COMPOSITION FOR PREVENTING OR TREATING CANCER

Abstract

The present invention relates to a composition capable of preventing or treating cancer, particularly pancreatic cancer. The composition can effectively inhibit the growth or proliferation of cancer cells, and furthermore, effectively prevent the resistance of cancer cells to anticancer agents, or the metastasis or recurrence of cancer, by using an inhibitor of the activity of at least one of interleukin-10 receptor subunit beta (IL-10RB, IL-10R2) and interleukin-22 (IL-22); or an inhibitor of expression of a gene encoding at least one of the interleukin-10 receptor subunit beta and the interleukin-22.

Claims

1-8. (canceled)

9. A method for preventing or treating cancer comprising administering, to a subject in need of administration, a pharmaceutical composition containing, as an active ingredient: an inhibitor of activity of at least one of interleukin-10 receptor subunit beta (IL-10RB, IL-10R2) and interleukin-22 (IL-22); or an inhibitor of expression of a gene encoding at least one of the interleukin-10 receptor subunit beta and the interleukin-22, wherein the cancer is pancreatic cancer.

10. The method of claim 9, further comprising administering an inhibitor of activity of programmed death ligand 1 (PD-L1) or an inhibitor of expression of a gene encoding the programmed death ligand 1 to the subject.

11. The method of claim 9, wherein the interleukin-10 receptor subunit beta or the interleukin-22 is present in CD45.sup.+ cells in the subject.

12. The method of claim 9, wherein the inhibitor of the activity comprises any one or more selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products, which bind specifically to the interleukin-10 receptor subunit beta or the interleukin-22.

13. The method of claim 9, wherein the inhibitor of the expression comprises any one or more selected from the group consisting of an antisense nucleotide, small interfering RNA (siRNA), short hairpin RNA (shRNA), and ribozyme, which bind complementarily to the gene encoding the interleukin-10 receptor subunit beta or the interleukin-22.

14. The method of claim 9, wherein the inhibitor of the activity binds specifically to an epitope of interleukin-10 receptor subunit beta represented by SEQ ID NO: 7.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0056] FIG. 1 is a graph showing the results of measuring the numbers of IL-10R2+CD45+ cells and IL-10R2-CD45+ cells in peripheral blood mononuclear cells derived from mice transplanted with pancreatic cancer cells in Experimental Example 1 of the present invention.

[0057] FIG. 2 is a graph showing the results of measuring the numbers of IL-10R2+CD45+ cells and IL-10R2-CD45+ cells in peripheral blood mononuclear cells derived from mice transplanted with pancreatic cancer cells after treatment of the mice with an anti-IL-10R2 antibody in Experimental Example 1 of the present invention.

[0058] FIG. 3 shows a photograph of pancreatic tumors in IL-22 gene knockout (K/O) mouse models transplanted with pancreatic cancer cells in Experimental Example 2 of the present invention.

[0059] FIG. 4 is a graph showing the results of measuring the weights of pancreatic tumors in IL-22 gene knockout (K/O) mouse models transplanted with pancreatic cancer cells in Experimental Example 2 of the present invention.

[0060] FIG. 5 is a graph showing the results of measuring the volumes of peripancreatic lymph nodes in IL-22 gene knockout (K/O) mouse models transplanted with pancreatic cancer cells in Experimental Example 2 of the present invention.

[0061] FIG. 6 is a photograph showing microscopic observation of pancreatic tumor tissue in IL-22 gene knockout (K/O) mouse models transplanted with pancreatic cancer cells in Experimental Example 2 of the present invention.

[0062] FIG. 7 is a graph showing the results of measuring the number of IL-10R2+7AAD− cells in PBMCs derived from IL-22 gene knockout (K/O) mice transplanted with pancreatic cancer cells in Experimental Example 3 of the present invention.

[0063] FIG. 8 is a graph showing the results of measuring the proportion of IL-10R2+CD11b+ cells in PBMCs derived from IL-22 gene knockout (K/O) mice transplanted with pancreatic cancer cells in Experimental Example 3 of the present invention.

[0064] FIG. 9 is a graph showing the results of measuring the number of IL-10R2+CD11b+7AAD− cells in PBMCs derived from IL-22 gene knockout (K/O) mice transplanted with pancreatic cancer cells in Experimental Example 3 of the present invention.

[0065] FIG. 10 depicts photographs showing microscopic observation after trichrome and Picrosirius red staining of the pancreatic tissues of IL-22 gene knockout (K/O) mice transplanted with pancreatic cancer cells in Experimental Example 4 of the present invention.

[0066] FIG. 11 is a graph showing the results of measuring the numbers of CD3+ cells, CD8+ cells and CD4+ cells separated from IL-22 gene knockout (K/O) mouse models transplanted with pancreatic cancer cells in Experimental Example 5 of the present invention.

[0067] FIG. 12 depicts graphs showing the results of measuring the absorbance after 24 hours, 48 hours or 72 hours of culture of pancreatic cancer cells in an IL-10RB+ or IL-10RB− conditioned medium supplemented with an anti-IL-10R2 antibody in Experimental Example 6 of the present invention.

[0068] FIG. 13 depicts graphs showing the results of measuring the number of cells after 48 hours of culture of pancreatic cancer cells in an IL-10RB+ or IL-10RB− conditioned medium supplemented with an anti-IL-10R2 antibody in Experimental Example 6 of the present invention.

[0069] FIG. 14 depicts graphs showing the results of measuring the number of cells after 72 hours of culture of pancreatic cancer cells in an IL-10RB+ or IL-10RB− conditioned medium supplemented with an anti-IL-10R2 antibody in Experimental Example 6 of the present invention.

[0070] FIG. 15 depicts graphs showing the results of measuring the number of cells after 48 hours or 72 hours of culture after adding IL-10R2+ cells or IL-10R2− cells to pancreatic cancer cells in Experimental Example 7 of the present invention.

[0071] FIG. 16 is a graph showing the results of measuring the number of cells after treating pancreatic cancer cells with anti-IL-10R2 antibody, anti-IL-22R1 antibody, anti-TNF-α antibody, anti-IFN-γ antibody, anti-IL-2 antibody and anti-IL-6 antibody in Experimental Example 8 of the present invention.

[0072] FIG. 17 schematically shows an experimental design of Experimental Example 9 of the present invention.

[0073] FIG. 18 depicts photographs of pancreatic tissue after an anti-PD-L1 antibody and/or isotype antibody was administered to the IL-22 gene knockout (K/O) mouse models and wild-type (WT) mice transplanted with pancreatic cancer cells in Experimental Example 9 of the present invention.

[0074] FIG. 19 is a graph showing the results of measuring the weight of pancreatic tissue including cancer after an anti-PD-L1 antibody and/or isotype antibody was administered to the IL-22 gene knockout (K/O) mouse models and wild-type (WT) mice transplanted with pancreatic cancer cells in Experimental Example 9 of the present invention.

[0075] FIG. 20 depicts graphs showing the proportion of CD3+CD8+ cells in immune cells that infiltrated into pancreatic tissue, measured after an anti-PD-L1 antibody and/or isotype antibody was administered to the IL-22 gene knockout (K/O) mouse models and wild-type (WT) mice transplanted with pancreatic cancer cells in Experimental Example 10 of the present invention.

[0076] FIG. 21 is a graph showing the number of CD3+CD8+ cells in immune cells that infiltrated into pancreatic tissue, measured after an anti-PD-L1 antibody was administered to the IL-22 gene knockout (K/O) mouse models and wild-type (WT) mice transplanted with pancreatic cancer cells in Experimental Example 10 of the present invention.

BEST MODE

[0077] One embodiment of the present invention is directed to a pharmaceutical composition for preventing or treating cancer containing, as an active ingredient: an inhibitor of the activity of at least one of interleukin-10 receptor subunit beta (IL-10R2, IL-10RB) and interleukin-22 (IL-22); or an inhibitor of expression of a gene encoding at least one of the interleukin-10 receptor subunit beta and the interleukin-22.

[0078] The composition of the present invention may further contain an inhibitor of the activity of programmed death ligand 1 (PD-L1) or an inhibitor of expression of a gene encoding the programmed death ligand 1, which may exhibit a synergistic effect on the prevention or treatment of cancer.

[0079] In the present invention, the interleukin-10 receptor subunit beta, interleukin-22 or programmed death ligand 1 may be present in peripheral blood mononuclear cells (PBMCs), preferably CD45.sup.+ cells, more preferably white blood cells. Accordingly, in the present invention, the inhibitor of the activity or the inhibitor of expression may inhibit the activity of the interleukin-10 receptor subunit beta, interleukin-22 or programmed death ligand 1 present in peripheral blood mononuclear cells (PBMCs), preferably CD45.sup.+ cells, more preferably white blood cells, or inhibit expression of the gene encoding the protein.

[0080] In the present disclosure, the “cancer” may be pancreatic cancer, thyroid cancer, breast cancer, biliary tract cancer, gallbladder cancer, colorectal cancer, uterine cancer, esophageal cancer, gastric cancer, brain cancer, rectal cancer, lung cancer, bladder cancer, kidney cancer, ovarian cancer, prostate cancer, head and neck cancer, skin cancer, blood cancer or liver cancer. Preferably, the cancer may be pancreatic cancer.

MODE FOR INVENTION

[0081] Hereinafter, the present invention will be described in more detail with reference to examples. It will be apparent to those of ordinary skill in the art that these examples serve merely to describe the present invention in more detail, and the scope of the present invention according to the subject matter of the present invention is not limited by these examples.

EXAMPLES

Experimental Example 1

[0082] The pancreatic ductal adenocarcinoma cell line (Pan02) was injected directly into the pancreases of 8-week old wild-type (WT) mice (C57BL/6, OrientBio) at a concentration of 2×10.sup.6 cells/20 μL. After 14 days, peripheral blood mononuclear cells (PBMCs) were collected from the mice, and IL-10R2+CD45+ cells and IL-10R2-CD45+ cells were separated using a flow cytometer and the number of the cells was measured. The results of the measurement are shown in FIG. 1. In addition, after administration of an anti-IL-10R2 antibody (R&D Systems, Catalog number: MAB874) to the mice, PBMCs were collected, and IL-10R2+CD45+ cells and IL-10R2-CD45+ cells were separated using a flow cytometer and the number of the cells was measured. The results of the measurement are shown in FIG. 2.

[0083] As shown in FIG. 1, it could be confirmed that IL-10R2+CD45+ cells were highly expressed in the mice injected with the pancreatic cancer cells.

[0084] In addition, as shown in FIG. 2, as a result of administering the anti-IL-10R2 antibody to the mice injected with the pancreatic cancer cells, it could be confirmed that the number of IL-10R2+CD45+ cells significantly decreased.

Example 2

[0085] The pancreatic ductal adenocarcinoma cell line (Pan02) was injected directly into the pancreases 8-week old IL-22 gene knockout (K/O) mouse models (B6; 129S5-ll22tm1lex/Mmucd, Genentech) and 8-week old wild-type (WT) mice (C57BL/6, OrientBio) at a concentration of 2×10.sup.6 cells/20 μL. After 14 days, the pancreatic tumors were photographed and the results are shown in FIG. 3. Also, the weights of the pancreatic tumors were measured and the results are shown in FIG. 4. In addition, the volumes of the peripancreatic lymph nodes are measured and the results are in FIG. 5. Furthermore, photographs showing microscopic observation of the pancreatic tumor tissues are shown in FIG. 6.

[0086] As shown in FIGS. 3 and 4, it could be confirmed that the size of the pancreatic tumor in the IL-22 gene knockout mouse models injected with the pancreatic cancer cells decreased compared to that in the wild-type mice.

[0087] As shown in FIGS. 5 and 6, it could be seen that, in the IL-22 gene knockout mouse models, the lymph nodes were activated, but in the wild-type mice, the lymph nodes were atrophied and the tumor cells infiltrated the lymph nodes.

Experimental Example 3

[0088] An experiment was conducted in the same manner as in Experimental Example 2. On day 14 after injection of the pancreatic cancer cells, PBMCs were collected from the mice, and IL-10R2+7AAD− cells, IL-10R2+CD11b+ cells and IL-10R2+CD11b+7AAD− cells were separated using a flow cytometer and the number of the cells was measured. The results of the measurement are shown in FIGS. 7 to 9, respectively.

[0089] As shown in FIG. 7, it could be confirmed that the number of IL-10R2+7AAD− cells in the IL-22 gene knock mouse models significantly decreased compared to that in the wild-type mice.

[0090] As shown in FIG. 8, it could be confirmed that the number of IL-10R2+CD11b+ cells increased in the mice injected with the pancreatic cancer cells, but the number of IL-10R2+CD11b+ cells decreased in the IL-22 gene knockout mouse models compared to the wild-type mice.

[0091] As shown in FIG. 9, it could be confirmed that the number of IL-10R2+CD11b+7AAD− cells significantly increased in the mice injected with the pancreatic cancer cells, but the number of IL-10R2+CD11b+7AAD− cells in the IL-22 gene knockout mouse models decreased to that in the mice not injected with the pancreatic cancer cells, compared to the wild-type mice.

Experimental Example 4

[0092] An experiment was conducted in the same manner as in Experimental Example 2. On day 14 after injection of the pancreatic cancer cells, the mice were euthanized, and the pancreas tissue was taken from each mouse, stained with trichrome and Picrosirius red, and then observed with a microscope. Photographs of the observation are shown in FIG. 10.

[0093] As shown in FIG. 10, it could be confirmed that the wild-type mice (B6) injected with the pancreatic cancer cells were much thicker and had more fibrosis than the IL-22 gene knockout mouse models (IL-22KO).

Experimental Example 5

[0094] An experiment was conducted in the same manner as in Experimental Example 2. On day 14 after injection of the pancreatic cancer cells, the tumor-infiltrating cells, CD3+ cells, CD8+ cells, and CD4+ cells, were separated using a flow cytometer, and the number of the cells was measured. The results of the measurement are shown in FIG. 11.

[0095] As shown in FIG. 11, it could be confirmed that the number of CD8+ cells in the IL-22 gene knockout mouse models increased compared to that in the wild-type mice, and the number of CD4+ cells decreased in the IL-22 gene knockout mouse models. Thereby, it could be seen that, in the IL-22 gene knockout mouse models, cancer immune evasion was restored and cytotoxic immunity corresponding to the cancer cells was restored.

Experimental Example 6

[0096] PanO2 cells were dispensed into a 96-well plate at a density of 5×10.sup.3 cells (100 μl/well) (n=5), and then pre-cultured in a humidified incubator at 37° C. under 5% CO.sub.2. The PanO2 cells were cultured for 24 hours, 48 hours or 72 hours in 200 μl of conditioned medium (IL-10RB+, IL-10RB−) supplemented with 2 μk/ml of an anti-IL-10R2 neutralizing antibody (R&D Systems, Catalog number: MAB874) or 1 μk/ml of an anti-IL-10R2 neutralizing antibody (Novus Biologicals, Catalog number: NBP211654), which binds to the epitope represented by SEQ ID NO: 7. 10 μl of CCK-8 solution was added to each well of the plate, and then each well was incubated in an incubator for 3 hours. The absorbance at 450 nm was measured using a microplate reader, and the results are shown in FIG. 12. In addition, number of cells based on the IL-10RB− conditioned medium was measured, and the results are shown in FIGS. 13 and 14.

[0097] As shown in FIG. 12, it could be confirmed that the number of the pancreatic cancer cells further increased in the IL-10RB+ conditioned medium compared to the IL-10RB− conditioned medium.

[0098] As shown in FIGS. 13 and 14, it could be confirmed that treatment with the anti-IL-10R2 antibody significantly decreased the number of the pancreatic cancer cells.

Experimental Example 7

[0099] 3.0×10.sup.6 Pano2 cells were labeled with CellTracker™ Green CMFDA (5-chloromethylfluorescein diacetate), and then the Pano2 cells were cultured at a density of 1×10.sup.5 cells per well (n=3). Thereafter, 1×10.sup.5 IL-10R2+ cells or IL-10R2− cells were added to each well, and then cultured for 48 hours or 72 hours. The cell number of the Pano2 cells was measured using a hemocytometer, and the results of the measurement are shown in FIG. 15.

[0100] As shown in FIG. 15, it could be confirmed that the number of the pancreatic cancer cells further increased when the IL-10R2+ cells were added compared to when the IL-10R2− cells were added.

Experimental Example 8

[0101] 3.0×10.sup.6 Pano2 cells were labeled with CellTracker™ Green CMFDA (5-chloromethylfluorescein diacetate), and then the Pano2 cells were cultured at a density of 1×10.sup.5 cells per well (n=3). Thereafter, the cells were treated with neutralizing antibodies specific to IL-10R2, IL-22R1, TNF-α, IFN-γ, IL-2 and IL-6, and cultured for 48 hours. The cell number of the Pano2 cells was measured using a hemocytometer, and the results of the measurement are shown in FIG. 16.

[0102] As shown in FIG. 16, it could be confirmed that the number of the pancreatic cancer cells significantly decreased only when the cells were treated with the anti-IL-10R2 antibody.

Experimental Example 9

[0103] FIG. 17 schematically shows a design of the following experiment. 2×10.sup.6 cells/20 μL of Pan02 PDACs (pancreatic ductal adenocarcinoma cell line (Pan02) were injected directly into the pancreases of 8-week-old IL-22 gene knockout (K/O) mouse models (B6; 129S5-ll22tm1lex/Mmucd, Genentech) and 8-week-old wild-type (WT) mice (C57BL/6, OrientBio). Days 3, 6, 8, and 10 after surgery, each of an anti-PD-L1 antibody (250 μg i.p.) and an anti-mouse IgG2 antibody (250 μg i.p.) (isotype: a negative control for PD-L1 antibody) as an isotype antibody was injected into the wild-type mice and IL-22 K/0 mouse group to which the Pan02 cells have been administered. As a control, PBS and an anti-mouse IgG2 antibody (250 μg i.p.) were injected into 8-week-old wild-type (WT) mice (C57BL/6, OrientBio) on days 3, 6, 8 and 10. On day 14, the mice were sacrificed, the pancreatic tumors were photographed, and the results are shown in FIG. 18. In addition, the weight of the pancreas including the tumor for each group was measured, and the results are shown in FIG. 19.

[0104] As shown in FIG. 18, it could be confirmed that, when both IL-22 gene knockout according to the present invention and administration of the anti-PD-L1 antibody were performed, the tumor suppression effect was better than when only IL-22 gene knockout or only administration of the anti-PD-L1 antibody was performed, and thus the tumor disappeared.

[0105] In addition, as shown in in FIG. 19, it could be confirmed that the weight of the pancreas including the tumor significantly decreased when both IL-22 gene knockout and administration of the anti-PD-L1 antibody were performed compared to when only IL-22 gene knockout was performed.

Experimental Example 10

[0106] The immune cells infiltrating into the pancreas tissue isolated for each group in Experimental Example 9 above were separated, stained with the cytotoxic T cell markers CD3 antibody and CD8 antibody, and subjected to FACS analysis. The results of the analysis are shown in FIGS. 20 and 21.

[0107] As shown in FIGS. 20 and 21, it could be confirmed that, when both IL-22 gene knockout according to the present invention and administration of the anti-PD-L1 antibody were performed, the number of CD3+CD8+ cells significantly decreased compared to when only IL-22 gene knockout was performed.

[0108] Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and it will be apparent to one of ordinary skill in the art that various modifications and variations are possible, without departing from the technical spirit of the present invention as defined in the appended claims.

INDUSTRIAL APPLICABILITY

[0109] The present invention is intended to provide a composition capable of preventing or treating cancer, particularly pancreatic cancer.