Composition for enhancing effect of antibody drugs

Abstract

Provided is a composition capable of enhancing the effect of an antibody drug such as an immune checkpoint inhibitor. [beta]-glucan is used as an active ingredient of a composition for enhancing the effect of an antibody drug. The composition is suitable for use in an antibody drug having an active effect of inhibiting cancer proliferation by inhibiting an immune checkpoint, for example, an antibody drug comprising a monoclonal antibody against PD-L1.

Description

[0001] The present invention is a divisional application of Chinese patent application No. 202080061449.2, filed on September 10, 2020, and entitled “Composition for Enhancing the Effect of Antibody Drugs.” This application claims priority to Japanese Patent Application No. 2019-165722 filed in Japan on September 11, 2019. Technical Field

[0002] The present invention relates to a composition for enhancing the effect of an antibody drug. Background Art

[0003] Molecular targeted drugs targeting immune checkpoints are attracting attention as a new type of cancer treatment. Immune checkpoints are a braking mechanism that prevents activated immune cells from attacking normal tissues and cells, and cancer cells use this mechanism to avoid attacks from immune cells. This is one of the functions that cancer cells have to prolong their lifespan, and it is also a part that has not yet been clarified. With the progress of this research, in recent years, it has been confirmed that antibody drugs that inhibit the functions of PD-1 or CTLA4, which are immune checkpoint molecules, have achieved effective anti-cancer effects in clinical practice, and further research and development of immune checkpoint inhibitors have been carried out more actively (see non-patent documents 1 to 3).

[0004] On the other hand, the inventors of the present application have used a culture of Aureobasidium pullulans, a fungus commonly known as a black yeast that produces abundant β-glucan, to disclose a skin moisturizer (Patent Document 1), a constipation-relieving agent (Patent Document 2), an immunostimulant (Patent Document 3), an immune adjuvant (Patent Document 4), a bio-healing promoter for reducing the side effects of anticancer drugs (Patent Document 5), a bio-healing promoter for promoting burn healing (Patent Document 6), a composition for preventing / treating bovine mastitis (Patent Document 7), a composition for promoting cytokine production by macrophages (Patent Document 8), a therapeutic agent for influenza virus infection (Patent Document 9), a TRAIL expression enhancer (Patent Document 10), a composition for preventing / improving anemia (Patent Document 11), a fat accumulation inhibitor (Patent Document 12), and the like.

[0005] Prior art literature

[0006] Non-patent literature

[0007] Non-patent document 1: Ishida, Y., Agata, Y., Shibahara, K. and Honjo, T. "Induced expression of PD-1, a novel member of the immunoglobulin genesuperfamily, upon programmed cell death." EMBO J. 11 3887-3895 (1992)

[0008] Non-patent literature 2: Freeman, GJ, Long, AJ, Iwai, Y., Bourque, K., Chernova, T., Nishimura, H., Fitsz, LJ, Malenkovich, N., Okazaki, T., Byrne, MC, Horton, HF, Fouser, L., Carter, L., Ling, V., Bowman, MR, Carreno, BM, Collins, M., Wood, CR and Honjo, T. “Engagement of the PD-1immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation.” J. Exp. Med. 192 1027-1034 (2000)

[0009] Non-Patent Literature 3: Spencer C. Wei, Colm R. Duffy, and James P. Allison, “Fundamental Mechanisms of Immune Checkpoint Blockade Therapy,” CANCERRESEARCH, doi: 10.1158 / 2159-8290.CD-18-0367, August 16, 2018

[0010] Patent Literature

[0011] Patent Document 1: Japanese Patent No. 4000078

[0012] Patent Document 2: Japanese Patent No. 4054697

[0013] Patent Document 3: Japanese Patent No. 4369258

[0014] Patent Document 4: Japanese Patent No. 5242855

[0015] Patent Document 5: Japanese Patent No. 5331482

[0016] Patent Document 6: Japanese Patent No. 5715659

[0017] Patent Document 7: Japanese Patent No. 5554221

[0018] Patent Document 8: Japanese Patent No. 5559173

[0019] Patent Document 9: Japanese Patent No. 5560472

[0020] Patent Document 10: Japanese Patent No. 5937029

[0021] Patent Document 11: Japanese Patent No. 6293187

[0022] Patent Document 12: Japanese Patent No. 6380968 Summary of the Invention

[0023] (1) Technical issues to be resolved

[0024] The object of the present invention is to provide a composition that utilizes Aureobasidium pullulans to enhance the effects of antibody drugs such as immune checkpoint inhibitors.

[0025] (2) Technical solution

[0026] To achieve the above objectives, the inventors of the present application conducted intensive research and discovered that combining β-glucan contained in cultures of Aureobasidium pullulans with antibody drugs such as immune checkpoint inhibitors enhances the effects of these drugs, leading to the completion of the present invention. Specifically, the present invention is as follows.

[0027] [1] A composition for enhancing the effect of an antibody drug, characterized in that it contains β-glucan as an active ingredient and is administered simultaneously with the antibody drug.

[0028] [2] The composition for enhancing the effect of an antibody drug according to [1] above, wherein the antibody drug has an effect of inhibiting cancer growth by inhibiting immune checkpoints.

[0029] [3] The composition for enhancing the effect of an antibody drug according to [1] above, wherein the antibody drug comprises a monoclonal antibody against PD-L1.

[0030] [4] The composition for enhancing the effect of an antibody drug according to [1] above, wherein the antibody drug has an effect of inhibiting melanoma proliferation.

[0031] (3) Beneficial effects

[0032] According to the present invention, by using β-glucan as an active ingredient and co-administering it with an antibody drug, the effect of the antibody drug can be enhanced. Therefore, it is suitable for use as a potentiator for immune checkpoint inhibitors, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a graph showing an overview of the administration schedule of each test substance in Test Example 1.

[0034] Figure 2 This is a graph showing the results of examining the tumor volume after a predetermined number of days have passed since the transplantation of tumor cells in Test Example 1.

[0035] Figure 3 This is a graph showing the results of multiplex staining of splenic lymphocytes using anti-mouse CD4 antibody or anti-mouse CD8 antibody and anti-mouse Ki-67 (lymphocyte activation marker) antibody and FACS analysis in Experimental Example 2.

[0036] Figure 4 This is a graph showing the results of multiplex staining of tumor-infiltrating lymphocytes (TILs) using anti-mouse CD4 antibody or anti-mouse CD8 antibody and anti-mouse Ki-67 (lymphocyte activation marker) antibody and FACS analysis in Experimental Example 2.

[0037] Figure 5 This is a graph showing the results of multiplex staining of spleen lymphocytes using anti-mouse CD4 antibody or anti-mouse CD8 antibody and anti-mouse INF-γ antibody and FACS analysis in Experimental Example 2.

[0038] Figure 6 This is a graph showing the results of multiplex staining of tumor-infiltrating lymphocytes (TILs) using anti-mouse CD4 antibody or anti-mouse CD8 antibody and anti-mouse INF-γ antibody and FACS analysis in Experimental Example 2.

[0039] Figure 7 This is a graph showing an overview of the administration schedule of each test substance in Test Example 3.

[0040] Figure 8This is a graph showing the results of examining the tumor volume after a predetermined number of days have passed since the transplantation of tumor cells in Test Example 3. DETAILED DESCRIPTION

[0041] In the present invention, β-glucan is used as an active ingredient of a composition for enhancing the effect of an antibody drug.

[0042] β-glucan is a polysaccharide formed by the polymerization of β-glucose via glycosidic bonds. β-glucan is found in cereals such as barley and oats; yeasts such as brewer's yeast and baker's yeast; deuteromycetes such as black yeast; basidiomycetes such as shiitake mushrooms, griseo mushrooms, versicolor, schizophyllum, hydrangea, and ganoderma; and seaweed such as kelp and wakame. Therefore, β-glucan can be obtained from these natural products through extraction using appropriate solvents. The extraction method can be carried out according to conventional methods and is not particularly limited. For example, extraction can be performed under heating and / or pressure by adding an extraction solvent such as water or aqueous alcohol to the dried and pulverized raw materials, as needed. Typical heating conditions include 105-135°C, and typical pressurization conditions include 1.8-2.1 standard atmospheres. Furthermore, to improve extraction efficiency, extraction can be performed while undergoing acid or alkali treatment, or enzymatic treatment with an enzyme that reduces the molecular weight of the polysaccharide. After extraction, the solvent may be distilled off and concentrated, or the product may be dried into a powder by a drying method such as spray drying. In recent years, various β-glucan raw materials have become commercially available, and commercially available products of such β-glucans may also be used.

[0043] The β-glucan used in the present invention is preferably polymerized using β-1,3 glycosidic bonds, β-1,4 glycosidic bonds, β-1,6 glycosidic bonds, or the like. More preferably, it is a homopolymer containing β-1,3 glycosidic bonds as the main chain, or a heteropolymer containing β-1,3 glycosidic bonds and β-1,6 glycosidic bonds as the main chain. Even more preferably, it is a homopolymer containing β-1,3 glycosidic bonds as the main chain and β-1,6 glycosidic bonds as side chains. β-glucan may also have functional groups such as sulfate and phosphate groups. The molecular weight is preferably within the range of 5,000 to 1,000,000, and more preferably within the range of 200,000 to 500,000. The molecular weight of β-glucan can be measured by gel filtration, for example.

[0044] As shown in the examples below, the β-glucan used in the present invention may be a culture composition containing β-glucan derived from a culture of Aureobasidium pullulans or a culture composition containing β-glucan derived from shiitake mushrooms.

[0045] β-glucan derived from microorganisms belonging to the genus Aureobasidium sp. is described in further detail below. Culture compositions containing β-glucan include not only cultures obtained by culturing microorganisms belonging to Aureobasidium pullulans (hereinafter sometimes referred to as "Aureobasidium pullulans microorganism"), cultures obtained by separating and removing bacterial cells by centrifugation, concentrated solutions of such cultures, diluted solutions of such cultures, or solid materials obtained by removing water from such cultures, but also those obtained by desalting such materials to increase the content of specific components such as β-glucan.

[0046] The pullulans microorganism used in the present invention may be any microorganism belonging to the genus Aureobasidium pullulans and having the ability to produce β-glucan, and suitable examples include Aureobasidium pullulans M-1 (Aureobasidium pullulans M-1, depository registration number: FERM BP-08615, deposit registration date: February 10, 2004, transformed from deposited FERM P-19213 on February 14, 2003) (National Institute for Product Evaluation and Technology Patent Organism Depository, Room 120, 2-5-8 Kazusa Kamata, Kisarazu-shi, Chiba Prefecture, Japan, postal code 292-0818) or Aureobasidium pullulans M-2 (Aureobasidium pullulans M-2, depository registration number: FERM BP-10014, deposit registration date: April 22, 2004) (Independent Administrative Institution for Product Evaluation and Technology, Patent Organism Depository, Room 120, 2-5-8 Kazusa Kamata, Kisarazu-shi, Chiba Prefecture, Japan, postal code 292-0818). Furthermore, structural analysis by NMR measurements (13C NMR: Varian UNITY INOVA 500, 1H NMR: Varian UNITY INOVA 600) revealed that the β-glucan produced by these strains is a β-1,3-1,6-glucan structure with glucose side chains based on β-1,6 glycosidic bonds within a main chain composed of glucose linked by β-1,3 glycosidic bonds.

[0047] The above-mentioned Aureobasidium pullulans microorganism can be cultured according to known methods (see Japanese Patent Application Laid-Open No. 57-149301, etc.). Specifically, the fungus is sown in a culture medium (pH 5.2-6.0) supplemented with 0.5-5.0% by mass of a carbon source (sucrose), 0.1-5.0% by mass of a nitrogen source (e.g., rice bran), and other trace substances (e.g., vitamins, inorganic substances). The culture is then aerated and cultured at a temperature of 20-30°C for 2-14 days, preferably with aeration and agitation. As β-glucan is produced, the viscosity of the culture increases, forming a high-viscosity gel. The resulting culture typically contains 0.6-10% by mass of solids, of which 5-80% by mass is β-glucan.

[0048] The culture containing β-glucan obtained by the above-mentioned culture is preferably heated or pressurized and sterilized before use. In addition, it can also be used after sterilization after separation and removal of bacterial cells by centrifugation or the like. In addition, it is also possible to use a culture that has been concentrated or dried as needed. Furthermore, it is also possible to use a substance obtained by extracting a component rich in β-glucan or a substance that has been desalted and purified. In addition, a culture of a microorganism belonging to Aureobasidium pullulans is used as a food additive such as a thickening stabilizer and is highly safe.

[0049] The composition of the present invention can be used in a manner of simultaneous administration with an antibody drug to enhance the effect of the antibody drug. In other words, it can be used to enhance the effect of the antibody drug when the antibody drug is exerting an effect for the purpose of, for example, anticancer effect.

[0050] Here, "simultaneous administration with an antibody drug" means that the two components, the β-glucan or β-glucan-containing culture composition, and the antibody drug, can be brought into contact with the target tissue, cell, or other biological component within a timely manner, within the range in which their effectiveness is demonstrated. It is not necessary to administer the two components in a single formulation. In other words, the effects of the present invention can be achieved by formulating the two components into separate formulations and administering them sequentially or at predetermined intervals within the range in which their effectiveness is demonstrated.

[0051] As the antibody drug of the composition of the present invention, there is no particular limitation. For example, it can be an antibody drug comprising an antibody having an effect of suppressing cancer proliferation by suppressing immune checkpoints. The type of disease or cancer to which the antibody drug can be applied is also not particularly limited. As cancer, for example, lung cancer, lymphoma, melanoma, leukemia, renal cell carcinoma, renal pelvis / ureteral cancer, nasopharyngeal carcinoma, osteosarcoma, gastric cancer, malignant mesothelioma, ovarian cancer, cervical cancer, pancreatic cancer, microsatellite high instability (MSI-High) colon / rectal cancer, esophageal cancer, hepatocellular carcinoma, bile duct cancer, etc. can be exemplified. As an antibody, it is known that there are, for example, anti-PD-L1 antibodies, anti-PD-1 antibodies, anti-LAG3 antibodies, anti-CTLA4 antibodies, anti-TIM3 antibodies, anti-TIGIT antibodies, anti-VISTA antibodies, etc., but it is not limited thereto. The antibody can be a polyclonal antibody, a monoclonal antibody, or a form of an antiserum comprising an antibody. Furthermore, antibodies may be directed against human proteins or against proteins from animals such as mice, rats, rabbits, goats, cattle, and monkeys. Furthermore, antibodies may be human antibodies or antibodies from animals such as mice. Alternatively, antibodies may be prepared by raising antisera from animals such as mice, rats, rabbits, goats, cattle, and monkeys.

[0052] The administration route of the composition of the present invention is not particularly limited, and a known formulation form can be appropriately selected for oral administration, intraperitoneal administration, intramuscular administration, transnasal administration, transpulmonary administration, vaginal administration, intravenous administration, rectal administration, and the like.

[0053] The dosage of the composition of the present invention can be appropriately determined based on the type of antibody drug, the health status, symptoms, age of the person or animal being administered, the method of administration, the number of doses, and the timing of administration. Typical dosages include, for example, oral administration of 0.025 to 4000 mg / kg (body weight) of the β-glucan or culture composition containing β-glucan, as calculated on a solid basis. Intraperitoneal administration of 0.05 to 5 mg / kg (body weight) of the β-glucan or culture composition containing β-glucan, as calculated on a solid basis, can be used.

[0054] The composition of the present invention can typically be used in various product forms, such as pharmaceuticals, quasi-drugs, functional foods, nutritional supplements, supplements, health foods, animal pharmaceuticals, quasi-drugs, functional foods, nutritional supplements, supplements, and health foods for animals. Alternatively, these products can be used in combination. Furthermore, the composition can be used in combination with various foods and beverages.

[0055] Example

[0056] The present invention will be described in detail below with reference to the following examples, but these examples are not intended to limit the scope of the present invention.

[0057] [Production Example 1]

[0058] Aureobasidium pullulans M-2 (National Institute for Product Evaluation and Research, Japan Patent Collection, registration number: FERM BP-10014) was seeded in liquid culture and cultured with shaking at 24.5°C for four days to allow β-glucan to form in the culture medium. The β-glucan concentration was estimated to be approximately 6 mg / mL using the phenol-sulfuric acid method and enzymatic methods.

[0059] <Test Example 1>

[0060] According to conventional methods, tumor cell lines are transplanted into cancer-bearing mice to investigate whether the administration of the test substance will affect the growth of their tumors.

[0061] Specifically, tumor-bearing mice were prepared by the following method: 3×10 5 Mouse melanoma cell line B16F10 (Riken BioBank) was inoculated into the flank of mice, and the tumors were allowed to grow for 16 days.

[0062] The experimental animals were divided into four groups: an untreated group, an anti-PD-L1 antibody alone group, a β-glucan alone group, and a combination of an anti-PD-L1 antibody and β-glucan group. The N number was set to 26 animals in the untreated group, 18 animals in the PD-L1 antibody alone group, 6 animals in the β-glucan alone group, and 9 animals in the combination of an anti-PD-L1 antibody and β-glucan group. Hereinafter, for convenience, the untreated group will be referred to as the "control group," the anti-PD-L1 antibody alone group as "test group 1," the β-glucan alone group as "test group 2," and the combination of an anti-PD-L1 antibody and β-glucan group as "test group 3."

[0063] As for the administration of the test substance, for the anti-PD-L1 antibody alone administration group ("Test Group 1"), anti-mouse PD-L1 antibody (clone: ​​MIH5) was intraperitoneally administered at a dose of 200 μg / mouse on the 8th and 12th days after tumor cell transplantation. For the β-glucan alone administration group ("Test Group 2"), the β-glucan-containing culture composition prepared in Preparation Example 1 was intraperitoneally administered at a dose of 30 mg / kg, calculated as the amount of β-glucan, on the 8th, 11th, and 14th days after tumor cell transplantation. For the anti-PD-L1 antibody and β-glucan combined administration group ("Test Group 3"), the anti-mouse PD-L1 antibody and the β-glucan-containing culture composition prepared in Preparation Example 1 were intraperitoneally administered to mice at the same time and in the same dose as the respective single-administration groups. Figure 1 The summary of the administration schedule of each test substance is shown in FIG.

[0064] On the 6th, 8th, 12th, 14th and 16th day after inoculation of melanoma cell lines, the long and short diameters of the tumors were measured, and the tumor volumes (mm) were recorded according to the following formula: 3 ).

[0065] [Mathematical formula 1]

[0066]

[0067] The results of tumor volume after a certain number of days from the transplantation of tumor cells are shown in Table 1 and Figure 2 In addition, statistical analysis was performed using the Brown-Forsythe Test to examine whether there were significant differences among the groups when comparing tumor volumes on the 16th day from tumor cell transplantation. The results of this examination are shown in Table 2.

[0068] [Table 1]

[0069]

[0070] [Table 2]

[0071]

[0072] The results showed that the group receiving a combination of an anti-PD-L1 antibody and β-glucan ("Test Group 3") showed a significant reduction in tumor volume compared to the untreated group ("Control Group"). This reduction was also statistically significant when compared to the group receiving the anti-PD-L1 antibody alone ("Test Group 1") or the group receiving β-glucan alone ("Test Group 2") (see comparison results on Day 16).

[0073] <Test Example 2>

[0074] Sixteen days after tumor cell transplantation, tumor-bearing mice were dissected, and splenic lymphocytes and tumor-infiltrating lymphocytes (TILs) were isolated and purified from the tumor-bearing mice in each experimental group. The collected tissues were hemolyzed with sterile distilled water, and lymphocytes were prepared using a lymphocyte isolation kit (trade name "lympholyte-M" (Cedarlane Laboratories).

[0075] Individual lymphocytes were stained and analyzed by FACS using anti-mouse CD4 antibody (Biolegend Japan, Inc.), anti-mouse CD8 antibody (Biolegend Japan, Inc.), anti-mouse Ki-67 (lymphocyte activation marker) antibody (Thermo Fisher Scientific KK), and anti-mouse INF-γ antibody (Becton Dickinson Japan). After staining for cell surface antigens, intracellular antigens were stained. Fixation Buffer and Permeabilization Wash Buffer (Biolegend Japan, Inc.) were used for intracellular antigen staining.

[0076] Figure 3 The figure shows the results of multiplex staining of spleen lymphocytes using anti-mouse CD4 antibody or anti-mouse CD8 antibody and anti-mouse Ki-67 (lymphocyte activation marker) antibody and FACS analysis.

[0077] Figure 4 Figure 3 shows the results of multiplex staining of tumor-infiltrating lymphocytes (TILs) using anti-mouse CD4 antibody or anti-mouse CD8 antibody and anti-mouse Ki-67 (lymphocyte activation marker) antibody and FACS analysis.

[0078] Figure 5 The figure shows the results of multiplex staining of spleen lymphocytes with anti-mouse CD4 antibody or anti-mouse CD8 antibody and anti-mouse INF-γ antibody and FACS analysis.

[0079] Figure 6 The figure shows the results of multiplex staining of tumor-infiltrating lymphocytes (TIL) using anti-mouse CD4 antibody or anti-mouse CD8 antibody and anti-mouse INF-γ antibody and FACS analysis.

[0080] according to Figure 3The results showed that regarding the Ki-67 expression of splenic lymphocytes, no significant difference was observed in the anti-PD-L1 antibody alone administration group ("experimental group 1"), the β-glucan alone administration group ("experimental group 2"), or the anti-PD-L1 antibody and β-glucan combined administration group ("experimental group 3") compared with the untreated group ("control group") in both CD4-positive T cells and CD8-positive T cells.

[0081] according to Figure 4 The results showed that regarding the expression of Ki-67 on tumor-infiltrating lymphocytes, a significant increase trend was observed in both CD4-positive T cells and CD8-positive T cells in the group that received a combination of anti-PD-L1 antibody and β-glucan ("experimental group 3") compared with the group that received no treatment ("control group"), the group that received anti-PD-L1 antibody alone ("experimental group 1"), or the group that received β-glucan alone ("experimental group 2"). Figure 4 In the data, “**” indicates p<0.01 and “***” indicates p<0.001. ).

[0082] according to Figure 5 The results showed that regarding the expression of INF-γ in splenic lymphocytes, there was no significant difference in CD4-positive T cells and CD8-positive T cells in the group where the anti-PD-L1 antibody was administered alone ("Test Group 1"), the group where β-glucan was administered alone ("Test Group 2"), or the group where the anti-PD-L1 antibody and β-glucan were administered in combination ("Test Group 3"), compared with the untreated group ("Control Group").

[0083] according to Figure 6 The results showed that regarding the expression of INF-γ in tumor-infiltrating lymphocytes, in CD8-positive T cells, there was a trend of significantly higher expression in the anti-PD-L1 antibody and β-glucan combination group ("Test Group 3") compared to the untreated group ("Control Group"), the anti-PD-L1 antibody alone group ("Test Group 1"), or the β-glucan alone group ("Test Group 2"). On the other hand, in CD4-positive T cells, no significant difference was observed between the untreated group ("Control Group") and the anti-PD-L1 antibody and β-glucan combination group ("Test Group 3"). Figure 6 In the data, “**” indicates p<0.01 and “***” indicates p<0.001. ).

[0084] In summary, the administration of anti-PD-L1 antibodies and β-glucan activated cell-infiltrating T cells and enhanced their cytosuppressive activity. However, there was no significant effect on splenic lymphocytes. Furthermore, the combined use of the two components significantly enhanced their activation effect compared to individual administration. These results were achieved through a favorable synergistic effect on tumor volume reduction in cancer-bearing mice.

[0085] <Test Example 3>

[0086] Tumor-bearing mice transplanted with tumor cell lines were prepared using the same method as in Experimental Example 1, and the effect of administration of the test substance on tumor growth was examined. In this case, β-glucan from Lentinus edodes ("Micelleglucan," manufactured by RL-JP Co., Ltd.) (hereinafter referred to as "Micelle β-glucan") was used instead of β-glucan from Aureobasidium pullulans.

[0087] Specifically, tumor-bearing mice were prepared by the following method: 3×10 5 Mouse melanoma cell line B16F10 (Riken BioBank) was inoculated into the flank of mice, and the tumors were allowed to grow for 13 days.

[0088] The experimental animals were divided into four groups: an untreated group, a Micelle β-glucan alone group, an anti-PD-L1 antibody alone group, and a combination of an anti-PD-L1 antibody and Micelle β-glucan group. The N number was set to 4 animals in the untreated group, 2 animals in the Micelle β-glucan alone group, 3 animals in the PD-L1 antibody alone group, and 3 animals in the combination of an anti-PD-L1 antibody and Micelle β-glucan group. Hereinafter, for convenience, the untreated group will be referred to as "Control Group A," the Micelle β-glucan alone group as "Test Group 1A," the anti-PD-L1 antibody alone group as "Test Group 2A," and the anti-PD-L1 antibody and Micelle β-glucan combination group as "Test Group 3A."

[0089] As for the administration of the test substance, for the untreated group ("Control Group A"), physiological saline was administered intraperitoneally on the 8th, 12th, and 14th day after tumor cell transplantation. For the group administered with Micelle β-glucan alone ("Test Group 1A"), Micelle β-glucan was administered intraperitoneally at a dose of 100 mg / kg, calculated based on the amount of β-glucan. For the group administered with anti-PD-L1 antibody alone ("Test Group 2A"), anti-mouse PD-L1 antibody (clone: ​​MIH5) was administered intraperitoneally at a dose of 200 μg / mouse on the 8th and 12th day after tumor cell transplantation. For the group receiving a combination of anti-PD-L1 antibody and Micelle β-glucan ("Test Group 3A"), anti-mouse PD-L1 antibody was administered using the same dosing regimen as described above on days 8 and 12 after tumor cell transplantation, and Micelle β-glucan was administered intraperitoneally on days 8 and 11 after tumor cell transplantation at a dose of 100 mg / kg, calculated based on the β-glucan content. Figure 7 The summary of the administration schedule of each test substance is shown in FIG.

[0090] On the 6th, 8th, 11th, and 12th day after inoculation of the melanoma cell line, the long and short diameters of the tumors were measured, and the tumor volumes (mm) were recorded in the same manner as in Experimental Example 1. 3 ).

[0091] The results of tumor volume after a certain number of days from the transplantation of tumor cells are shown in Table 3 and Figure 8 In addition, statistical analysis was performed using the Brown-Forsythe Test to examine whether there were significant differences among the groups when comparing tumor volumes on the 12th day after tumor cell transplantation. The results of this examination are shown in Table 4.

[0092] [Table 3]

[0093]

[0094] [Table 4]

[0095]

[0096] The results showed that the group receiving a combination of an anti-PD-L1 antibody and Micelle β-glucan ("Test Group 3A") showed a significant reduction in tumor volume compared to the untreated group ("Control Group A"). This reduction trended toward greater effect compared to the group receiving an anti-PD-L1 antibody alone ("Test Group 2A") (see comparison on Day 12).

Claims

1. Use of a culture of Aureobasidium pullulans containing β-glucan in the preparation of a medicament for treating melanoma and for simultaneous use with a monoclonal antibody against PD-L1, wherein the β-glucan from the culture of Aureobasidium pullulans is used to enhance the immune checkpoint inhibitory effect of the antibody.

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