Pharmaceutical Composition Comprising Hepatitis B Virus-Derived Polypeptide for Prevention or Treatment of Cancer

Abstract

One aspect relates to a pharmaceutical composition for the prevention or treatment of cancer or an anticancer immune vaccine composition, including a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. The composition can enhance anticancer immunity by activating dendritic cells and T cells, and furthermore, can exhibit a remarkably excellent synergistic anticancer immune effect through administration in combination with an immune checkpoint inhibitor.

Claims

1-20. (canceled)

21. A method of preventing or treating cancer, comprising administering a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to a subject in need thereof.

22. The method of claim 21, wherein the polypeptide activates dendritic cells and T cells.

23. The method of claim 21, wherein the polypeptide increases the expression of at least one selected from the group consisting of TNF-α and iNOS in dendritic cells.

24. The method of claim 21, wherein the cancer is at least one selected from the group consisting of colon cancer, melanoma, lung cancer, liver cancer, kidney cancer, gastric cancer, pancreatic cancer, rectal cancer, breast cancer, thyroid cancer, head and neck cancer, brain tumor, kidney cancer, bladder cancer, and prostate cancer.

25. The method of claim 21, further comprising administering an immune checkpoint inhibitor to the subject.

26. The method of claim 21, wherein the polypeptide is administered in combination with an immune checkpoint inhibitor.

27. The method of claim 25, wherein the immune checkpoint inhibitor is anti-PD-L1.

28. A method of enhancing anticancer immunity, comprising administering a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to a subject in need thereof.

29. The method of claim 28, wherein the polypeptide activates dendritic cells and T cells.

30. The method of claim 28, wherein the polypeptide increases the expression of at least one selected from the group consisting of TNF-α and iNOS in dendritic cells.

31. The method of claim 28, wherein the cancer is at least one selected from the group consisting of colon cancer, melanoma, lung cancer, liver cancer, kidney cancer, gastric cancer, pancreatic cancer, rectal cancer, breast cancer, thyroid cancer, head and neck cancer, brain tumor, kidney cancer, bladder cancer, and prostate cancer.

32. The method of claim 28, further comprising administering an immune checkpoint inhibitor to the subject.

33. The method of claim 28, wherein the polypeptide is administered in combination with an immune checkpoint inhibitor.

34. The method of claim 33, wherein the immune checkpoint inhibitor is anti-PD-L1.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0095] FIG. 1 is a graph showing the expression of TNF-α cytokine secreted in a concentration-dependent manner by poly6 stimulation in mouse-derived dendritic cells (statistical significance was tested by Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001; and ****, P<0.0001).

[0096] FIG. 2 illustrates the results of confirming the increase in NOS2 and nitric oxide in a type 1 interferon-dependent manner in dendritic cells by poly6 treatment, in which, specifically, FIG. 2(A) illustrates the results of confirming the increase in NOS2 through Western blot assay, after mouse-derived dendritic cells were treated with poly6 for 24 hr, and FIG. 2(B) illustrates the results of confirming through Nitrite/Nitrate kit assay that the nitrate level was increased in WT mice in a concentration-dependent manner, and was not increased in IFN K.O mice (statistical significance was tested by Student's t-test. *, P<0.05; **, P<0.01; and ***, P<0.001).

[0097] FIG. 3 is a graph showing the results of confirming the ability to differentiate into TNF-α/iNOS-producing DCs from bone marrow-derived dendritic cells (statistical significance was tested by Student's t-test. *, P<0.05; **, P<0.01; and ***, P<0.001).

[0098] FIG. 4 illustrates the expression patterns of molecular markers involved in maturation, by poly6 treatment in mouse-derived dendritic cells (statistical significance was tested by Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001; and ****, P<0.0001).

[0099] FIG. 5 is a graph showing the expression patterns of molecular markers involved in maturation by poly6 treatment in DC2.4 cells (statistical significance was tested by Student's t-test. *, P<0.05; **, P<0.01; and ***, P<0.001).

[0100] FIG. 6 illustrates the results of confirming the cytotoxicity of various cancer cell lines (MC38 murine colon cancer cell, B16F10 murine melanoma cancer cell, E0771 murine breast cancer cell, PanO2 murine pancreatic cancer cell, and MDA231 human breast cancer cell) by DC2.4 cells treated with poly6 at different concentrations, through FACS analysis (statistical significance was tested by Student's t-test and one-way-ANOVA. *, P<0.05; **, P<0.01; ***, P<0.001; and ****, P<0.0001).

[0101] FIG. 7 illustrates the results of confirming that the cytotoxicity of various cancer cell lines (MC38, B16F10, and EO771) was reduced when iNOS formation was inhibited by L-NAME (5 mM) treatment while stimulating DC2.4 cells with poly6 for 24 hours (statistical significance was tested by Student's t-test. *, P<0.05; **, P<0.01; and ***, P<0.001).

[0102] FIG. 8 illustrates the results of confirming that 3-nitrotyrosine was accumulated in cancer cells when MC38 cancer cells were treated with a culture medium in which nitric oxide had been accumulated, for 4 hours after DC2.4 cells were stimulated with poly6 for 48 hours (statistical significance was tested by Student's t-test. *, P<0.05; **, P<0.01; and ***, P<0.001).

[0103] FIG. 9 illustrates the results confirmed through confocal microscopy after DC2.4 cells were stimulated with poly6 for 48 hours, and MC38 cancer cells were treated with a culture medium in which nitric oxide had been accumulated, for 4 hours, followed by fixation and permeabilization of the cancer cells and staining with a 3-nitrotyrosine antibody for 30 minutes, and illustrates the results of analyzing fluorescence mean intensity through lmagej program, through which it was confirmed that the 3-nitrotyrosine level was statistically significantly increased in MC38 cancer cells by the culture medium of poly6-treated dendritic cells (statistical significance was tested by Student's t-test. *, P<0.05; **, P<0.01; and ***, P<0.001).

[0104] FIG. 10 illustrates the results of confirming the degree of accumulation of 3-nitrotyrosine in tumor tissue, the results of evaluation using a confocal microscopy 63× lens, and the results of measuring fluorescence mean intensity by using lmagej program (statistical significance was tested by Student's t-test. *, P<0.05; **, P<0.01; and ***, P<0.001).

[0105] FIG. 11 illustrates the results of confirming an anticancer effect in C57BL/6 mice into which MC38 cancer cells were injected, in which, specifically, (A) illustrates an in vivo animal experiment schedule, (B) illustrates the results of confirming the tumor growth rate through tumor size measurement, (C) is an image illustrating tumors extracted on day 16 after cancer cell injection, and (D) is a graph showing the results of measuring tumor weight on day 16 (statistical significance was tested by Student's t-test. *, P<0.05; **, P<0.01; and ***, P<0.001).

[0106] FIG. 12 illustrates the results of confirming an anticancer effect in melanoma B16F10, in which, specifically, (A) illustrates an in vivo animal experiment schedule, (B) illustrates the results of confirming the tumor growth rate through tumor size measurement, (C) is an image illustrating tumors extracted on day 12 after cancer cell injection, and (D) is a graph showing the results of measuring tumor weight on day 12 (statistical significance was tested by Student's t-test. *, P<0.05; **, P<0.01; and ***, P<0.001).

[0107] FIG. 13 illustrates the Hematoxylin and eosin staining results of MC38 colon cancer tissue.

[0108] FIG. 14 illustrates the results of confirming an anticancer effect in C57BL/6 mice into which MC38 cancer cells were injected, in which, specifically, (A) illustrates an in vivo animal experiment schedule, (B) illustrates the results of confirming the tumor growth rate through tumor size measurement, (C) is an image illustrating tumors extracted on day 23 after cancer cell injection, and (D) is a graph showing the results of measuring tumor weight on day 23 (statistical significance was tested by Student's t-test. *, P<0.05; **, P<0.01; and ***, P<0.001).

[0109] FIG. 15 illustrates the results of confirming the apoptotic cell death of C57BL/6 mice into which MC38 cancer cells were injected, through TUNEL assay (statistical significance was tested by Student's t-test. *, P<0.05; **, P<0.01; and ***, P<0.001).

[0110] FIG. 16 illustrates the results of confirming CD8 T cell-mediated CTL response in MC38 tumor tissue (statistical significance was tested by Student's t-test. *, P<0.05; **, P<0.01; and ***, P<0.001).

[0111] FIG. 17 illustrates the results of confirming effector T cells by using a flow cytometer, in which, specifically, tumor tissue was dissociated and separated into single cells, and surface T cell markers were stained with CD3, CD4, or CD8, followed by fixation and permeabilization, staining with a TNF- or IFN-γantibody through an intracellular cytokine staining method, and analysis using a flow cytometer (statistical significance was tested by Student's t-test. *, P<0.05; **, P<0.01; and ***, P<0.001)

[0112] FIG. 18 illustrates the results of analyzing T cells in MC38 tumor tissue through a flow cytometer, in which, specifically, (A) illustrates the results of confirming the increase in CD4 and CD8 T cells in tumor tissue according to poly6 injection, and (B) illustrates the results of confirming the increase in CD44/CD25-activated CD4/CD8 T cells in tumor tissues of poly6 groups (statistical significance was tested by Student's t-test. *, P<0.05; **, P<0.01; and ***, P<0.001).

[0113] FIG. 19 illustrates NK cell population, in which, specifically, the population of NK cells was confirmed through FACS analysis (statistical significance was tested by Student's t-test. *, P<0.05; **, P<0.01; and ***, P<0.001).

[0114] FIG. 20 illustrates the results of analyzing activated T cells in B16F10 tumor tissue by using a flow cytometer (statistical significance was tested by Student's t-test. *, P<0.05; **, P<0.01; and ***, P<0.001).

[0115] FIG. 21 illustrates the results of confirming increased dendritic cells in tumor tissue and spleen cells, in which, specifically, tumors and spleens were separated and dissociated, and CD11b- and CD11c-positive cells were confirmed using a flow cytometer (statistical significance was tested by Student's t-test. *, P<0.05; **, P<0.01; and ***, P<0.001).

[0116] FIG. 22 illustrates the results of confirming Tip-DC population in MC38 tumor tissue and spleen, in which, specifically, the induction of Tip-DCs was confirmed in poly6-stimulated groups by flow cytometry (*, P<0.05; **, P<0.01; and ***, P<0.001.).

[0117] FIG. 23 illustrates the results of confirming Tip-DC population in B16F10 tumor tissue.

[0118] FIG. 24 illustrates the results of confirming the degree of maturation through maturation markers of dendritic cells in tumor tissues and lymph nodes, in which, specifically, tumor tissues and lymph nodes were dissociated and separated into single cells, and then dendritic cells were stained with maturation markers, and the degree of maturation of dendritic cells was evaluated by flow cytometry (*, P<0.05; **, P<0.01; and ***, P<0.001.).

[0119] FIG. 25 is a graph confirming the number of macrophages in MC38 tumors by flow cytometry (*, P<0.05; **, P<0.01; and ***, P<0.001).

[0120] FIG. 26 illustrates graph confirming an anticancer effect by poly6 in a model in which HBV W4P large surface proteins-expressing NIH-3T3 cells (1×10.sup.8) were injected into balb nu/nu mice, in which reduced tumor size and reduced tumor weight were confirmed (*, P<0.05; **, P<0.01; and ***, P<0.001).

[0121] FIG. 27 illustrates the results of confirming anticancer effects when anti PD-L1 as an immune checkpoint inhibitor and poly6 as an adjuvant were used in combination, in which, specifically, (A) illustrates an in vivo animal experiment schedule, (B) is a graph confirming the tumor growth rate through tumor size measurement, (C) is an image showing tumors extracted on day 21 after cancer cell injection, and (D) is a graph showing the results of measuring tumor weight on day 21 (statistical significance was tested by Student's t-test. *, P<0.05; **, P<0.01; and ***, P<0.001).

MODE OF DISCLOSURE

[0122] Hereinafter, the present disclosure will be described in further detail with reference to the following examples. However, these examples are provided for illustrative purposes and are not intended to limit the scope of the present disclosure.

Example

1. Differentiation into TNF-α/NOS2-producing Dendritic Cells by Poly6 in Type 1 Interferon-Dependent Manner

(1) Measurement of TNF-α Cytokine Expression

[0123] Bone marrow-derived dendritic cells were obtained from C57BL/6 mice and interferon knockout mice.

[0124] Mouse-derived dendritic cells were treated with poly6 peptide (GRLVFQ, SEQ ID NO: 1) at different concentrations (10 pM, 1 nM, 100 nM, and 10 μM), and then TNF-α cytokine secreted by the dendritic cells was measured by ELISA.

[0125] As a result, it was confirmed that the expression of TNF-α cytokine was increased by poly6 treatment in a concentration-dependent manner. It was also confirmed that, compared to C57BL/6 mice, the amount of secreted TNF-α was statistically significantly reduced in interferon knockout mice when treated with 100 nM or 10 μM of poly6 (FIG. 1).

(2) Confirmation of Increase in NOS2 and Nitric Oxide

[0126] Mouse-derived dendritic cells were treated with poly6 peptide at different concentrations, the protein was obtained by prep using RIPA lysis buffer from cell pellets, and it was confirmed through Western blotting assay that NOS2 increased in a concentration-dependent manner in WT C57BL/6 mice, whereas it was confirmed that NOS2 did not increase in IFN K.O mice, but rather decreased (FIG. 2A).

[0127] It was confirmed using a nitrite/nitrate kit that nitrate concentration in a cell medium was increased by poly6 treatment, and was not increased in IFN K.O mice (FIG. 2B).

[0128] Through these results, it was confirmed that dendritic cells increased TNF-α and iNOS by poly6 treatment in a type 1 interferon-dependent manner.

(3) Confirmation of Ability to Induce Differentiation into TNF-α/iNOS-Producing Dendritic Cells

[0129] Since it was confirmed that TNF-α and iNOS were increased in a type 1 interferon-dependent manner, the ability to induce differentiation into NF-α/iNOS-producing dendritic cells from bone marrow-derived dendritic cells was examined.

[0130] Bone marrow-derived dendritic cells were stimulated with poly6 for 24 hr, followed by fixation with 1% paraformaldehyde and permeabilization with 0.1% Triton X-100, and TNF-α/iNOS-producing dendritic cells were analyzed using a flow cytometer.

[0131] As a result, it was confirmed that Tip-DCs (TNF-α/iNOS-producing dendritic cells) were formed in bone marrow-derived dendritic cells of WT mice by poly6 treatment in a concentration-dependent manner. In contrast, it was confirmed that Tip-DCs did not differentiate in IFN K.O mice (FIG. 3).

2. Activation of Dendritic Cells by Poly6

(1) Measurement of Expression of CD40, CD80, CD86, and MHCII

[0132] Mouse-derived dendritic cells were treated with poly6 peptide (10 μM) for 24 hours, and then, the expression levels of CD40, CD80, CD86, and MHCII, which are molecular markers involved in the maturation of dendritic cells, were measured using a flow cytometer.

[0133] As a result, it was confirmed that the expression of CD40, CD80, CD86 and MHCII molecules in dendritic cells was increased by poly6 treatment (FIG. 4).

(2) Confirmation of Dendritic Cell Activation in DC2.4 Cells

[0134] A DC2.4 cell line was starved for 30 minutes, and cultured along with poly6 in a medium supplemented with 1% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin for 24 hours, and then dendritic cells in which CD40, CD80 and MHCII molecules involved in the maturation of dendritic cells were expressed were identified by flow cytometry (FIG. 5).

3. Nitric Oxide (NO)-Dependent Anticancer Effect of Poly6

(1) Cytotoxicity against Various Cancer Cell Lines by DC2.4 Cells Treated with Poly6 at Different Concentrations

[0135] Direct cytotoxicity was induced in several cancer cell lines by poly6-stimulated DC2.4 cells. It was confirmed that DC2.4 was stimulated by poly6 treatment in a concentration-dependent manner and cytotoxicity was induced in cancer cell lines (MC38, B16F10, EO771, PanO2, and MDA231). In particular, it was confirmed that, for the MC38 and B16F10 cancer cell lines, the cytotoxicity against cancer cells was statistically significantly increased in DC2.4 cells treated with 10 μM of poly6 compared to when treated with LPS (1 μg/ml) (FIG. 6).

(2) When iNOS Formation was Inhibited

[0136] It was also confirmed that cytotoxicity was reduced when poly6-treated DC2.4 cells and cancer cells were co-cultured, due to iNOS inhibition by L-NAME (FIG. 7).

(3) Whether 3-nitrotyrosine was Accumulated in Cancer Cells

[0137] When cancer cells were treated with DC2.4 cells stimulated with poly6 for 48 hours in a culture medium in which nitric oxide was accumulated, the increase in the level of 3-nitrotyrosine, which is an indicator of peroxynitrite, was confirmed by flow cytometry (FIG. 8). It was also confirmed through Immunofluorescence (FIG. 9).

[0138] Furthermore, the increase of 3-nitrotyrosine in tumor tissue was also confirmed using confocal microscopy through an immunofluorescence assay method (FIG. 10).

4. Function of Poly6 as Anticancer Vaccine

(1) Confirmation of Efficacy in Colcon Cancer

[0139] MC38 colon cancer cells (1×10.sup.6 cells) were injected subcutaneously into C57BL/6 mice to form tumors, and poly6 peptide (10 μg) was injected at a location away from the cancer cell injection site to confirm an anticancer immune effect.

[0140] As a result, t was confirmed that, compared to a PBS control, the tumor growth rate was reduced, and finally, tumor mass and tumor weight were reduced (FIG. 11).

(2) Confirmation of Efficacy in Melanoma

[0141] In addition, B16F10 melanoma cancer cells (1×10.sup.6 cells) were injected subcutaneously into C57BL/6 mice to form tumors, and poly6 peptide (10 μg) was injected at a location away from the cancer cell injection site to confirm an anticancer immune effect.

[0142] As a result, it was confirmed that, compared to a PBS control, the tumor growth rate was reduced, and finally, tumor mass and tumor weight were reduced (FIG. 12).

(3) Histological Staining Results

[0143] Tumors were extracted from MC38-bearing mice, fixed with 4% paraformaldehyde, and then subjected to hematoxylin and eosin staining through paraffin section.

[0144] As a result, it was confirmed that density in tumor tissue was reduced in a poly6-treated group, compared to a PBS control. Additionally, it was confirmed that immune cells gathered at the edge of tumors in the poly6-treated group (FIG. 13).

(4) Confirmation of Anticancer Effect according to Injection of Poly6 Peptide Before Injection of MC38 Cancer Cells

[0145] Poly6 peptide (10 μg) was additionally injected one day before cancer cell injection to induce immune enhancement as an anticancer immune vaccine in the mouse body, and then poly6 was additionally injected three times to confirm a long-term anticancer effect up to day 23.

[0146] As a result, it was confirmed that, compared to a PBS control, the tumor growth rate was reduced, and finally, tumor mass and tumor weight were reduced (FIG. 14).

(5) TUNEL Assay

[0147] The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was carried out using the ApopTag Peroxidase In Situ Apoptosis Detection Kit (Millipore).

[0148] As a result, greatly increased apoptotic cell death was confirmed in MC38 tumor tissue compared to a PBS control. Positive staining was observed not only at the edge of tissue but also inside the tissue. This was digitized through the HistoQuest (TissueGnostics) program of Tissue FAXS to analyze the Dab positive cell population (FIG. 15).

(6) Confirmation of CD8 T Cell-Mediated CTL Response

[0149] The mRNA levels of cell lytic proteins (granzymeB and perforin), pro-apoptotic proteins (Bax, bak, and cytochrome C), and death signal inducing ligands (TRAIL, Fas, and Fas L) were identified in MC38 tumor tissues.

[0150] Specifically, tumor tissue was sectioned, and total mRNA was prepped through the Trizol method, and analyzed by qRT-PCR using each primer set.

[0151] As a result, it was confirmed that the mRNA levels of cytolytic proteins, pro-apoptotic proteins, and death signal ligands were statistically significantly increased in the poly6-stimulated tumor tissue (FIG. 16).

5. Anticancer Immune Action by T Cell Activation and Tip-DC Formation in Mice In Vivo

(1) Confirmation of T Cell Activity

1) Identification of Effector T Cells

[0152] MC38 colon cancer cells (1×10.sup.6 cells) were injected subcutaneously into

[0153] C57BL/6 mice to form tumors, separated into single cells through tumor dissociation, and then TNF-α/IFN-α-producing CD4 and CD8 T cells were identified by flow cytometry.

[0154] As a result, it was confirmed that both CD4 and CD8 T cells producing TNF-α or IFN-γ, which function as effectors, were increased in poly6-stimulated tumor tissue (FIG. 17).

2) T Cell Analysis in MC38 Tumor Tissue

[0155] It was also confirmed that, compared to a control, the number of T cells increased in poly6-stimulated tumor tissue, and CD44- and CD25-positive cells, which are markers of activated T cells, were increased (FIG. 18).

3) Identification of NK Cells

[0156] In contrast, natural killer cells were increased in tumor tissue and spleen by poly6 stimulation, but statistically significant results were not obtained (FIG. 19).

[0157] 4) Confirmation of Increase in TNF-α+, CD4+ and TNF-α+, CD8+ T Cells in Melanoma

[0158] Additionally, it was confirmed that TNF-α+ CD4+ and TNF-α+ CD8+ T cells were increased in tumor tissue in B16F10 melanoma carcinoma, in addition to MC38 colon cancer (FIG. 20).

[0159] Through this result, it was confirmed that the ability to induce T cell activity by poly6 was not specific to colon carcinoma, but showed the ability to non-specifically induce immunity against various carcinomas.

(2) Confirmation of Tip-DC Formation

1) Identification of Number of Dendritic Cells

[0160] Specifically, it was confirmed that the number of dendritic cells statistically significantly increased in MC38 tumor tissue and spleen in a poly6-injected group (FIG. 21).

2) Confirmation of Ability to Induce Tip-DCs

[0161] Since it was confirmed that poly6 exhibited the ability to induce Tip-DCs in dendritic cells in vitro, the characteristics of increased dendritic cells were examined.

[0162] CD11b+, CD11c+, MHC2+, TNF-α+, and NOS2+ cells were analyzed in vivo using dendritic cell intracellular cytokine staining to confirm and evaluate the ability to induce differentiation into Tip-DCs.

[0163] As a result, statistically significant formation of the Tip-DC population was confirmed in both MC38 tumor tissue and spleen in a poly6-stimulated group (FIG. 22).

3) Confirmation of Tip-DC Induction Ability in Melanoma

[0164] Additionally, it was confirmed that Tip-DCs were increased in tumor tissue in B16F10 melanoma carcinoma, in addition to MC38 colon cancer (FIG. 23).

[0165] Through this result, it was confirmed that the ability to induce Tip-DCs by poly6 was not specific to colon carcinoma, and the ability to non-specifically induce immunity against various carcinomas was shown.

(3) Confirmation of Degree of Maturation of Dendritic Cells

[0166] It was confirmed that the maturation of dendritic cells was enhanced in MC38 tumor tissue and draining lymph nodes. It was confirmed that CD40, MHCII, and CD86 were increased in the tumor tissue, and CD40, MHC2, and CD80 were increased in the draining lymph nodes. In particular, the expression of CD40 showed a drastic increase in both tumor tissue and lymph nodes (FIG. 24).

[0167] It was expected to show the ability to induce T cell activation using the CD40/CD40L axis.

(4) Case of Macrophages

[0168] Among the innate immune cells in MC38 tumors, the number of macrophages statistically significantly decreased in tumor tissue and decreased in the spleen, but the results were not statistically significant (FIG. 25).

[0169] Taken together, these results indicate that poly6 induces T cell activity in vivo, increases the CTL response, and induces direct anticancer activity through the induction of differentiation into Tip-DCs.

(5) In Absence of T Cells

[0170] Additionally, the anticancer effect was also confirmed in balb/c nu/nu mice excluding T cells (FIG. 26), and this result supports the ability of Tip-DCs to directly induce anticancer activity, in that the anticancer effect is induced by the innate immune cells.

6. Induction of Enhanced Anticancer Immunity by Poly6 as Adjuvant when Treated in Combination with anti PD-L1 as Immune Checkpoint Inhibitor

[0171] Animal experiments were conducted to evaluate the enhanced ability to induce anticancer activity through the combined effect of poly6 and anti PD-L1, which is a commercially available immune checkpoint inhibitor.

[0172] As a result, statistically significant enhanced anticancer activity was observed compared to a PBS control, an anti PD-L1 alone group, or a poly6 alone group. This was confirmed by measuring the tumor growth rate and tumor mass and weight on day 21 after cancer cell injection (FIG. 27).