KR20230007688A - Adjuvant compositions comprising fucoidan from Ecklonia cavafor as an active ingredient - Google Patents

Abstract

The present invention relates to an adjuvant composition comprising fucoidan from Ecklonia cava as an active ingredient. Administration of fucoidan from Ecklonia cava through the nasal cavity increases the number of dendritic cells in a lymphatic gland and the activity of the dendritic cells and raises the activity of NK cells and T cells, thereby bringing excellent effects of cancer immunotherapy. Administration in combination with an anti-PD-L1 antibody as one of immune checkpoint inhibitors is found to have more excellent anticancer effects than single administration of the anti-PD-L1 antibody and completely inhibit the metastasis of cancer in vivo. Accordingly, fucoidan from Ecklonia cava can be provided as an adjuvant and a concomitant drug for treating cancer.

Description

Adjuvant compositions comprising fucoidan from Ecklonia cavafor as an active ingredient}

The present invention relates to an adjuvant composition comprising Ecklonia cava -derived fucoidan as an active ingredient.

Cancer is a disease characterized by abnormal local cell growth that has the potential to spread throughout the body, and is one of the representative incurable diseases that modern medicine has not conquered. Cancer types are very diverse, such as lung cancer, bladder cancer, prostate cancer, pancreatic cancer, cervical cancer, brain cancer, stomach cancer, colorectal cancer and melanoma, and various methods of treating cancer have been developed. Among them, surgery, radiation therapy, or chemotherapy drugs have been used as the most common treatment methods, but recently, immuno-anticancer therapy using the patient's immune system has been in the limelight.

Immuno-oncology therapy is a method of using immuno-oncology agents as a fourth-generation cancer treatment. Immuno-anticancer drugs are substances that enhance the immune response to cancer by activating the patient's immune cells rather than directly killing cancer cells. Immuno-anticancer drugs induce specific responses to cancer antigens or eliminate cancer through cytotoxicity by activating natural killer cells.

Regarding the immune response to cancer, immunity by dendritic cells and natural killer cells (NK cells) appears non-specific to antigens. When activated by external substances and pathogens, natural killer cells secrete various proteins that can exhibit cytotoxicity, which destroys the cell membrane of pathogens and induces natural cell death. Therefore, recently, various cancer treatment methods using the activity of natural killer cells have been developed.

However, cancer patients generally have lower immune activation ability than normal people, and have difficulties in treatment because effective immune activation against cancer is not well induced. This is a phenomenon that occurs because even if immune cells detect cancer, they evade the attack of immune cells by various immunosuppressive substances secreted by cancer cells. Therefore, there is a need for the development of a new method capable of overcoming such immune evasion and selectively killing cancer cells.

Recently, cancer treatment using immunity has been deriving treatment results for many patients with excellent efficacy. Immune anti-cancer drugs use a method of artificially activating the immune cells of cancer patients so that these cells selectively attack and kill cancer cells. However, cancer cells secrete and label various immunosuppressive substances to evade the attack of immune cells. This allows cancer cells to continuously grow by avoiding being attacked even if immune cells detect cancer cells. Therefore, as a method capable of overcoming such immune evasion and killing cancer cells, antibodies that block proteins expressed in cancer cells and immune cells in the middle are being developed as cancer therapeutics. These antibodies are referred to as immune checkpoint inhibitory antibodies and induce excellent anticancer effects even in terminal cancer patients.

Cancer treatment with immune checkpoint inhibitors is actively progressing, but there are also disadvantages. In particular, it has been reported that there is no significant effect in patients with relatively low expression of immune checkpoint proteins, and there are cases in which the death of cancer cells cannot be induced due to insufficient activation of immune cells in the body. Therefore, in this study, a novel immune enhancer (adjuvant) was developed to improve the efficacy of immune checkpoint inhibitors, and a new treatment method capable of exhibiting specific efficacy for lung cancer is being developed.

On the other hand, the immuno-anticancer adjuvant is a drug that can be administered in combination with a therapeutic agent administered for the treatment of cancer, and includes, for example, an immuno-anticancer agent administered together with a cell therapeutic agent for the treatment of cancer. The combined administration is not only useful when resistance to the anticancer drug is shown, but also has the advantage of reducing the amount of the anticancer drug administered by enhancing the efficacy of the anticancer drug even when the efficacy is shown. Through this, it is possible to increase the anticancer efficacy while minimizing the toxicity and side effects of the anticancer agent on each organ of the body. Although a number of effective combination therapy therapies have been identified over the past several decades, there is an ongoing need to identify effective therapies for use in anti-cancer therapy in light of the continuing high cancer deaths each year.

Korean Patent Publication No. 10-2020-0126334 (2020. 11. 06. notice)

An object of the present invention is to provide an immune enhancer composition for intranasal administration that induces the activity of immune cells.

Another object of the present invention is to provide a composition for combined administration for the prevention or treatment of cancer containing the above immune enhancer.

The present invention provides an adjuvant composition comprising Ecklonia cava -derived fucoidan as an active ingredient.

In addition, the present invention provides a composition for combined administration for the prevention or treatment of cancer comprising Ecklonia cava -derived fucoidan and an immune checkpoint inhibitor as active ingredients.

According to the present invention, when Ecklonia cava -derived fucoidan is administered intranasally, the number of dendritic cells in the lymph node increases, the activity of dendritic cells increases, and the activity of NK cells and T cells increases through this, so immunotherapy The effect is excellent, and as a result of combined administration with anti-PD-L1 antibody, one of the immune checkpoint inhibitors, it was confirmed that anti-cancer effect was superior to anti-PD-L1 antibody alone administration, and cancer metastasis was completely prevented in vivo. By confirming the inhibition, Ecklonia cava ) derived fucoidan can be provided as an adjuvant and a combined administration for the treatment of cancer.

1 is a schematic diagram of a combined administration method for immunity enhancement and cancer treatment of Ecklonia cava -derived fucoidan.
Figure 2 is a result of evaluating the effect on the activity of pulmonary lymph node dendritic cells according to nasal administration of Ecklonia cava -derived fucoidan. (A) is a schematic diagram of the method of administering 50 mg/kg of Ecklonia cava-derived fucoidan and lung lymph nodes, (B) is the result of separating pulmonary lymph node dendritic cells by flow cytometry, and (C) is the result of Ecklonia cava-derived fucoidan. (D) is the result of confirming the expression pattern of CC chemokine receptor type 7 (CCR7) in lung lymph node DCs. (E) is the result of confirming the expression of surface active proteins and MHC class I and II of dendritic cells by flow cytometry, and (F) is the result of confirming the cytokine secreted inside the lung.
Figure 3 is a result of evaluating the effect on the activity of natural killer cells according to nasal administration of Ecklonia cava -derived fucoidan. (A) is the result of partitioning pulmonary lymph node natural killer cells, and (B) is the result of confirming the ratio of pulmonary lymph node natural killer cells by intranasal administration of Ecklonia cava-derived fucoidan. (C) is the result of observing changes in the number of natural killer cells in the lung lymph nodes, and (D) is the result of confirming the degree of secretion of cytotoxic substances by active natural killer cells. (E) is the result of confirming the toxic substances secreted into the lungs following nasal administration of Ecklonia cava-derived fucoidan.
Figure 4 is a result of evaluating the effect on the activation of T cells according to the nasal administration of Ecklonia cava -derived fucoidan. (A) is the result of confirming the intracellular cytokine expression pattern and (B) the ratio of T cells expressing CD4 and CD8 in lung lymph nodes.
5 is a result of evaluating changes in activation of natural killer cells and T cells induced by fucoidan derived from Ecklonia cava according to the presence or absence of dendritic cells.
Figure 6 is a result of evaluating the anti-tumor effect of immune checkpoint inhibitors according to intranasal administration of Ecklonia cava -derived fucoidan. (A) is the result of confirming the survival rate of the lung metastasis model mouse of melanoma (B16 cell line) by administering anti-PD-L1 antibody, E. c. This is the result of confirming the weight change of rats during administration of . (C) is the result of confirming the extent of cancer penetration into the lungs 10 days after cancer administration (7 days after drug administration) using pathological techniques, and (D) is anti-PD in mice in which natural killer cells or CD8 T cells have been removed. -This is the result of confirming the survival rate of mice according to the mixed administration of L1 antibody and Ecklonia-derived fucoidan. (E) is the result of confirming the cancer growth inhibitory effect of anti-PD-L1 antibody and E. c.

The terms used in this specification have been selected from general terms that are currently widely used as much as possible while considering the functions in the present invention, but these may vary depending on the intention of a person skilled in the art, precedent, or the emergence of new technologies. In addition, in a specific case, there is also a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, not simply the name of the term.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in this application, it should not be interpreted in an ideal or excessively formal meaning. don't

Numerical ranges are inclusive of the values defined therein. Every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if such higher numerical limitations were expressly written. Every numerical limitation given throughout this specification will include every better numerical range within the broader numerical range, as if the narrower numerical limitations were expressly written.

Hereinafter, the present invention will be described in more detail.

The present invention provides an adjuvant composition comprising Ecklonia cava -derived fucoidan as an active ingredient.

The fucoidan may induce the activation of immune cells in lymph nodes, and the immune cells may be dendritic cells, NK cells, or T cells.

The immune enhancing composition of the present invention may additionally contain pharmaceutically acceptable carriers and diluents for formulation. The pharmaceutically acceptable carriers and diluents include excipients such as starch, sugar and mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose, gelatin, alginates, and polyvinyl fibres. binders such as rolidone, etc., lubricants such as talc, calcium stearate, hydrogenated castor oil and polyethylene glycol, disintegrants such as povidone, crospovidone, surfactants such as polysorbates, cetyl alcohol, and glycerol; don't The pharmaceutically acceptable carrier and diluent may be biologically and physiologically compatible with the subject. Examples of diluents include, but are not limited to, saline, aqueous buffers, solvents and/or dispersion media.

The immune enhancing composition of the present invention may be administered in an appropriate manner depending on the purpose, and most preferably used for intranasal administration.

The dosage of the immune enhancer composition of the present invention depends on the condition and weight of the patient, age, sex, health condition, dietary constitution specificity, the nature of the preparation, the severity of the disease, the administration time of the composition, the administration method, the administration period or interval, and the excretion rate. , And the range may vary depending on the type of drug, and may be appropriately selected by a person skilled in the art. For example, it may be in the range of about 0.1 to 10,000 mg/kg, but is not limited thereto, and may be divided and administered once or several times a day.

In addition, the present invention provides a composition for combined administration for the prevention or treatment of cancer comprising Ecklonia cava -derived fucoidan and an immune checkpoint inhibitor as active ingredients.

The immune checkpoint inhibitor may be an anti-CTLA-4 antibody, an anti-PD-1 antibody or an anti-PD-L1 antibody, but is not limited thereto.

The cancer is bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, squamous cell cancer, epithelial cell cancer, skin cancer, leukemia, acute lymphocytic leukemia, B-cell Lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma, Burkett's lymphoma, acute and chronic myeloid leukemia, promyelocytic leukemia, fibrosarcoma, rhabdomyosarcoma, melanoma, seminoma, teratocarcinoma, It may be any one selected from the group consisting of neuroblastoma, glioma, astrocytoma, neuroblastoma, schwannoma, osteosarcoma, xeroderma pigmentosa, keratoacanthoma, seminoma, thyroid follicular carcinoma and teratocarcinoma, wherein the cancer is metastatic cancer can

Hereinafter, experimental examples and examples will be described in detail to aid understanding of the present invention. However, the following experimental examples and examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following experimental examples and examples. Experimental examples and examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

<Experimental Example> Experimental Materials and Methods

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Animal Models

C57BL/6 (5-8 weeks old, female) and BLAB/c (5-8 weeks old, female) mice were purchased from Shanghai Public Health Clinical Center (SPHCC, Shanghai, China) or Hyochang Science (Daegu, Korea). Mice were bred at the Experimental Animal Center of SPHCC and Yeungnam University under specific pathogen-free conditions. Animal experiments were approved by Yeungnam University Laboratory Animal Center (#2019-029) and SPHCC (#2018-A049-01). All experiments were conducted in accordance with the ethical principles of animal experimentation and in accordance with the IACUC regulations of Yeungnam University and SPHCC.

2. Cancer Cell Lines

B16-F10 cells (ATCC, Manassas, VA, USA) were cultured with 1% penicillin-streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) and 10% FBS (Sigma-Aldrich, St. Louis, MO, USA). were cultured in RPMI-1640 (Merck KGaA, Darmstadt, Germany) at 37°C and 5% CO ₂ conditions. CT26.WT-iRFP-Neo (CT-26-iRFP; Imanis Life Sciences, CL091, Rochester, USA) cells were cultured in DMEM medium containing 0.4 mg/mL of G418 (Sigma-Aldrich).

3. Ecklonia fucoidan extraction

20 g of dried E. cava was ground and suspended in 1 L of 85% ethanol aqueous solution. It was heated under reflux conditions at 85° C. for 10 minutes, and ethanol-soluble substances were removed by filtration. The filtered residue was washed with methanol and centrifuged at 3,000 xg for 20 minutes to remove unhydrolyzed residue. Acetic acid and sodium acetate were added to the purified residue, and ethanol was added at a concentration of 42% (v/v) to prepare a concentrate. The concentrate was centrifuged at 10,000 xg at 4° C. for 30 minutes to remove the precipitate, and the supernatant was harvested and ethanol was added at a concentration of 60% (v/v) to prepare a concentrate. The concentrate was centrifuged at 10,000 xg at 4°C for 30 minutes to harvest the precipitate, resuspended in distilled water, dialyzed (MWCO, 10-12 kDa) at 4°C for 72 hours, and lyophilized. The dried powder was used as E. cava ) derived fucoidan (ECF) sample.

4. Reagents and Antibodies

Isotype control antibodies and Fc receptor antibodies (Abs) were purchased from BioLegend (San Diego, CA, USA). anti-CD3 (17A2), anti-CD11c (N418), anti-CD40 (HM40-3), anti-CD80 (16-10A1), anti-CD69 (H1.2F3), anti-CD86 (GL-1), Anti-CD253 (tumor necrosis factor-related apoptosis inducing ligand; TRAIL, N2B2), anti-granzyme B (GB11), anti-IFN-γ (R4-6A2), anti-interleukin (IL)-6 (MP5-20F3 ), anti-IL-12/23p40 (C17.8), anti-Zuo histocompatibility complex (MHC) class I (AF6-88.5), anti-MHC class II (M5/114.15.2), anti-NK1.1 (PK136), anti-perforin (S16009A), and anti-TNF-α (MP6-XT22) were purchased from BioLegend (San Diego, CA, USA). Anti-PD-L1 (programmed cell death-ligand 1) Abs were purchased from BioXcell (Lebanon, NH, USA). Lipopolysaccharide (LPS; O111:B4) was purchased from Sigma-Aldrich.

5. Preparation of single cell suspension of the mediastinal lymph node (mLN)

Mediastinal lymph node (mLN) cells were treated with collagenase IV and DNase I (Sigma-Aldrich) containing digestion buffer for 20 minutes. Cells were suspended in 5 mL of Histopaque-1077 (Sigma-Aldrich) and top layer was made with 5 mL of fresh histopaque. Cells were centrifuged at 2,000 xg for 10 minutes. Leukocytes were harvested after washing with PBS.

6. NK cell activation assay

Mediastinal lymph node (mLN) cells were incubated with an Fc receptor-binding antibody and then stained with an unconjugated isotype control antibody. After washing with PBS, cells were incubated with antibodies of CD3, CD69, NK1.1, and TRAIL. After staining with DAPI (4,6-diamidino-2-phenylindole) solution (Sigma-Aldrich), cells were analyzed by flow cytometry (ACEA Biosciences Inc., San Diego, CA, USA).

7. Analysis of intracellular cytokine and cytotoxic mediator production

Mediastinal lymph node (mLN) single cell suspensions were incubated with Golgistop™ (2 μM monesin solution; BioLegend) at 37° C. for 2 hours. Cells were harvested and stained using the Zombie Violet Fixable Viability Kit (BioLegend). After staining the cells with surface antibodies, they were fixed (BioLegend) at 4°C for 20 minutes. Cells were permeabilized using permeabilization wash buffer (BioLegend) and stained with intracellular antibodies for 30 min at 25°C. After washing, cells were resuspended in PBS and analyzed by flow cytometry (ACEA Biosciences Inc.).

8. Enzyme-linked immunosorbent assay (ELISA)

Levels of IFN-γ, IL-6, IL-12p70, and TNF-α were measured using ELISA kits (BioLegend). An ELISA kit for Perforin was purchased from Abbkine (Wuhan, China). An ELISA kit for granzyme B was purchased from LSBIO (Seoul, Korea). The concentration of cytokines was quantified in triplicate.

9. mLN DC assay

Mediastinal lymph node (mLN) cells were stained with lineage antibodies (anti-B220, anti-CD3, anti-CD49b, anti-CD90.1, anti-Gr-1, and anti-TER-119) and anti-CD11c antibody. CD11c ⁺ lineage ⁻ cells in viable leukocytes were defined as DC.

10. DC depletion

Mediastinal lymph node (mLN) cells were incubated for 15 minutes at 4°C with anti-CD11c-biotin antibody (BioLegend). Cells were further stained with anti-biotin-microbeads (Miltenyi Biotec) for 20 minutes. Cells were applied to LD columns (Miltenyi Biotec) and negative cells were harvested. The depletion efficiency of CD11c+ cells was greater than 95% as analyzed by flow cytometry (ACEA Biosciences).

11. Mouse lung cancer model and Ecklonia-derived fucoidan (ECF) administration

B16 (0.5×10 ⁶ ) cells were intravenously (iv) administered to C57BL/6 mice. Mice were randomly divided into PBSA, anti-PD-L1 antibody, ECF and anti-PD-L1 antibody + ECF groups. ECF (50 mg/kg) was injected intranasally (in) every 3 days from the 3rd day after tumor injection. Anti-PD-L1 antibody (10 mg/kg) was infused intraperitoneally (ip) every 3 days starting 5 days after tumor injection. Mice survival was monitored up to 30 days after tumor injection.

12. Histological analysis

Tissues were harvested after injecting 4% paraformaldehyde (1 ml) into the lungs on day 10 after tumor implantation. Lungs were fixed with 4% paraformaldehyde for 24 hours, followed by paraffin embedding. Lungs were cut at a thickness of 5 μm. After deparaffinization and rehydration of the sections, lung sections were stained with hematoxylin and eosin (Sigma-Aldrich).

13. NK cell and T cell depletion

NK or CD8 T cells were depleted by administration of 50 μg of anti-NK1.1 (PK136) or anti-CD8α (2.43) antibody (BioXcell) every 2 days in C57BL/6 mice, respectively.

14. In vivo fluorescence imaging

Metastatic CT-26 cancer of the lung was imaged using a fluorescence in vivo imaging system from FOBI (Cellgentek, Cheongju, Republic of Korea) 14 days after CT-26-iRFP challenge in BALB/c mice.

15. Statistics

The experiment was performed with two samples, three times each. Data are analyzed as mean ± standard deviation (SEM). All experimental data were analyzed using the Tukey multiple comparison test and the Mann-Whitney t-test. P < 0.05 was considered statistically significant.

Example 1. Effect of Ecklonia cava-derived fucoindan (ECF) on dendritic cells (DC) in lymph nodes

To evaluate the effect of Ecklonia cava-derived fucoidan (ECF) on mucosal immune stimulation, C57BL/6 mice were intranasally administered with Ecklonia cava-derived fucoidan (ECF) at a concentration of 50 mg/kg and endotoxin (LPS) at a concentration of 1 mg/kg. administered. After 18 hours, the spleen was removed and the activity of dendritic cells (DC) in the spleen was analyzed. As shown in FIG. 2B , splenic DC was defined as a live floating leukocyte that does not express Lineage and expresses CD11c using flow cytometry. In the case of mice administered with Ecklonia cava-derived fucoidan (ECF), the number and frequency of dendritic cells (DC) increased significantly in the mediastinal lymph node (mLN) after 18 hours (Fig. 2C), and mLN even when PBS was treated (Fig. 2D). showed an increase in the number of DCs. In particular, in the case of mice administered with Ecklonia cava-derived fucoidan (ECF), the expression of C-C chemokine receptor type 7 (CCR7), which can induce cell migration on the surface of dendritic cells, was rapidly increased compared to the control group. It is demonstrated that Ecklonia cava-derived fucoidan (ECF) promoted the migration of dendritic cells (DCs) to the lymph node (Fig. 2E). In addition, Ecklonia-derived fucoidan (ECF) induced upregulation of co-stimulators, MHC class I and II expression in dendritic cells (DC) in the mediastinal lymph node (mLN) (Fig. 2F). Concentration levels of IL-6, IL-12p70, and TNF-α in bronchoalveolar lavage fluid (BAL) were significantly increased by Ecklonia cava-derived fucoidan (ECF) compared to the control group (Fig. 2G). The above results demonstrate that Ecklonia cava-derived fucoidan (ECF) induces expression of surface-active protein expression factors of dendritic cells in lymph nodes, increases secretion of inflammatory cytokines, and induces dendritic cell activation.

Example 2. Effect of Ecklonia cava-derived fucoindan (ECF) on NK cells in lymph nodes

Mature dendritic cells (DCs) promote the activation of other immune cells such as natural killer cells (NK cells). NK cells are one of the powerful immune cells capable of selectively killing cancer cells and pathogens in an antigen-specific manner. It was confirmed that Ecklonia cava-derived fucoidan induces dendritic cell activation, and whether it induces NK cell activation was evaluated. To evaluate the effect of Ecklonia cava-derived fucoidan (ECF) on the activation of NK cells in the mediastinal lymph node (mLN), Ecklonia cava-derived fucoidan (ECF) was intranasally administered at a concentration of 50 mg/kg to C57BL/6 mice, and pulmonary lymph node NK cells in the liver were compartmentalized and compared to lactation. NK cells were defined as NK1.1 ⁺ CD3 ⁻ cells as shown in FIG. 3A . In the case of mice administered with Ecklonia cava-derived fucoidan (ECF), the number and density of NK1.1 ⁺ CD3 ^- cells increased (Fig. 3B, 3C), and the expression of NK cell surface activation markers CD69 and TRAIL appeared to increase (Fig. 3D). Ecklonia cava-derived fucoidan (ECF) also significantly increased the concentrations of IFN-γ and cytotoxic mediators (granzyme B and perforin) in BAL fluid (Fig. 3E). The above results demonstrate that Ecklonia cava-derived fucoidan (ECF) induces activation of NK cells in lymph nodes.

Example 3. Effect of Ecklonia cava-derived fucoindan (ECF) on T lymphocytes in lymph nodes

Mature dendritic cells (DCs) induce differentiation and activation of naive T cells. It was confirmed that Ecklonia cava-derived fucoidan induces dendritic cell activation, and its effect on T lymphocyte activation was evaluated. Ecklonia cava-derived fucoidan (ECF) was intranasally administered to C57BL/6 mice twice at 3-day intervals, and lung lymph nodes were removed to confirm T cell activity. Ecklonia cava-derived fucoidan (ECF) promoted effector T cell differentiation in the mediastinal lymph node (mLN). As shown in Figure 4, when Ecklonia cava-derived fucoidan (ECF) was administered, interferon-gamma (IFN-γ) production increased and tumor death factor-alpha (TNF-α)-expressing CD8 and CD4 T cells has increased. The above results demonstrate that Ecklonia cava-derived fucoidan (ECF) promoted the differentiation of naive T cells into IFN-γ and TNF-α-producing Th1 and Tc1 cells.

Example 4. T cell and NK cell activation mechanism of Ecklonia-derived fucoindan (ECF)

Activation of NK cells and T cells is controlled by mature dendritic cells. It was confirmed that dendritic cells stimulated with Ecklonia cava-derived fucoidan (ECF) in the mediastinal lymph node (mLN) are essential for the induction of T cell and NK cell activation. Dendritic cells were removed from the lymph node suspension using a biotin-coupled anti-CD11c antibody, and treated with Ecklonia cava-derived fucoidan (ECF). As shown in FIG. 5 , in the state in which dendritic cells were removed, upregulation of CD69 and TRAIL in NK cells was suppressed even when Ecklonia cava-derived fucoidan (ECF) was treated. In addition, it was shown that the upregulation of granzyme B, IFN-γ, and perforin was inhibited in the state in which dendritic cells were removed. The above results demonstrate that the activation of NK cells and T cells induced by Ecklonia cava-derived fucoidan (ECF) appears only when dendritic cells are present.

Example 5. Evaluation of anti-tumor effect of fucoidan derived from Ecklonia cava in vivo

As it was confirmed that Ecklonia cava-derived fucoidan (ECF) promotes the activation of immune cells in the mediastinal lymph node (mLN), Ecklonia cava-derived fucoidan (ECF) improves the effect of anti-PD-L1 antibody, a checkpoint immunosuppressant, as a cancer treatment. evaluated. Lung metastases were induced by intravenous administration of melanoma cells, B16 cells, into the tail vein of C57BL/6 mice. From 3 days after cancer administration, saline (negative control), anti-PD-L1 antibody, Ecklonia cava-derived fucoidan (ECF) or a mixture of anti-PD-L1 antibody and Ecklonia cava-derived fucoidan (ECF) were intranasally administered every 3 days. At this time, as shown in FIG. 1, the anti-PD-L1 antibody was intraperitoneally injected, and Ecklonia cava-derived fucoidan (ECF) was administered intranasally.

As shown in FIG. 6A, when the anti-PD-L1 antibody was administered alone, all mice died on day 23, and when Ecklonia cava-derived fucoidan (ECF) was administered alone, all mice died on day 17. When both anti-PD-L1 antibody and Ecklonia cava-derived fucoidan (ECF) were administered, 80% of the mice survived up to 30 days. Treatment with the anti-PD-L1 antibody delayed death of the mice compared to administration of saline (control), but all mice died within 23 days. Mice administered with both the anti-PD-L1 antibody and Ecklonia cava-derived fucoidan (ECF) survived up to 30 days.

In addition, as a result of measuring the weight change of the mice, in the case of mice administered with both the anti-PD-L1 antibody and Ecklonia cava-derived fucoidan (ECF), the body weight increased over time, whereas in the other experimental groups and the control group, the body weight gradually increased. It was found to decrease to (Fig. 6B). In addition, in the case of mice administered with both the anti-PD-L1 antibody and Ecklonia cava-derived fucoidan (ECF), the infiltration of B16 cells into the lungs was suppressed, whereas in the other experimental groups and the control group, B16 cells infiltrated into the lungs (FIG. 6C). ). The above results demonstrate that Ecklonia cava-derived fucoidan (ECF) promoted the activation of NK cells and T cells, and that the activated NK cells and T cells induced antitumor effects.

In order to confirm that the anti-cancer effect of the combined administration of anti-PD-L1 antibody and Ecklonia cava-derived fucoidan (ECF) requires the activation of NK cells and T cells, an antibody that removes immune cells in the body is administered to induce NK cells and T cells. After removing the cells, it was confirmed whether anti-cancer effects were observed even when the anti-PD-L1 antibody and Ecklonia cava-derived fucoidan (ECF) were co-administered. As shown in Figure 6D, despite the combined administration of anti-PD-L1 antibody and Ecklonia cava-derived fucoidan (ECF) in mice in which NK cells and T cells were removed, the injection of B16 tumor cells did not prevent the death of the mice. appear. Compared to other control groups, mice in which both NK cells and T cells were removed showed the shortest survival period. The above results demonstrate that the anticancer effect of the combined administration of the anti-PD-L1 antibody and Ecklonia cava-derived fucoidan (ECF) appears through activation of NK cells and T cells.

In addition, the anticancer effect of Ecklonia cava-derived fucoidan (ECF) was evaluated in various other cancers in addition to cancers caused by B16 tumor cells. Tumors were induced in BALB/c mice by administering CT-26 cells, which are epithelial carcinoma cells, through the tail vein. Anti-PD-L1 antibody and fucoidan (ECF) derived from Ecklonia cava were administered in the same manner as above. As shown in Fig. 5E, the combined administration of the anti-PD-L1 antibody and Ecklonia cava-derived fucoidan (ECF) completely inhibited the growth of metastatic CT-26 tumors in the lung. These results demonstrate that Ecklonia cava-derived fucoidan (ECF) enhances the anti-tumor effect of anti-PD-L1 antibodies by activating T cells and NK cells, and this effect is shown in various cancers.

Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. Do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (9)

An immune enhancer (adjuvant) composition comprising Ecklonia cava -derived fucoidan (Fucoidan) as an active ingredient. The immune enhancer composition according to claim 1, wherein the adjuvant composition is for intranasal administration. The immune enhancer composition according to claim 1, wherein the fucoidan induces the activation of immune cells in the lymph nodes. The immune enhancer composition according to claim 3, wherein the immune cells are dendritic cells, NK cells, or T cells. A composition for combined administration for the prevention or treatment of cancer, comprising Ecklonia cava -derived fucoidan and an immune checkpoint inhibitor as active ingredients. The composition for combined administration according to claim 5, wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody, an anti-PD-1 antibody or an anti-PD-L1 antibody. The method of claim 5, wherein the cancer is bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, squamous cell cancer, epithelial cell cancer, skin cancer, leukemia, acute lymphatic cancer leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma, Burkett's lymphoma, acute and chronic myelogenous leukemia, promyelocytic leukemia, fibrosarcoma, rhabdomyosarcoma, melanoma, characterized by at least one selected from the group consisting of seminoma, teratocarcinoma, neuroblastoma, glioma, astrocytoma, neuroblastoma, schwannoma, osteosarcoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer and teratocarcinoma A composition for concomitant administration. The composition for combined administration according to claim 5, wherein the cancer is metastatic cancer. The composition for combined administration according to claim 5, wherein the Ecklonia cava -derived fucoidan is for nasal administration, and the immune checkpoint inhibitor is for intravenous injection.

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