary language ("eg," "suchas," etc.) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0129] No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0130] Here, the word "about" has its ordinary meaning. The term "about" is used to indicate that a value includes the inherent error variation of the device or method used to determine the value, or includes values ​​close to the stated value, such as within 10% of the stated value (or range of values).

[0131] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0132] The scope of the claims should not be limited by the preferred embodiments described in the examples, but should be given the broadest interpretation consistent with the entire description.

[0133] The present invention includes any and all combinations and sub-combinations of the embodiments and features disclosed herein.

[0134] In the studies described herein, the inventors have demonstrated in two mouse tumor models that crude extracts from the berries of Myrtle Bark (Camu Camu, CC) are able to induce anti-tumor responses and enhance the anti-tumor responses of immune checkpoint inhibitors, but are also able to restore the anti-tumor response of immune checkpoint inhibitors in resistant tumors. The inventors also provide convincing evidence that the effects of Camu Camu extracts are mediated at least in part by modulation of the intestinal microbiota and involve T cell-mediated immune responses. Further characterization of the Camu Camu extracts identified castanein as the main active ingredient responsible for the extract's anti-tumor response effects.

[0135] Therefore, in the first aspect, the present invention provides a method for inducing or restoring a subject with a cancer resistant to immunotherapy (e.g., immune checkpoint inhibitor treatment) to respond to immunotherapy (e.g., immune checkpoint inhibitor treatment), comprising administering an effective therapeutic amount of chestnut ellagitannin or its analog to the subject. The present invention also provides chestnut ellagitannin or its analogs for inducing anti-tumor and / or improving or restoring response to immunotherapy (e.g., immune checkpoint inhibitor treatment) alone in a subject with a cancer resistant to immunotherapy (e.g., immune checkpoint inhibitor treatment). The present invention also provides chestnut ellagitannin or its analogs for the preparation of a drug for inducing or restoring a subject with a cancer resistant to immunotherapy (e.g., immune checkpoint inhibitor treatment) to respond to immunotherapy (e.g., immune checkpoint inhibitor treatment). The present invention also provides chestnut ellagitannin or its analogs for inducing or restoring a subject with a cancer resistant to immunotherapy (e.g., immune checkpoint inhibitor treatment) to respond to immunotherapy (e.g., immune checkpoint inhibitor treatment).

[0136] In another aspect, the present invention provides a method for treating a subject with a cancer resistant to immunotherapy (e.g., immune checkpoint inhibitor treatment), comprising administering to a subject an effective therapeutic amount of castanea ellagitannin or its analog in combination with an immunotherapy (e.g., an immune checkpoint inhibitor). The present invention also provides the use of castanea ellagitannin or its analog in combination with an immunotherapy (e.g., an immune checkpoint inhibitor) for treating a subject with a cancer resistant to such immunotherapy (e.g., an immune checkpoint inhibitor treatment). The present invention also provides the use of castanea ellagitannin or its analog in combination with an immunotherapy (e.g., an immune checkpoint inhibitor) for the preparation of a drug for treating a cancer patient resistant to such immunotherapy (e.g., an immune checkpoint inhibitor treatment). The present invention also provides a combination therapy comprising a combination of castanea ellagitannin or its analog and an immunotherapy (e.g., an immune checkpoint inhibitor) for treating a subject with a cancer resistant to such immunotherapy (e.g., an immune checkpoint inhibitor treatment).

[0137] On the other hand, the present invention provides a method for enhancing the immune response (e.g., anti-tumor immune response) of a subject in need thereof, the method comprising administering to the subject an effective therapeutic amount of chestnut ellagitannin or an analog thereof. The present invention also provides the use of chestnut ellagitannin or an analog thereof for enhancing the immune response (e.g., anti-tumor immune response) of a subject. The present invention also provides the use of chestnut ellagitannin or an analog thereof in the preparation of a medicament for enhancing the immune response (e.g., anti-tumor immune response) of a subject. The present invention also provides chestnut ellagitannin or an analog thereof for enhancing the immune response (e.g., anti-tumor immune response) of a subject.

[0138] In one embodiment, the above treatment increases the level of immune cells such as T cells (e.g., tumor infiltrating lymphocytes, or TILs) in the tumor. In one embodiment, the T cells are CD4 + and / or CD8 + T cells, such as activated (ICOS+) and / or memory CD4+ or CD8+ T cells (central memory (T CM )CD4 + cells and / or CD8 + In another embodiment, the above treatment increases CD8 + T cells / Foxp3 + CD4 + In another embodiment, the above treatment increases ICOS + Foxp3 - CD4 + T cells.

[0139] In another aspect, the present invention provides a method for increasing the intestinal Fig.14The method of the bacterial level of the family or genus shown, such as Acetatifactor, Lachnospiraceae FCS020 group, Acetitomaculum, Lachnospira GCA-900066225, Akkermansia, Lachnospira GCA-900066575, Alistipes, Lachnospira UCG-001, Anaeroplasma Lachnospira UCG-004, Anaerosporobacter, Lachnospira UCG-006, Anaerovorax,Lactobacillus, Oscillibacter, Bifidobacteriaceae, Oscillospiraceae UCG-005, Bifidobacterium, Prevotella, Bilophila, Parasutterella, Blautia, Peptococcaceae, Butyricicoccus, Peptostreptococcaceae, Rikenellaceae, Candidatus soleaferrea, Porphyromonadaceae, Carnobacteriaceae, Christensenaceae, Romboutsia, Christensenia R-7 group, Roseburia, Clostridiales vadinBB60 group, Ruminiclostridium, Clostridium sensu stricto 1 1), Clostridium rumenii 5, Coprococcus 3, Clostridium rumenii 9, Eisenbergiella, Ruminococcaceae, Enterobacteriaceae, Ruminococcus UBA1819, Enterococcaceae, Ruminococcus UCG-005, Enterococcus, Ruminococcus UCG-009, Erysipelotrichaceae, Ruminococcus 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.,UBA1819), Parasutterella, Clostridium sensu stricto 1, Christensenaceae, Alternaria, Ruminococcus, Akkermansia and / or Anaerobic Pseudomonas, comprising administering to the subject an effective amount of chestnut ellagitannin or an analog thereof. In another aspect, the present invention provides a use of chestnut ellagitannin or an analog thereof for increasing the level of bacteria of the following families or genera in the intestine of a subject, the family or genus being Acetogenes, Lachnospira FCS020 group, Polyacetogenes, Lachnospira GCA-900066225, Akkermansia, Lachnospira GCC-900066575, Alternaria, Lachnospira UCG-001, Anaerovorax, Lachnospira UCG-004, Anaerobacillus, Lachnospira UCG-006, Anaerovorax, Lactobacillus, Angelakisella, Monoglobus, Asaccharospora, Oscillobacterium, Bifidobacterium, Oscillating Spirulina UCG-005, Bifidobacterium, Paraprevotella, Bilophila, Parasartella, Eubacterium, Peptococcus, Butyricococcus, Peptostreptococcus, Candidatus soleaferrea, Porphyromonadaceae, Carnobacteriaceae, Rikenellaceae, Christensenaceae, Rombutzia spp., Christensenella R-7 group, Roseburia spp., Clostridium spp., Clostridium spp. sensu stricto 1, Clostridium spp. 5, Coprococcus spp. 3, Clostridium spp. 9, Eisenbergiella, Ruminococcaceae, Enterobacteriaceae, Ruminococcus UBA1819, Enterococcaceae, Ruminococcus UCG-005, Enterococcus spp., Ruminococcus UCG-009, Erysipelothrixaceae, Ruminococcus UCG-014, Escherichia / Shigella spp., Ruminococcus spp., Family XIII, Sporacetigenium, Family XIIIUCG-001, Staphylococcus, Flavobacterium, Staphylococcus, Herbinix, Tannerella, Isobaculum, Turicibacter, Lachnospiraceae, Tyzzerella3 and / or Lachnospiraceae A2, preferably Turicibacter, Bilophila, Ruminococcaceae (e.g., Ruminococcus UBA1819), Parasartella, Clostridium sensu stricto 1, Christensenaceae, Alternaria, Ruminococcus, Akkermansia and / or Anaerobe. In another aspect, the present invention provides a use of chestnut ellagitannin or an analog thereof in the preparation of a composition for increasing the level of bacteria of the following families or genera in the intestinal tract of a subject, wherein the family or genera is Acetogenes, Lachnospira FCS020 group, Polyacetogens, Lachnospira GCA-900066225, Akkermansia, Lachnospira GCC-900066575, Alternaria, Lachnospira UCG-001, Anaeroproma, Lachnospira UCG-004, Anaerobic Bacillus,Lachnospira UCG-006, Anaerovorax, Lactobacillus, Angelakisella, Monoglobus, Asaccharospora, Oscillobacterium, Bifidobacteraceae, Oscillatory Spirulina UCG-005, Bifidobacterium, Paraprevotella, Bilophila, Parasartella, Eubacterium, Peptococcus, Butyricoccus, Peptostreptococcus, Candidatus soleaferrea, Porphyromonadaceae, Carnobacteriaceae, Rikenellaceae, Christensenaceae, Rombutzia spp., Christensenella R-7 group, Roseburia spp., Clostridium spp., Clostridium spp. sensu stricto 1, Clostridium spp. 5, Coprococcus spp. 3, Clostridium spp. 9, Eisenbergiella, Ruminococcaceae, Enterobacteriaceae, Ruminococcus UBA1819, Enterococcaceae, Ruminococcus UCG-005, Enterococcus spp., Ruminococcus UCG-009, Erysipelothrixaceae, Ruminococcus UCG-014, Escherichia / Shigella spp., Ruminococcus spp., Family XIII, Sporacetigenium, Family XIIIUCG-001, Staphylococcus, Flavobacterium, Staphylococcus, Herbinix, Tannerella, Isobaculum, Turicibacter, Lachnospiraceae, Tyzzerella3 and / or Lachnospiraceae A2, preferably Turicibacter, Bilophila, Ruminococcaceae (e.g., Ruminococcus UBA1819), Parasartella, Clostridium sensu stricto 1, Christensenaceae, Alternaria, Ruminococcus, Akkermansia and / or Anaerobe. ,

[0140] The present invention also provides chestnut ellagitannin or its analogues for increasing the bacterial level of the following families or genera in the intestinal tract of a subject, wherein the families or genera are acetobacter, Lachnospira FCS020 group, Polyacetogenic bacteria, Lachnospira GCA-900066225, Akkermansia, Lachnospira GCC-900066575, Alternaria, Lachnospira UCG-001, Anaeroproteus, Lachnospira UCG-004, Anaerobacillus, Lachnospira UCG-006, Anaerovorax, Lactobacillus, Angelakisella, Monoglobus, Asaccharospora, Oscibacter, Bifidobacterium, Oscibacter UCG-005, Bifidobacterium, Paraprevotella, Bilophila, Parasartella, Eubacterium, Peptococcus, Butyricococcus, Peptostreptococcus, Candidatus soleaferrea, Porphyromonadaceae, Carnobacteriaceae, Rikenellaceae, Christensenaceae, Rombutzia spp., Christensenella R-7 group, Roseburia spp., Clostridium spp., Clostridium spp. sensu stricto 1, Clostridium spp. 5, Coprococcus spp. 3, Clostridium spp. 9, Eisenbergiella, Ruminococcaceae, Enterobacteriaceae, Ruminococcus spp. UBA1819, Enterococcaceae, Ruminococcus spp. UCG-005, Enterococcus spp., Ruminococcus spp. UCG-009, Erysipelothrixaceae, Ruminococcus spp. UCG-014, Escherichia / Shigella spp., Ruminococcus spp., Family XIII, Sporacetigenium, Family XIII UCG-001, Staphylococcaceae, Flavobacterium, Staphylococcus, Herbinix, Tannerellaceae, Isobaculum, Turicibacter, Lachnospiraceae, Tyzzerella 3 and / or Lachnospiraceae A2, preferably Turicibacter, Bilophila, Ruminococcaceae (e.g., Ruminococcus UBA1819), Parasartella, Clostridium sensu stricto 1, Christensenaceae, Alternaria, Ruminococcus, Akkermansia and / or Anaerobe.

[0141] In one embodiment, the above methods or uses increase the level of bacteria of the Turicibacter family or genus. In one embodiment, the above methods or uses increase the level of bacteria of the Bilophila family or genus. In one embodiment, the above methods or uses increase the level of bacteria of the Ruminococcus (e.g., Ruminococcus UBA1819) family or genus. In one embodiment, the above methods or uses increase the level of bacteria of the Parasartella family or genus. In one embodiment, the above methods or uses increase the level of bacteria of the Fusobacterium sensu stricto 1 family or genus. In one embodiment, the above methods or uses increase the level of bacteria of the Akkermansia family or genus. In one embodiment, the above methods or uses increase the level of bacteria of the Anaerobes family or genus. In another embodiment, the bacteria of the Akkermansia family or genus is Akkermensia muciniphilia.

[0142] In another aspect, the present invention provides a method for reducing the level of Lactobacillus and / or Pseudoflavonifractor family or genus bacteria in the intestinal tract of a subject, comprising administering an effective amount of chestnut ellagitanni or its analog to the subject. On the other hand, the present invention provides the use of chestnut ellagitanni or its analog for reducing the level of Lactobacillus and / or Pseudoflavonifractor family or genus bacteria in the intestinal tract of a subject. On the other hand, the present invention provides the use of chestnut ellagitanni or its analog in the preparation of a medicament for reducing the level of Lactobacillus and / or Pseudoflavonifractor family or genus bacteria in the intestinal tract of a subject. The present invention also provides chestnut ellagitanni or its analog for reducing the level of Lactobacillus and / or Pseudoflavonifractor family or genus bacteria in the intestinal tract of a subject.

[0143] The present invention also provides a combination therapy and immunotherapy including castanea ellagitanni or its analogs, such as immune checkpoint inhibitors. The present invention also provides a combination therapy and immunotherapy (i.e., immunotherapeutic agent) comprising castanea ellagitanni or its analogs, such as immune checkpoint inhibitors, for treating subjects with cancer (e.g., cancer resistant to immunotherapy such as immune checkpoint inhibitor monotherapy). The present invention also provides a combination therapy including castanea ellagitanni or its analogs and immunotherapy (such as immune checkpoint inhibitors) for the preparation of a drug for treating a subject with cancer (e.g., cancer resistant to immunotherapy (e.g., immune checkpoint inhibitor monotherapy). The present invention also provides a method for treating a subject with cancer (e.g., cancer resistant to immunotherapy such as immune checkpoint inhibitor monotherapy), the method comprising administering to the subject an effective amount of a combination therapy and immunotherapy comprising castanea ellagitanni or its analogs, such as an immunological checkpoint inhibitor.

[0144] Castanopsis ellagitannin (molecular weight 934.63, CAS No. 24312-00-3) has the following structure:

[0145]

[0146] It is the (33β) isomer of ellagitannin (molecular weight 934.63, CAS No. 36001-47-5) and has the following structure:

[0147]

[0148] Castanea ellagitannins and Quercus ellagitannins belong to a special group of ellagitannins consisting of a series of highly water-soluble C-glucoside variants.

[0149] Therefore, the analogs of chestnut ellagitannin include chestnut ellagitannin glycosides, such as grandinin (lyxose) and roburin E (xylose), casuarinin (casuarinin) and chestnut glaucine. As described in WO2014 / 071438, chestnut ellagitannin analogs can also be ethoxylated chestnut ellagitannin. Chestnut ellagitannin analogs retain or share the biological activity of chestnut ellagitannin, more specifically, have the ability to improve immune response (anti-tumor immune response) and restore the subject's response to immunotherapy such as immune checkpoint inhibitors. In one embodiment, chestnut ellagitannin analogs are chestnut ellagitannin salts, preferably pharmaceutically acceptable salts. The term "salt" used herein means an acidic salt formed with an inorganic acid and / or an organic acid, and an alkaline salt formed with an inorganic base and / or an inorganic base. The salt used in the pharmaceutical composition will be a pharmaceutically acceptable salt. As used herein, the term "pharmaceutically acceptable salt" refers to salts of castanea ellagitanni that retain the biological activity of castanea ellagitanni and are not biologically or otherwise undesirable.

[0150] For example, the salt of castanea ellagitannin can be an acid addition salt, such as 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. It can also be a base addition salt, such as sodium hydroxide or potassium hydroxide, triethylamine or tert-butylamine. Such salts can be formed very easily by those skilled in the art using standard techniques. In fact, chemical modification of drug compounds (i.e., chestnut ellagitannin) into salts is a technique well known to pharmaceutical chemists (e.g., see 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) 33201-217; Anderson et al., The Practice of Medicinal Chemistry (1996), Academic Press, New York; and in the Orange Book on its website (Food & Drug Administration, Washington, DC). For example, a salt of castanea ellagitanni can be formed by reacting castanea ellagitanni with an amount of an acid or base (e.g., an equal amount) in a medium (e.g., a medium in which the salt is precipitated) or in an aqueous medium, followed by freeze drying.

[0151] Chestnut ellagitannins can be found or isolated from a variety of sources, including fruit and / or plant extracts, such as extracts from Myrtle Bark (Camu camu) berries, Lythrum salicaria extract (see, e.g., WO / 2016 / 102874), Quercus plant extracts (Quercus sp.), chestnut extracts (Castanes p.), stem bark extracts from Anogeissus leiocarpus and Terminalia avicennoides (Shuaibu MN et al., Parasitology Research. 103(6): 1333-8), Syzygium samarangense (Blume) leaf extract (Kamada et al., Fitoterapia, Volume 129, September 2018, Pages 94-101). Methods for isolating ellagitannins and / or other C-glucoside ellagitannins are well known in the art and are notably described in some of the above references as well as in Araujo et al., Rsc Advances, 2015, 5, 96151-96157 and Stine et al., Methods in Molecular Biology (Clifton, NJ), 2011, 670, 13-32).

[0152] For the methods, uses and treatments described herein, chestnut ellagitannins or its analogs can be used in the form of extracts (fruit and / or plant extracts) containing a suitable amount of chestnut ellagitannins or its analogs, including crude extracts or partially purified extracts rich in chestnut ellagitannins or its analogs, or can be purified forms (isolated or synthesized from natural sources). Therefore, in one embodiment, an extract containing chestnut ellagitannins or its analogs is used or administered. In another embodiment, purified or separated chestnut ellagitannins or its analogs are used or administered. In one embodiment, purified or separated chestnut ellagitannins or its analogs are used or administered.

[0153] Those skilled in the art will appreciate that the extract or purified castanea ellagitannin or its analogs may 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.

[0154] "Excipient" as used herein has its normal meaning in the art and is any component that is not the active ingredient (drug) itself. Excipients include, for example, buffers, adhesives, lubricants, diluents, fillers, thickeners, disintegrants, plasticizers, coatings, barrier layer preparations, stabilizers, sustained release agents and other components. "Pharmaceutically acceptable excipient" as used herein refers to any excipient that does not interfere with the effectiveness of the biological activity of the active ingredient and is non-toxic to the subject, that is, it is an excipient and / or is used in an amount that is non-toxic to the subject. Excipients are well known in the art, and the compositions of the present invention are not limited in these respects. Carriers / excipients may be suitable for, for example, intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, intraocular, intraventricular, intracapsular, intraspinal, intrathecal, epidural, intracerebral, intraperitoneal, intranasal or pulmonary (e.g., aerosol) administration. By mixing the active ingredient having the desired purity with one or more optional pharmaceutically acceptable carriers, excipients and / or stabilizers (see Remington: The Science and Practice of Pharmacy, by Loyd V Allen, Jr, 2012, 22 nd edition,Pharmaceutical Press;Handbook of Pharmaceutical Excipients,by Rowe etal.,2012,7 th edition,Pharmaceutical Press).

[0155] In one embodiment, a castanea ellagitannin or an analog thereof is prepared for oral administration. Formulations suitable for oral administration may include (a) a liquid solution, such as an effective amount of active agent / composition suspended in a diluent (such as water, saline or PEG 400); (b) capsules, sachets or tablets, each containing a predetermined amount of active ingredient, such as a liquid, solid, granules or gelatin; (c) a suspension in an appropriate liquid; and (d) a suitable emulsion. Tablet forms may include lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearates and other excipients, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavoring agents, pigments, disintegrants, preservatives and / or mixtures thereof One or more, and a pharmaceutically compatible carrier. Lozenge forms may contain the active ingredient in an inert matrix containing, in addition to the active ingredient, a flavoring such as sucrose and such carriers as are known in the art such as emulsions, gels, and the like of gelatin and glycerin or sucrose and acacia.

[0156] In one embodiment, the chestnut ellagitannin or its analog is formulated for parenteral administration (e.g., injection). For example, parenteral formulations may include excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, or hydronaphthalene. Biocompatible, biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compound. Other potentially useful parenteral delivery systems for chestnut ellagitannin or its analogs include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Inhalation formulations 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 administered in the form of nasal drops or gels.

[0157] In one embodiment, chestnut ellagitannin or its analog is formulated for enteral delivery, i.e., delivered to the intestine. This can be achieved by methods well known in the art. For example, chestnut ellagitannin or its analog can be coated or encapsulated with enteric solvents or materials. For example, enteric solvents allow release at certain pH or in the presence of degradative enzymes or bacteria, and the degradative enzymes or microorganisms are characteristically present in a specific position (e.g., small intestine, large intestine, or a specific region thereof) of the gastrointestinal tract that needs to be released. In one embodiment, the enteric material is pH sensitive and is affected by the pH changes encountered in the gastrointestinal tract (pH sensitive release). The enteric material is usually kept insoluble at gastric pH, and then the active ingredient is allowed to be released in a higher pH environment of the downstream gastrointestinal tract (e.g., usually the duodenum, sometimes the colon). In another embodiment, the enteric material includes an enzymatically degradable polymer, which is degraded by bacterial enzymes (e.g., carbohydrate processing enzymes, such as glycosidases, polysaccharide lyases, and carbohydrate esterases) present in the lower gastrointestinal tract, particularly in the colon. Such enteric materials include, for example, cellulosic polymers such as hydroxypropylcellulose, hydroxyethylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate succinate, hydroxypropylmethylcellulose phthalate, methylcellulose, ethylcellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate, and sodium carboxymethylcellulose; acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate, and / or ethyl methacrylate, and other methacrylic acid resins, which are marketed under the trade name acrylic acid; (Colorcon, USA), (RohmPharma; Westerstadt, Germany), including L30D-55 and L100-55 (soluble at pH 5.5 and above), L-IOO (soluble at pH 6.0 and above), S (soluble at pH 7.0 and above due to a higher degree of esterification), and NE, RL and RS (water-insoluble polymers with different permeability and swelling properties); vinyl polymers and copolymers, such as polyvinyl pyrrolidone, vinyl acetate, vinyl acetate-vinyl phthalate, vinyl acetate-crotonic acid copolymer and ethylene-vinyl acetate copolymer; enzyme-degradable polymers, such as azo polymers, pectin, chitosan, amylose and guar gum; zein and shellac. Combinations of different enteric materials can also be used. Methods for colon-targeted drug delivery are well known in the art (e.g., see Philip et al., Oman Med J. 2010 Apr; 25(2):79–87; 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, microbially triggered drug delivery systems (e.g., pressure-controlled colon delivery capsules (drug release induced by the higher pressures encountered in the colon), osmotic-controlled drug delivery, and any combination of these methods (e.g., colon-targeted delivery system (CODESE™) using a combination of pH-dependent and microbially triggered drug delivery methods).

[0158] In one embodiment, castanea ellagitannin or an analog thereof is formulated in a capsule made of enteric material (enteric-coated capsule).

[0159] Any suitable amount of castanea ellagitannin or its analogue can be administered to a subject. The dosage depends on many factors, including the mode of administration. Generally, the amount of castanea ellagitannin or its analogue contained in a single dose will be an amount that is effective in preventing, delaying or treating cancer without inducing significant toxicity.

[0160] In order to prevent, treat or reduce the severity of a given disease or condition (cancer), the appropriate dose of chestnut ellagitannin or its analogs will depend on the type of disease or condition to be treated, the severity and course of the disease or condition, whether chestnut ellagitannin or its mimetics are administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to chestnut ellagitannin or its analogs, and the judgment of the attending physician. Chestnut ellagitannin or its analogs are appropriately administered to the patient in one or a series of treatments. Preferably, it is desirable to determine the dose-response curve in vitro and then determine it in a useful animal model before human trials. The present invention provides a dose of chestnut ellagitannin or its analogs and a composition comprising the same. For example, depending on the type and severity of the disease, the body weight is about 1 μg / kg to 1000 mg / kg (mg / kg) per day. In addition, effective dose can be 0.5mg / kg, 1mg / kg, 5mg / kg, 10mg / kg, 15mg / kg, 20mg / kg, 25mg / kg, 30mg / kg, 35mg / kg, 40mg / kg, 45mg / kg, 50mg / kg, 55mg / kg, 60mg / kg, 70mg / kg, 75mg / kg, 80mg / kg, 90mg / kg, 100mg / kg, 125mg / kg, 150mg / kg, 175mg / kg, 200mg / kg and can be increased to 1000mg / kg with an increment of 25mg / kg or can be between any two values ​​mentioned above. According to the above factors, typical daily dose may be in 1 μ g / kg to 100mg / kg or larger range. For repeated administration for several days or longer, depending on the state of an illness, continuous treatment is continued until the desired disease symptoms are suppressed (for example, tumor volume or tumor cell number are reduced). However, other dosage regimens may be useful. The progress of this treatment is easily monitored by conventional techniques and assays. In one embodiment, the dose administered to a human subject corresponds to a dose of at least 0.8 mg of castanea ellagitannin per kg in mice.

[0161] The term immunotherapy used herein refers to an anti-tumor therapy that enhances or strengthens the immune response for tumor cells.Immunotherapy includes cell-based immunotherapy, such as administering immune cells (such as chimeric antigen receptors (CAR) T cells and NK cells) that can recognize tumor cells, or T cells with tumor antigen-specific TCR, or antigen presenting cells (APCs, such as dendritic cells) that can express tumor antigens on their surface.Immunotherapy also includes administering antigens that recognize tumor cell expression and targeting them for specific antibodies that the immune system destroys, or administering cytokines (interferons, interleukins) that stimulate immune responses.Another type of immunotherapy includes administering immune checkpoint inhibitors.It is possible to use a combination of different types of immunotherapies, such as the combined administration of immune cells (CAR T or NK cells) and immune checkpoint inhibitors.

[0162] As used herein, the term "immune checkpoint inhibitor" (ICI) or "immune checkpoint blocker" (ICB) refers to an agent that blocks or inhibits 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 attenuator (BTLA or CD272), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene-3 (LAG3), immunosuppressive factor (CTLA) and immunosuppressive factor, nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2 (NOX2), programmed death 1 (PD-1) receptor, 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-type lectin 7 (SIGLEC7 or CD328) and SIGLEC9 (CD329). In one embodiment, the immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-1, or PD-L1. In one embodiment, the immune checkpoint inhibitor is an inhibitor of PD-1, such as an anti-PD-1 antibody. In one embodiment, the immune checkpoint inhibitor is an inhibitor of PD-L1, such as an anti-PD-L1 antibody. In one embodiment, the immune checkpoint inhibitor is an inhibitor of CTLA-4, such as an anti-CTLA-3 antibody.

[0163] Cancer can be any type of cancer, including primary (or original) cancer, recurrent cancer or metastatic cancer. Examples of cancer include cardiac sarcoma, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer, bronchial cancer (squamous cell carcinoma, undifferentiated small cell carcinoma, undifferentiated large cell carcinoma, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma (such as Ewing sarcoma, Kaposi's sarcoma), lymphoma, chondromatous hamartoma, mesothelioma; cancers of the gastrointestinal system, such as esophageal (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (stomach) (carcinoma, lymphoma, leiomyosarcoma), gastric (gastric), pancreatic (ductal adenocarcinoma, insulinoma, pancreatic hyperplasia) cancer. Glucagonoma, gastrinoma, carcinoid tumor, VIPoma, small intestine (adenocarcinoma, lymphoma, carcinoid, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); genitourinary tract cancer, for example, kidney cancer (adenocarcinoma, Wilms' tumor, lymphoma, leukemia), bladder and / or urethra cancer (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate cancer (adenocarcinoma, sarcoma), testicular cancer (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma), tumors, mesenchymal cell carcinomas, fibromas, fibroadenomas, adenomatoid tumors, lipomas); liver cancers, for example, liver cancer (hepatocellular carcinoma, HCC), bile duct cancer, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, pancreatic endocrine tumors (such as pheochromocytoma, insulinoma, heme intestinal peptide tumor, islet cell tumor and glucagonoma); bone cancers, for example, osteosarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, malignant lymphoma (reticular cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochondroma (osteocartilaginous exostosis), benign enchondroma , chondroblastoma, chondromyxofibroma, osteoid osteoma, and giant cell tumor; nervous system cancers, for example, central nervous system (CNS) tumors, primary CNS lymphoma, skull bone cancer (osteomas, hemangiomas, granulomas, xanthomas, osteitis deformans), meningiomas (meningiomas, meningosarcomas, gliomatosis), brain cancer (astrocytomas, medulloblastomas, gliomas, ependymomas, germ cell tumors [pineal], glioblastoma multiforme, oligodendrogliomas, schwannomas, retinoblastomas, and congenital tumors), spinal neurofibromas, meningiomas, gliomas, sarcomas);Reproductive system cancers, e.g. gynecological cancers, uterine cancer (endometrial cancer), cervical cancer (cervical cancer, precancerous cervical dysplasia), ovarian cancer (ovarian cancer [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa cell tumor, testicular Leydig cell tumor, dysgerminoma, malignant teratoma), vulvar cancer (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vaginal cancer (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tube cancer (carcinoma); placental cancer, penile cancer, prostate cancer, testicular cancer; hematological cancers, e.g. Blood cancers (acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), myeloproliferative disorders, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; oral cancers, such as lip cancer, tongue cancer, gum cancer, palate cancer, oropharyngeal cancer, nasopharyngeal cancer, sinus cancer; skin cancers, such as malignant melanoma, cutaneous melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles dysplastic dysplastic nevi), lipoma, hemangioma, dermatofibroma and keloid; adrenal cancer: neuroblastoma; and cancers of other tissues, including connective and soft tissue, retroperitoneum and peritoneum, eye cancer, intraocular melanoma and adnexa, breast cancer (e.g. ductal breast cancer), head and neck cancer (head and neck squamous cell carcinoma), anal cancer, thyroid cancer, parathyroid cancer; secondary and non-specific malignancies of the lymph nodes, secondary malignancies of the respiratory and digestive systems, and secondary malignancies of other sites. ;

[0164] Immune checkpoint inhibitors have been approved or are currently in Phase III and IV clinical trials for several cancers, including lung cancer (e.g., non-small cell lung cancer (NSCLC) and small cell lung cancer, squamous cell lung cancer), head and neck cancer (e.g., head and neck squamous cell carcinoma, renal cell carcinoma, gastric adenocarcinoma, nasopharyngeal tumors, urothelial carcinoma, colorectal cancer, mesothelioma (e.g., pleural mesothelioma), breast cancer (e.g., triple-negative breast cancer, TNBC), esophageal cancer, multiple myeloma, gastric and gastroesophageal junction cancer, gastric cancer, 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 tumors, bladder cancer, transitional cell carcinoma, prostate tumors, and biliary tract tumors (see, e.g., Darvin et al., Experimental & Molecular Medicine volume 50, Article number:165(2018)). Thus, in one embodiment, the cancer is one of the above cancers for which an immune checkpoint inhibitor has been approved or is currently being tested in Phase III and Phase IV clinical trials.

[0165] Currently approved immune checkpoint inhibitors include anti-CTLA-4 Ipilimumab (Ipilimumab) (melanoma and lung cancer), anti-PD-1 Nivolumab (Nivolumab) (melanoma, lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck cancer, colon cancer and liver cancer) Pembrolizumab (Pembrolizumab) (melanoma, lung cancer, head and neck cancer, Hodgkin's lymphoma, renal cell tumor and gastric cancer), and Cemiplimab (Cemiplimab) (squamous cell skin cancer, myeloma and lung cancer), and anti-PD-L1 Atezolizumab (Atezolizumab) (NSCLC, small cell lung cancer, TNBC), Avelumab (Avelumab) (non-small cell lung cancer, MCC) and Durvalumab (Durvalumab) (urothelial carcinoma, lung cancer). Therefore, in one embodiment, the cancer is one of the above-mentioned cancers for which immune checkpoint inhibitors have been approved. In another embodiment, 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 skin cancer, or myeloma.

[0166] In one embodiment, the above-mentioned treatment includes the use / application of more than one (i.e., combination) active / therapeutic agent, castanea ellagitannin or its analog in combination with an immune checkpoint inhibitor (i.e., combination therapy). The combination of medicaments can be administered or co-administered (e.g., continuously, simultaneously, at different times) in any conventional dosage form. In the context of the present invention, co-administration refers to the administration of more than one therapeutic agent during coordinated treatment to achieve improved clinical outcomes. This co-administration can also be co-extended, i.e., occurring during overlapping time periods. For example, the first active agent can be administered to the patient before, simultaneously, before and after, or after the administration of the second active agent. In one embodiment, the medicaments can be combined / prepared in a single composition, and therefore administered simultaneously. Alternatively, they can be formulated with a separate composition, and therefore administered separately (simultaneously or at different times).

[0167] In one embodiment, the dosage of castanea ellagic acid or its analogs and / or immune checkpoint inhibitors used / administered in the methods, uses, compositions, and combined therapies of the present invention is a suboptimal dosage. As used herein, "suboptimal dosage" refers to the dosage of one of the compounds of the combination described herein (castanea ellagic acid or its analogs and / or immune checkpoint inhibitors), which, when used in the absence of other compounds of the combination, produces 50% or less of the biological effect, in one embodiment 40% or less, in another embodiment 30% or less, in another embodiment 20% or less, in another embodiment 10% or less. Therefore, using a combination of compounds described herein, wherein one or more compounds in the combination are used at a suboptimal dosage, an improved efficacy / biological effect can be achieved relative to the use of the compound at a comparable suboptimal dosage in the absence of other compounds.

[0168] As used herein, when the effect of the combined compound is greater than the theoretical sum of the effect of each agent in the absence of another agent, a synergistic effect is achieved. A potential advantage of the combined therapy with a synergistic effect is that one or both drugs or treatments at lower doses (e.g., suboptimal doses) can be used to achieve high therapeutic activity with low toxicity. In one embodiment, the combined therapy (chestnut ellagitannin or its analogs and immune checkpoint inhibitors) results in an increase of at least 5% in the effect of the predicted theoretical additive effect relative to the agent. In another embodiment, the combined therapy results in an increase of at least 10% in the effect of the predicted theoretical additive effect relative to the agent. In another embodiment, the combined therapy results in an increase of at least 20% in the effect of the predicted theoretical additive effect relative to the agent. In another embodiment, the combined therapy results in an increase of at least 30% in the effect of the predicted theoretical additive effect relative to the agent. In another embodiment, the combined therapy results in an increase of at least 50% in the effect of the predicted theoretical additive effect relative to the agent. Another advantage of using these drugs in combination is that in the case where any of the drugs alone will not produce an effect, for example, for cancers or tumors resistant to immune checkpoint inhibitors, efficacy can be achieved. Drug resistance means that the administration of immune checkpoint inhibitors alone does not result in significant therapeutic effects, such as a significant reduction in tumor volume or number of tumor cells. Examples of cancers that have been reported to be resistant to immune checkpoint inhibitors in patients and / or animal models include lung cancer (eg, NSCLC), pancreatic cancer, prostate cancer, melanoma, ovarian cancer, urothelial carcinoma, 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).

[0169] Castanopsis or its analogs and / or immune checkpoint inhibitors can be administered / used in combination with one or more additional active agents or treatments (chemotherapy, radiotherapy, surgery, vaccines, immunotherapy, etc.) for treating targeted diseases / disorders (cancer) or for treating one or more symptoms of targeted diseases / disorders (such as analgesics, anti-nausea agents, etc.). In one embodiment, castanopsis or its analogs and / or immune checkpoint inhibitors are used in combination with one or more chemotherapeutic agents, immunotherapy (e.g., using CART cells or CARNK cells), antibodies, cell-based therapies, etc. Examples of chemotherapeutic agents suitable for use in combination with castanea ellagitanni or its analogs and / or immune checkpoint inhibitors include, but are not limited to, vinca alkaloids, drugs that disrupt microtubule formation (such as colchicine and its derivatives), anti-angiogenic agents, therapeutic antibodies, EGFR targeting agents, tyrosine kinase targeting agents (such as tyrosine kinase inhibitors), transition metal complexes, proteasome inhibitors, antimetabolites (such as nucleoside analogs), alkylating agents, platinum-based preparations, anthracyclines, topoisomerase inhibitors, macrolides, retinoids (such as all-trans retinoic acid or its derivatives); geldamycin or its derivatives (such as 17-AAG) and other cancer therapeutics recognized in the art. In some embodiments, chemotherapeutic agents used in combination with castanea or its analogs 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, cholesteryl phenylacetic acid mustard, taxanes and their derivatives (e.g., paclitaxel and its derivatives, taxotere and its derivatives, etc.), topetecan, vinblastine, vincristine, tamoxifen, piposafen, nab-5404, nab-5800, nab-5801, irinotecan, HKP, ortataxel, gemcitabine, oxaliplatin, Vinorelbine, Capecitabine, One or more of lapatinib, sorafenib, erlotinib, erbital and their derivatives.

[0170] The subject can be any animal, in particular a mammal, such as a mouse, a rat, a dog, and a human. In one embodiment, the subject is a human.

[0171] Mode for Carrying Out the Invention

[0172] The invention is illustrated in more detail by the following non-limiting examples.

[0173] Materials and methods

[0174] Mouse studies. All animal studies were approved by the Institutional Animal Care Committee (CIPA) and performed in accordance with Canadian Council on Animal Care guidelines. Mouse experiments were performed using 7-week-old female C57BL / 6 mice from Charles River, Canada. Germ-free female C57BL / 6 mice were purchased from the International Center for Microbiology germ-free facility (University of Calgary, Canada) and maintained in the CR-CHUM germ-free facility.

[0175] Cell culture, reagents, and tumor cell lines. MCA-205 fibrosarcoma cells and E0771 breast cancer cells were used in this study. MHC class I H-2 b Syngeneic cell lines. 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). 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 (WISEST) in the presence of 5% CO2.

[0176] Subcutaneous models of MCA-205 sarcoma and E0771 breast cancer. Syngeneic C57BL / 6 mice were implanted subcutaneously with 0.8×10 6 MCA-205 or 0.5×10 6 E0771. When the tumor size reaches 20 to 35 mm 2Mice were treated intraperitoneally (ip) every three days four times (or twice for flow cytometry analysis, see below) with anti-PD-1 monoclonal antibody (mAb) (250 μg / mouse; clone RMP1-14, BioXcell) or isotype control (clone 2A3, BioXcell). After the start of treatment, mice received oral gavage of the following products daily: C. camu camu (CC) crude extract (Sunfood) (200 mg / kg per mouse), fractions from round 1 extraction (P, INT, NP, Insol) (40.18 mg / kg per mouse), fractions from round 2 extraction (P1, P2, P3, and P4) (equivalent to a dose of fraction P of 40.18 mg / kg per mouse), ellagic acid (extracted from CC, see isolation procedure below) (0.85 mg / kg per mouse), ellagic acid (0.85 mg / kg per mouse) (Sigma-Aldrich), urolithin A (0.5 mg / kg per mouse) (Sigma-Aldrich), and castanein (0.5 mg / kg per mouse). Sigma-Aldrich) and different concentrations of chestnut ellagitannin: 1 / 8, 1 / 6, 1 / 4, 1 / 2 of the standard concentration, standard concentration (0.85 mg / kg per mouse), 1.5-fold (1.25 mg / kg per mouse), and 3-fold increased concentration (2.55 mg / kg per mouse) ( Sigma-Aldrich or isolated from food grade Quercus, see below for isolation procedures). In the control group, mice were orally gavaged with water (100 μL) daily, and tumor area was routinely monitored every three days by caliper. In depletion experiments, we used anti-CD8 monoclonal antibody (150 μg / mouse; clone 53-6.7, BioXcell) or isotype control (clone 2A3, BioXcell).

[0177] Antibiotic treatment. For antibiotic (ATB) experiments, mice were treated by adding an ATB solution containing ampicillin (1 mg / ml), streptomycin (5 mg / ml), and colistin (1 mg / ml) to their 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 by weekly culturing of fecal pellets resuspended in brain heart infusion (BHI) medium + 15% glycerol (0.1 g / ml) on COS (Columbia agar with 5% sheep blood) plates at 37°C under aerobic and anaerobic conditions for 48 h. For fecal microbiota transplantation (FMT) experiments in SPF-housed mice, mice received the same ATB combination for 3 days before FMT.

[0178] Fecal microbiota transplantation (FMT) experiments. FMT was performed by thawing fecal material from five different patients with non-small cell lung cancer (NSCLC) after obtaining appropriate ethical approval at the Centre de Recherche du Centre hospitalier de l'Université de Montréal (CRCHUM) as previously published by Routy et al. Patient records were retrospectively reviewed to determine response status. Two weeks after FMT, mice were treated with anti-PD-1 mAb or isotype control + / - CC, castanea glabra, or water by subcutaneous injection of tumor cells as described above.

[0179] Flow cytometry analysis. Tumors and spleens were harvested 9 days after the first injection of anti-PD-1 mAb into mice bearing MCA-205 tumors and 11 days after the first injection of anti-PD-1 mAb into mice bearing E0771 tumors. The excised tumors were cut into small pieces and lysed in a 25 μg / mL Liberase TM The spleen was digested in RPMI medium containing 150 UI / mL of DNase I (Roche) at 37°C for 30 minutes, and then crushed and filtered twice with 100 and 70 μm cell strainers (Fisher Scientific). The spleen was crushed in RPMI medium and then filtered through a 100 μm cell strainer. Two million tumor cells or spleen cells were pre-incubated with purified anti-mouse CD16 / CD32 (clone 93; eBioscience) for 30 minutes at 4°C before membrane staining with anti-mouse antibodies against 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), ICO (7E.17G9) (BD, BioLegend, R&D and eBioscience). For intracellular staining, the Foxp3 staining kit (eBioscience) was used. Dead cells were excluded using the Live / Dead Fixable Aqua Dead Cell Stain Kit (Life Technologies). Samples were acquired on a BD Fortessa 16-color cytometer (BD) and analyzed using FlowJo software (BD).

[0180] Immunofluorescence staining. Mouse tumors preserved in optimal cutting temperature (OCT) compound were cut into 5 μm thick sections) and adhered to microscope slides and stored at -80 ° C. At the beginning of the experiment, the slides were air-dried and washed with cold acetone. To prevent nonspecific binding to biotin, an endogenous anti-biotin-biotin blocking kit (ThermoFisher) was used. In addition, the tissue was 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 conjugated to AF-488 were used as secondary antibodies, and the slides were incubated with Cy3 streptavidin to detect biotinylated antibodies. Cell nuclei were displayed by counterstaining with DAPI (ThermoFisher). Images were generated using a full slide scanner Olympus BX61VS (20x 0.75NA objective lens with a resolution of 0.3225mm). Images were analyzed using Visiomorph software (Visiopharm).

[0181] HPLC and LC-MS systems. A 1260 Infinity LC system connected to a 6120 quadrupole LC / MS mass spectrometer from Agilent Technologies was used for reverse phase chromatography (C18) and mass spectrometry (MS), respectively. X-Select SCH and HSS columns (Waters) were used for HPLC. For chromatography, a MilliQ TM The two-component solvent system of used water (solvent A) and acetonitrile (ACN) (solvent B), each acidified with 0.1% formic acid (FA). Only negative ionization data are reported, as this polarity is optimal for polyphenols in acidified solutions.

[0182] Camu camu (CC) extraction. Polyphenols in CC were extracted according to the procedure of Fracassetti et al. (Food Chem 201315;139(1-4):578-88) with slight modifications. Freeze-dried crude CC extract (SunFood) was extracted with 50% aqueous methanol (MeOH) at a ratio of 1:15 (g:mL) (analytical experiments) or 1:8 (preparative experiments). The suspension was vortexed, sonicated, and incubated at room temperature for 60 min. The suspension was centrifuged and the supernatant recovered. The sediment was subjected to a second extraction using 90% aqueous methanol. The supernatants of the two extracts were combined and filtered before analysis.

[0183] Camu camu (CC) extract analysis and LC-MS peak identification. After extraction of polyphenols from CC, the combined extracts were injected into the LC-MS system for analysis. The solvent gradient used to resolve the components in the sample was modified from Fracassetti et al., 2013. The relative retention times at 254 nm and negative ion mass spectra from the LC-MS analysis were compared with the CC polyphenol characterization results from Fracassetti et al., 2013. The identities of the peaks were tentatively determined based on the agreement between the current data and the data reported by Fracassetti et al., 2013.

[0184] Identification of the active fraction P (fractionation cycle 1) isolated from Camu camu (CC). To assess which components of CC are responsible for its activity, four fractions were generated by reverse phase chromatography and sequential extraction: polar (P), intermediate polar (M), non-polar (NP), and insoluble (INS). Polyphenols were extracted from CC and concentrated to dryness before being redissolved in a 40% ACN:10% MeOH water mixture to dissolve most of the polyphenols. Insoluble material was separated by filtration and discarded. The same solvent gradient used for CC peak identification (above) was used for preparative HPLC. Fractions were manually collected every 10 minutes for a total of 60 minutes. The fractions were then frozen to -80°C and lyophilized. The three fractions from 30-60 minutes were combined to produce fraction NP. A 0-10 minute HPLC column breakthrough was used as the starting point for the generation of fraction P. Briefly, four Strata C18-E solid phase extraction (SPE) columns (Phenomenex) were set up in parallel and conditioned with methanol. The lyophilized 0-10 minute HPLC penetration was dissolved in MilliQ water at a concentration of 10 mg / mL. 5 ml of sample (10 mg / mL) was added to each column and flow was collected. Then, 9 ml of 5% ACN was added to the column and flow was collected. The flow collected from each column was combined and freeze-dried to produce fraction P. By continuous extraction of the CC crude extract in water, to remove highly polar compounds, 50% MeOH and 90% MeOH, fractions M and INS were produced. Fraction M consists of 50% methanol extract, which is evaporated and then freeze-dried. The INS fraction consists of the dry sediment after all extraction steps are completed.

[0185] Characterization of the active fraction P3 (fractionation cycle 2). In order to evaluate which components of fraction P are responsible for its activity, 4 fractions (P1, P2, P3 and P4) were produced. A new 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, 100% B at 36-46 min. For the production of fraction P, the HPLC flowthrough from 0-10 min (fractionation cycle 1) dissolved in MilliQ water was used as the starting point for fractionation cycle 2. Fractions were collected manually every minute for 30 min. Fractions from each run were analyzed by LC-MS, then lyophilized and combined to produce fractions P1, P2, P3, and P4 as follows: 0-5 minutes were combined to produce fraction P1, 5-17 minutes were combined to form fraction P2, 18-19 minutes were combined to make fraction P3, and 20-30 minutes were combined to make fraction P4.

[0186] Characterization of fraction P3. The purity of the chestnut ellagitannin peak in fraction P3 was determined by peak integration of the LC-MS chromatogram at 254 nm. The chestnut ellagitannin analytical standard dissolved in MilliQ water was used to compare the retention time and negative ion mass spectrum at 254 nm. Fraction P3 and chestnut ellagitannin analytical standard were dissolved in D2O for NMR analysis. 1 H. 1 H- 1 H correlation (COSY) and 1H-13C heteronuclear single quantum coherence (HSQC) NMR spectra were recorded on a Bruker AviiiiHD 500MHz NMR spectrometer. The peaks were compared with the reassignment of the chestnut ellagitannin structure by Matsuo et al., 2015 (Org Lett 2015 Jan 2;17(1):46-9. Epub 2014 Dec 12).

[0187] Separation of chestnut ellagitannins and oak ellagitannins from Camu camu (CC) and food grade Quercus plants. 20 grams of freeze-dried CC powder were extracted as described above. The crude extract was pre-fractionated using a Strata C18-E SPE column. In brief, 3 ml of the re-dissolved 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 was collected in 3 ml batches. Chestnut ellagitannins and oak ellagitannins were subsequently purified from the flow by HPLC. The isolates were analyzed by LC-MS and their purity was assessed.

[0188] Synthesis, purification and characterization of fluorescent chestnut ellagitannin. Chestnut ellagitannin was monofunctionalized with fluorescein by transesterification with 5 / 6-carboxyfluorescein succinimidyl ester (fluorescein NHS). In brief, chestnut ellagitannin was dissolved in DMF and then reacted with fluorescein NHS (2 equiv) in the presence of triethylamine (2 equiv) and 4-dimethyliminopyridine. The crude mixture was analyzed by LC-MS after treatment with DOWEX50WX8 resin. The peak corresponding to the monofunctionalized fluorescent chestnut ellagitannin was separated by HPLC. Ruminococcus braunii, Escherichia coli and Bacteroides polymorpha bacteria were stained in the presence of fluorescein-bound chestnut ellagitannin at 37°C and 0°C and in the presence of 100-fold concentration of unbound chestnut ellagitannin.

[0189] Inverted Epi-fluorescence Microscope An inverted optical microscope (Ti2, Nikon, Inc.) configured for epi-fluorescence and equipped with a high-sensitivity CCD camera (C14440-20UP, Hamamatsu, Inc.) was used to acquire images of fluorescent chestnut ellagitannin staining.

[0190] Genomic DNA was extracted from mouse feces. Total genomic DNA was extracted from fecal pellets using the Zymobomics DNA Miniprep Kit (Zymo Research) and immediately stored at -80°C. The protocol included a beadbeating step to ensure complete recovery of bacterial DNA. DNA concentration and quality were determined using Nanodrop ND-1000 (ThermoFisher).

[0191] Quantitative real-time PCR (qRT-PCR). Quantitative real-time PCR was performed to assess the relative levels of total bacterial DNA and to amplify the V6 region of the 16S rRNA gene using primer set 891F (5'-TGGAGCATGTGGTTTAATTCGA-3', SEQ ID NO: 1) and 1033R (5'-TGCGGGACTTAACCCAACA-3', SEQ ID NO: 2) (Anhê et al., Diabetologia. 2018 Apr; 61(4): 919-931), and to specifically assess the relative levels of Ruminococcaceae DNA using specific primers F (5'-TGCGGGACTTAACCCAACA-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 500 nM of the above primer mixture and 1X qPCRBIO-SyGreen-blue-mix-Hi-ROX (PCRBIOSystems). The qPCR reaction was performed at 95°C for 3 minutes on the Applied Biosystems StepOnePlus real-time PCR system (ThermoFisher Scientific) to denature the DNA, amplify at 95°C for 5 seconds, 60°C for 30 seconds, 40 cycles, and completed at the melting curve stage. The original threshold cycle (Ct) value was compared with the bacterial standard curve generated by Escherichia coli DNA for 16s analysis and compared with Ruminoccocusbicirculans of the Ruminococcaceae family to roughly estimate the bacterial load.

[0192] 16SrRNA gene sequence processing and mouse fecal sample analysis. The isolated DNA was analyzed using the 16S ribosomal RNA (rRNA) gene sequence to investigate the microbial composition in the fecal samples. The V3-V4 region of the 16S rDNA gene was amplified by PCR, and primers Bakt_341F (5′-CCTACGGGNGGCWGCAG-3′, SEQ ID NO: 5) and Bakt_805R (5′-GACTACHVGGGTATCTAATCC-3′, SEQ ID NO: 6) were suitable for combining the transposon-based IlluminaNextera adapter (Illumina) and the sample barcode sequence that allows multiple double-end sequencing. The PCR mixture contained 1× Q5 buffer (NEB), 1× Q4 enhancer (NEB), 200 μM dNTPs (VWR International), 0.2 μM forward and reverse primers ((Integrated DNA Technologies), 1 unit Q5 (NEB), and 1 μl template DNA in a 50 μl reaction. PCR cycling conditions included an initial denaturation at 98°C for 30 s, followed by a first set of 15 cycles (98°C for 10 s, 55°C for 30 s, and 72°C for 30 s), followed by a second step of 15 cycles (98°C for 10 s, 65°C for 30 s, and 72°C for 30 s), and a final extension at 72°C for 2 min followed by indefinite cooling to 4°C. PCR products were purified using 35 μl magnetic beads (AxyPrep Mag PCR Cleanup Kit; Axygen Biosciences) per 50 μl PCR reaction. DNA was purified using a DNA 7500 chip (Agilent Technologies). Amplification was controlled on a Bioanalyzer 2100 (Life Technologies). Samples were pooled in equimolar ratios; the pooled samples were repurified as described previously and quality checked on a Bioanalyzer 2100 using a DNA High Sensitivity chip. Pooled samples were quantified using picogreen (Life Technologies) and loaded onto a MiSeq system (Illumina). High-throughput sequencing was performed at IBIS (Institut de Biologie Intégrative et des Systèmes-Université Laval).

[0193] Gene sequence processing and analysis were performed using R v4.0.0. The DADA2 R 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 Illumina amplicon sequence errors were corrected, deduplicated, chimeras were removed, and the double-end reads were merged with 260 bases of forward reads and 190 bases (reverse reads). Taxonomic assignment was performed according to the SILVA reference database v138 (Quast et al., Nucleic Acids Res. 2013 Jan; 41 (Database issue): D590-6). Residual sequences of archaea and eukaryotes were removed. Downstream analysis was performed at the genus level using the phyloseq R package v1.32.0 (McMurdie et al., 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 test.

[0194] Statistical analysis. Statistical analysis was performed using R v4.0.0. Significant differences between groups were determined using the Mann–Whitney U test using alpha diversity, which shows the diversity measured for each sample. DESeq2 (Love et al., Genome Biol. 2014;15(12):550) was used for differential abundance analysis at the genus level. Spearman rank correlation tests were obtained using Graphpad. Prism 8 was used to compare continuous variables between flow cytometry analysis parameters and to compare important bacteria identified in water by differential abundance analysis for water vs. CC, water / αPD-1 vs. CC / αPD-1, water / αPD-1 vs. ellagitanni / αPD-1, and water / IsoPD-1 vs. ellagitanni / αPD-1 groups. Unless otherwise stated, all p-values ​​are reported after Bonferroni correction when questions involved more than two experimental conditions. p values ​​are two-sided with a confidence interval of 95%, *p<0.05, **p<0.01, ***p<0.001.

[0195] Example 1: Administration of Camu Camu (CC) extract enhances or restores anti-PD-1 anti-tumor activity in a mouse tumor model.

[0196] To find ways to enhance or restore the antitumor activity of ICBs (e.g., anti-PD-1 antibodies), a crude extract from the Amazonian fruit Camu camu (Myrtles camu) with a unique phytochemical profile was prepared according to Figure 1AThe regimen shown was co-administered with anti-PD-2 in syngeneic C57BL / 6 mice implanted with MCA-205 sarcoma cells (anti-PD-1 sensitive). The camu camu extract used in the experiments described herein was commercialized camu camu raw powder from Sunfood, which was obtained from camu camu berries from the South American rainforest, dried at low temperatures and ground into a fine powder.

[0197] Figure 1B-1C The results showed that daily oral gavage with CC extract alone exhibited anticancer activity (similar to anti-PD-1 monotherapy) and enhanced the anticancer activity of anti-PD-2 antibody, as evidenced by a reduction in tumor size.

[0198] Next, according to Figure 2A The protocol shown tests the antitumor effect of CC in mice implanted with anti-PD-1 resistant tumors (E0771 breast cancer). As expected, administration of anti-PD-1 alone did not result in a significant reduction in tumor size in this model, confirming the resistance of E0771 breast cancer cells to anti-PD-2 monotherapy ( Figure 2B-2C ). Similarly, administration of CC extract alone failed to significantly reduce E0771 tumor size. However, after administration of anti-PD-1 and CC extract, a significant reduction in E0771 tumor size was obtained ( Figure 2B -C), providing evidence that CC extract has the ability to restore anti-PD-2 antitumor responses.

[0199] Example 2: Camu camu extract exerts its effects by regulating intestinal microbiota

[0200] according to Figure 3A The experiments were performed according to the protocol shown in order to better understand the mechanism by which CC extract exerts its antitumor effect. Figure 3B-Figure 3C It was shown that in the mouse MCA-205 tumor model, the administration of broad-spectrum antibiotics (ATB), which affect the intestinal microbiota, completely abolished the antitumor effect of CC extract. Secondly, 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 implanted with MCA-205 tumors, and the effect on tumor size ( Figure 4A ).like Figure 4B As shown, microbial transfer from mice previously treated with CC extract was sufficient to restore CC activity in monotherapy or in combination with anti-PD-1. To explore the therapeutic potential of CC, ATB-treated mice were re-cloned by FMT in two non-small cell lung cancer (NSCLC) responder (R) patients and two non-responder (NR) patients. MCA-205 tumors were inoculated into these “avatar” mouse models, and mice were treated with CC or water with or without the addition of αPD-1 ( Figure 5A ). FMT from NR patients conferred resistance to αPD-1, whereas FMT from R patients restored the anti-tumor effects of αPD-2 ( Figure 5A and 5B ). The baseline microbiota of mice receiving FMT was characterized after FMT implantation (before CC+ / -αPD-1). FMT from R was associated with greater α diversity ( Figure 5C When analyzing the β diversity R compared to the NR group, two target clusters were also found ( Figure 5D Interestingly, in mice that received FMT from R and had tumors that were sensitive to water / αPD-1, Bilophila and Ruminococcaceae UBA1819 were overexpressed ( Figure 5E ).

[0201] Oral supplementation of CC / isoPD-1 restored the antitumor effects of CC in FMTNR avatar mice ( Figure 4B ). Moreover, the combination of CC / αPD-1 restored the impaired αPD1 efficacy in FMT NR mice treated with water / αPD-2. In contrast, in FMTR avatar mice, CC alone or in combination with anti-PD-1 did not show any further enhancement of the antitumor response compared with water. At the microbiome level, CC / isoPD-1 increased α diversity in FMT NR avatars but did not affect diversity in FMT R avatar mice in water / isoPD-2 ( Fig. 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).

[0202] Next, 16s rRNA sequencing was used to Figure 1A Fecal samples were subjected to microbiota analysis for the experiments described in . The V3-V4 region of the 16S rDNA gene was amplified by PCR using primers Bakt_341F and Bakt-805R, which have been adapted to bind to transposon-based Illumina Nextera adapters (Illumina). High-throughput sequencing was performed at the Institut debiologie intégrative et des système (IBIS). After dataset filtering (from low to high read length), rRNA sequences that successfully passed the pre-processing steps and had ≥97% nucleotide sequence identity were merged into operational taxonomic units (OTUs) using USEARCH 61 (version 6.1.544). These experiments showed that CC treatment was associated with an increase in α-diversity compared to water / isoPD-1, independent of αPD-1 treatment ( Fig. 6AQuantitative real-time (qRT PCR) PCR using specific primers based on the 16S rRNA gene confirmed that the bacterial abundance in the CC group was increased compared with water ( Figure 6B ).

[0203] Beta diversity measured by Bray-Curtis showed that oral CC resulted in the formation of separate bacterial communities compared with before CC supplementation (p < 0.001)( Figure 6C ), while oral gavage of water / αPD-1 showed that the microbiota did not separate into different groups (p = 0.16) ( Figure 6C-D ).

[0204] Differential abundance analysis showed that specific bacteria at the genus level were particularly enriched in the CC group compared with the water group. Ruminococcus (adjusted p < 0.05) was the most differentially abundant bacterium, followed by Turicibacter and Oscillatory Spirospira UCG 005 (unadjusted p < 0.01) ( Fig. 6E In addition, Ruminococcus was the only bacterium that consistently increased in the CC / isoPD1 and CC / αPD-1 groups compared to the corresponding water groups ( Fig. 6F -G).

[0205] Profiling of the gut microbiota using 16S rRNA sequencing in the αPD-1-resistant E0771 tumor model showed an overrepresentation of Turicibacter, Bilophila, Ruminococcaceae UBA1819, Parasutterella, Clostridium sensu stricto 1, Ruminococcus, Akkermansia, and Anaerobicus in CC / αPD-3-treated mice compared with water / αPD-2 (adjusted p < 0.05). Figure 6H Interestingly, Akkermansia and Ruminococcus were also overexpressed in mice treated with CC / isoPD-1 compared to water / ISO-1 ( Fig.6I ). Taken together, these results reveal specific associations between bacterial species and the anticancer effects of CC.

[0206] These results provide convincing evidence that the antitumor activity of CC extract is at least partially dependent on the gut microbiota.

[0207] Example 3: Effects of CC administration on immune cells

[0208] Immunotherapy was performed in mouse tumor models to evaluate the effects of treatment on immune cells. Administration of CC extract alone or in combination with anti-PD-1 resulted in central memory (T) in the MCA-205 tumor model. CM )CD8 + The significant upregulation of Fig. 7A ), and CD8 + / Foxp3+ CD4 + The ratio of T(Treg) increased significantly ( Figure 7B ), that is, CC / isoPD-1, CC / αPD-1 or water / αPD-1 increased relative to water / isoPD-1. In addition, CD8 + Significant increase in ICOS expression on T cells ( Figure 7C ).

[0209] To verify that the antitumor activity associated with CC is mediated by CD8 + T cell-mediated, MCA-205-bearing mice received CC and anti-CD8 + Monoclonal antibodies to deplete CD8 + subsets and showed increased tumor growth compared with CC / isoCD8 (control), indicating that the antitumor effect of CC is CD8 + T cell-dependent ( Fig.7D ).

[0210] Next, we investigated the effects of CC on the gut microbiota, tumor size, and immune profile of tumors and spleens in the MCA-205 mouse model. Pairwise comparisons between CC / isoPD-1-enriched bacteria and water / isoPD-2 groups using nonparametric Spearman correlations were performed using intratumoral cell count immune markers and tumor size. + T cell infiltration ratio, CD8 + T cell PD-L1 expression, CD8 + TCM cells and CD8 + The increase in the ratio of Tregs was associated with CC-enriched bacteria such as Ruminococcus and down-regulated Lactobacillus and Pseudomonas aeruginosa ( Fig. 7E Similarly, in the CC / isoPD-1 group, CD8 + Cells and CD8+ / Treg ratios were associated with Ruminococcus and Oscillatory Spirulina UCG 005.

[0211] In parallel, TILs in E0771 tumors were analyzed after CC+ / -αPD-1. CC combined with αPD-1 induced intratumoral CD8 + T cell activation, ICOS compared with water / αPD-1 + CD8 + The MFI of T cells increases ( Figure 7C). Spearman rank correlation analysis was then performed between upregulated bacteria, immune markers, and tumor size in water / αPD-1 and CC / αPD-1, further demonstrating that the CD8 / Treg ratio was associated with ICOS+Foxp3-CD4 + A positive correlation between the upregulation of T cell infiltration, the abundance of Ruminococcus, Bilophila, and Akkermansia resulted in a decrease in tumor size ( Figure 7F ).

[0212] Example 4: Isolation of Castanea ellagitannin polyphenol extract as a bioactive compound for CC antitumor activity

[0213] To identify specific compounds in CC extracts that confer the antitumor activity observed in mouse tumor models, Fig. 8A Fractionation workflow diagram shown for HPLC separation of CC extracts. Figure 8B A representative plot of HPLC retention times for the intact Camu camu extract is depicted in Figure 1, followed by the HPLC retention times for the polar fraction and fraction P3, as well as for Castanea ellagitannins extracted from Quercus plants. Using this technique, the CC extract was first separated into 4 fractions (P-polar, M-intermediate / medium polar, NP-nonpolar, INS-insoluble), and each fraction was tested in the MCA-205 tumor model. Figure 8C The results presented indicate that only the polar fraction (P) was able to mimic the effects of the CC extract tested in parallel in this experiment at a dose of 200 mg / kg.

[0214] The active polar fraction P was then further separated into different sub-fractions based on retention time (P1-P4) and subjected to HPLC analysis. The P1 fraction consisted mainly of ascorbic acid, P2 consisted of the polyphenols oak ellagitannins and gallic acid, P3 consisted of the polyphenols chestnut ellagitannins, and P4 consisted of different impurities. These four sub-fractions were tested in the MCA-205 model and it was found that P3, which consisted mainly of chestnut ellagitannins, was the only fraction associated with an antitumor effect similar to that of CC ( Fig.8D ).

[0215] To confirm that the active ingredient in subfraction D was indeed castanein, a commercial source (MilliporeSigma) was used. Purified chestnut ellagitannins obtained from the reference material (reference material) and chestnut ellagitannins extracted from CC and Quercus were additionally tested by HPLC. HPLC of chestnut ellagitannins from different sources confirmed that the retention time was similar to that of the P3 fraction extracted from CC. or castanea ellagitannins from the genus Quercus, at similar concentrations in CC (0.85 mg / kg per mouse), achieved similar tumor inhibition in MCA-205 and in combination with αPD-1 in E0711 ( Figure 8E-8FTo determine whether castanea ellagitannin is dose-dependent, six different concentrations (1 / 8 to 3 times the standard dose) were administered orally to mice. Some anticancer activity was observed at 1 / 2 the dose (0.42 mg / kg), and only at the standard dose (0.85 mg / kg, Fig. 8E ). On the other hand, a 3-fold increase in concentration (2.55 mg / kg per mouse) did not reach the standard dose. In conclusion, the results suggest that castanea ellagitannins are bioactive compounds of CC with a dose-dependent effect and a potential plateau.

[0216] Example 5: Supplementation with chestnut ellagitannins increases bacterial diversity of the intestinal microbiota and enhances T cell-mediated ICI responses

[0217] Proof-of-principle experiments were performed to determine the microbiota-dependent effects of ellagic acid from chestnuts under aseptic conditions. Fig.9A As shown, performing the experiment under sterile conditions abolished the antitumor effect of ellagitannin. The next step was to evaluate the impact of ellagitannin on the microbiome of SPF mice treated with ellagitannin / isoPD-1. 16S microbiome analysis showed an increase in α diversity after ellagitannin ( Fig. 9B ) and the significant group formation observed in β diversity ( Fig. 9C At the taxonomic level, ellagitannins resulted in the enrichment of Akkermansia, Ruminococcus UBA1819, Ruminococcus, Staphylococcus, Escherichia / Shigella, Eubacterium, and Alternaria, whereas feces from the water group were enriched in Lachnospiraceae UCG-001 ( Fig.9D ). In addition, in the NR FMT experiment, an increase in the relative abundance of Ruminococcus, Alternaria, Christensenella R7, and Paraprevotella in the ellagitannin group was observed relative to the water group after 16S sequencing analysis, while no difference in Lachnoclostridium was observed between the water and ellagitannin groups in the NR FMT experiment ( Figure 9E-Figure 9I In the dose-dependent chestnut ellagitannin experiment, feces were subjected to qRT-PCR using primers specific for Ruminococcaceae ( Figure 9J Compared with the water control, 1 / 4 of the chestnut ellagitannin concentration (no antitumor effect) did not increase the abundance of Ruminococcaceae, while supplementation with the standard chestnut ellagitannin dose or three times the standard chestnut ellagitannin dose (with antitumor effect) significantly increased the abundance of Ruminococcus ( Figure 9J ).

[0218] To determine whether castanea ellagitannins have the same effects on systemic immune responses as CC, two techniques were used. First, flow cytometric analysis in the MCA-205 experiment again revealed that T CM CD8+ T cells, while no effect of CC was observed under sterile conditions ( Fig. 10A Second, immunofluorescence (IF) staining further demonstrated that CD8 + / Foxp3 + CD4 + The ratio of water / IsoPD-1 increases ( Figure 10B-Figure 10C In E0771 cells, the effects of castanea ellagitannin on the expression of memory CD8 in the tumor microenvironment and spleen cells were compared when αPD-1 was used with or without CC. + T cells (CD44 High CD62L - CD8 + T cells) Fig. 10D and 10E ).

[0219] As previously performed using surrogate mice, we tested the therapeutic effect of castanea ellagitannin in NRNSCLC patients after FMT under ATB and GF conditions. Addition of castanea ellagitannin alone was able to restore antitumor activity and had an additive effect when combined with αPD-1 ( Figure 11A-11B ).

[0220] To elucidate the mechanism by which castanein alters the composition of the intestinal microbiota and changes the microbiota, the metabolites of castanein and its isomer, quercetin, were analyzed. Castanein is hydrolyzed into ellagic acid and castanein, and ellagic acid is further converted into urolithin ( Figure 12A-12B Therefore, some of these metabolites (ellagic acid and urolithin A) were tested individually for their potential antitumor effects, as well as ellagic acid from quercetin. In contrast to ellagic acid from quercetin, no antitumor effects were observed for downstream metabolites or isomers ( Fig. 12B ).

[0221] Using fluorescein-conjugated castanea ellagitannin ( Fig. 12C ), we next evaluated whether chestnut ellagitannins could interact with Ruminococcaceae. Fig.12D and 12E The results shown show that co-cultivation of fluorescently labeled chestnut ellagitannins with Ruminococcus braunii resulted in labeling of the majority (~80%) of the bacteria, whereas labeling rates for E. coli and Bacteroides multilineata were 10% and 34%, respectively, after co-cultivation under the same conditions. Incubation in the presence of excess (100x) unlabeled chestnut ellagitannins reduced the proportion of fluorescently labeled Ruminococcus braunii ( Fig.12E ).

[0222] Figure 12G and 12Hshowed that daily administration of 1.5 mg of camu camu for 3 weeks in two non-cancer human patients resulted in an increase in diversity (16s) and the presence of Ruminococcaceae in fecal samples, which is consistent with the results obtained in mice.

[0223] Fig.13 Figure 2 shows the results of repeated qRT-PCR experiments performed on feces in the chestnut ellagitannin experiment using primers specific for Ruminococcaceae, confirming the increase of Ruminococcaceae in the chestnut ellagitannin treatment group ( Fig.13 ).

[0224] Although the present invention has been described above by specific embodiments of the present invention, modifications may be made to the present invention without departing from the spirit and nature of the present invention as defined in the appended claims. In the claims, the word "including" is used as an open-ended term, substantially equivalent to the phrase "including but not limited to". The singular forms "a", "an" and "the" include the corresponding plural forms unless the context clearly dictates otherwise. Sequence Listing <110> Royal Society for the Advancement of Science / McGill University Val-Chaim Partners Ltd. Laval University <120> Use of castanea ellagitannin or its analogs for anticancer efficacy and increased response to immune checkpoint inhibitors <130> G17004-00008 <150> US 62 / 979,327 <151> 2020-02-20 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 1 tggagcatgt ggtttaattc ga 22 <210> 2 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 2 tgcgggactt aacccaaca 19 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 3 actgagaggt tgaacggcca 20 <210> 4 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 4 cctttacacc cagtaawtcc gga 23 <210> 5 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> misc_feature <222> (9)..(9) <223> n is a, c, g, or t <400> 5 cctacgggng gcwgcag 17 <210> 6 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 6 gactachvgg gtatctaatc c 21

Claims

1. Use of chestnut ellagitannin in the preparation of a medicament for treating a subject suffering from a cancer resistant to treatment with an anti-programmed death-1 (PD-1) antibody, wherein the medicament is used in combination with an anti-PD-1 antibody, and the cancer is a sarcoma or breast cancer.

2. The use according to claim 1, wherein the anti-PD-1 antibody is nivolumab, pembrolizumab or cemiplimab.

3. The use according to claim 1, wherein the chestnut ellagitannin is present in a plant extract.

4. The use according to claim 3, wherein the castanea ellagitannin is present in a fruit extract.

5. The use according to claim 4, wherein the extract is a Myrtlesia scabra extract.

6. The use according to claim 1 or 2, wherein the castanea ellagitannin is present in a pharmaceutical composition.

7. The use according to claim 3, wherein the extract is formulated for delivering the castanea ellagitannins to the intestine.

8. The use according to claim 6, wherein the pharmaceutical composition is formulated for delivering the castanea ellagitannin to the intestine.

9. The use according to claim 7, wherein the extract is formulated into capsules.

10. The use according to claim 8, wherein the pharmaceutical composition is formulated into a capsule.

11. The use according to any one of claims 1 or 2, wherein the cancer is breast cancer.

12. The use according to claim 11, wherein the breast cancer is triple negative breast cancer (TNBC).

13. The use according to claim 1 or 2, wherein the cancer is a sarcoma.

14. Use of chestnut ellagitannin in the preparation of a medicament for enhancing the anti-tumor immune response of a subject suffering from cancer resistant to treatment with an anti-programmed death-1 (PD-1) antibody, wherein the medicament is used in combination with an anti-PD-1 antibody, and the cancer is a sarcoma or breast cancer.

15. The use according to claim 14, wherein the anti-tumor immune response is an anti-tumor T cell response.

16. The use according to claim 14, wherein the anti-PD-1 antibody is nivolumab, pembrolizumab or cimiprilimab.

17. Use according to any one of claims 14 to 16, wherein the castanea ellagitannin is present in a plant extract.

18. The use according to claim 17, wherein the castanea ellagitannins are present in a fruit extract.

19. The use according to claim 18, wherein the extract is a Myrtlesia scabra extract.

20. The use according to any one of claims 14 to 16, wherein the castanea ellagitannin is present in a pharmaceutical composition.

21. The use according to claim 17, wherein the extract is formulated for delivery of the castanea ellagitannins to the intestine.

22. The use according to claim 20, wherein the pharmaceutical composition is formulated for delivering the castanea ellagitannin to the intestine.

23. The use according to claim 21, wherein the extract is formulated into capsules.

24. The use according to claim 22, wherein the pharmaceutical composition is formulated into a capsule.

25. The use according to any one of claims 14 to 16, wherein the subject suffers from breast cancer.

26. The use according to claim 25, wherein the breast cancer is triple negative breast cancer (TNBC).

27. The use according to any one of claims 14 to 16, wherein the subject has a sarcoma.

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