Compositions for the activation of cytolytic T cells (CTL)

Abstract

Provided is a composition for activating cytotoxic T cells (CTLs) containing a spirulina extract. A composition for proliferating cytotoxic T cells (CTLs) containing a spirulina extract. In addition, a pharmaceutical composition for activating CTLs containing the composition for activating CTLs.

Description

Technical Field

[0001] This invention relates to a CTL activation composition containing spirulina extract for promoting the proliferation of cytotoxic T cells (CTLs), and a CTL activation pharmaceutical composition containing the CTL activation composition. Background Technology

[0002] Spirulina ( It is a genus of cyanobacteria rich in protein, carbohydrates, various vitamins, minerals, and plant pigments.

[0003] It has been reported that oral administration of spirulina (spirulina extract) can induce the activation of natural killer (NK) cells (e.g., non-patent literature 1).

[0004] In addition, various immunotherapies have been extensively researched in recent years. For example, research on cancer treatment using immunotherapy is also actively underway.

[0005] Normally, organisms possess immune surveillance mechanisms to eliminate cancer cells, thus suppressing cancer development. This so-called "cancer immunity" is handled by various immune-active cells. Besides NK cells, cytotoxic T cells (CTLs) are also immune-active cells that attack cancer cells. CTLs recognize and attack cancer cells by recognizing and specifically expressing various molecules within them, thereby contributing to cancer immunity.

[0006] Existing technical documents

[0007] Non-patent literature

[0008] Non-patent literature 1: Hirahashi T, et al. "Activation of the human innateimmune system by spirulina: Augmentation of interferon gamma production andNK cytotoxicity by oral administration of spirulina." International Immunopharmacology 2 (2002) 423-434. Summary of the Invention

[0009] The problem the invention aims to solve

[0010] However, it was previously unknown that spirulina extract, as a spirulina extract, could activate cytotoxic T cells (CTLs) that specifically attack tumors and cause tumor shrinkage in a CTL-dependent manner.

[0011] The purpose of this invention is to provide a composition for activating cytotoxic T cells (CTLs) containing spirulina extract.

[0012] Solution for solving the problem

[0013] The inventors conducted repeated studies on mice given CTL-sensitive tumors and spirulina extract. The results showed that CTLs that specifically attack tumors are spontaneously activated by spirulina extract, and tumors shrink in dependence on CTLs, thus completing the present invention.

[0014] That is, the present invention includes the following methods.

[0015] [1] A composition for activating cytotoxic T cells (CTLs) to promote the proliferation of cytotoxic T cells (CTLs), comprising spirulina extract.

[0016] [2] The CTL activation composition described in [1] above is for oral use.

[0017] [3] A pharmaceutical composition for CTL activation, comprising the CTL activation composition described in [1] or [2] above.

[0018] [4] The CTL activation pharmaceutical composition described in [3] above is used for the treatment of any cancer among malignant melanoma, non-small cell lung cancer, Hodgkin lymphoma, head and neck cancer, gastric cancer, kidney cancer, and bladder cancer.

[0019] [5] The combination of the pharmaceutical composition for CTL activation described in [3] or [4] above with an immune checkpoint inhibitor.

[0020] [6] A treatment method for any cancer including malignant melanoma (melanoma), non-small cell lung cancer, Hodgkin lymphoma, head and neck cancer, gastric cancer, renal cancer, and bladder cancer, said method using the aforementioned [3] or [4] pharmaceutical composition for CTL activation and immune checkpoint inhibitors.

[0021] The effects of the invention

[0022] According to the present invention, a composition for activating cytotoxic T cells (CTLs) containing spirulina extract can be provided. Attached Figure Description

[0023] Figure 1 A schematic diagram illustrating the experimental procedure performed in Example 1.

[0024] Figure 2 The figure illustrates the increase in MO5 tumor cells when administered orally with H2O and spirulina extract in Example 1.

[0025] Figure 3 The figure shows the increase in EG7 tumor cells when administered orally with H2O and spirulina extract in Example 2.

[0026] Figure 4 To demonstrate oral administration Figure 3 The results of spirulina extract in mice show tumor inhibition in mice with particularly significant tumor shrinkage.

[0027] Figure 5 The figure illustrates the increase in EG7 tumor cells when administered orally with H2O, spirulina extract, or spirulina extract plus anti-CD8β antibody in Example 3.

[0028] Figure 6 A schematic diagram illustrating the experimental procedure performed in Example 4.

[0029] Figure 7 A schematic diagram illustrating the co-cultivation steps performed in Example 4.

[0030] Figure 8 A graph showing the results of the proportion of cells that divided in CFSE-labeled OT-1 cells during Example 4.

[0031] Figure 9 The figure illustrates the increase in MO5 tumor cells when administered orally with H2O and spirulina extract in Example 5.

[0032] Figure 10 The figure shows the results of flow cytometry analysis of CD8+ T cells, CD4+ T cells, and NK cells performed in Example 5.

[0033] Figure 11 The figure shows the increase in MO5 tumor cells when administered orally with H2O + isotype control Ab or spirulina extract + anti-PD-L1 Ab in Example 6. Detailed Implementation

[0034] The composition for activating cytotoxic T cells (CTLs) of the present invention will be described in detail below. However, the following description of the constituent elements is an example of one embodiment of the present invention and is not limited to these contents.

[0035] (Composition for activating cytotoxic T cells (CTLs))

[0036] This invention relates to a composition for promoting the activation of cytotoxic T cells (CTLs), which is used to proliferate cytotoxic T cells (CTLs) that specifically target antigens of animals, including humans.

[0037] The CTL activation composition of the present invention contains spirulina extract.

[0038] As shown in the following examples, the CTL activation composition of the present invention can activate CTLs and inhibit tumor proliferation in tumors that are insensitive to NK cells but sensitive to CTLs without the addition of specific antigens.

[0039] Spirulina extract

[0040] Spirulina extract is an extract obtained by extracting spirulina with solvents such as water and ethanol, or an extract obtained by concentrating / drying the extract. There is no particular limitation on the extract used in manufacturing the spirulina extract, within the range that achieves the effects of this invention; for example, hot water can be used. In this invention, it is preferable to use an extract obtained by hot water extraction of spirulina, or an extract obtained by concentrating / drying the extract.

[0041] Spirulina is a fine spirulina belonging to the genus Spirulina in the family Oscillatiaceae of the order Nostocales of the class Cyanobacteria. It is rich in protein, carbohydrates, various vitamins, minerals, and plant pigments.

[0042] Examples of spirulina used in this invention include Spirulina platensis (Pentaphyllum). Spirulina macrophylla ( ) Spirulina gigantea () ), conjoined spirulina ( Spirulina macrophylla Spirulina salsa ( Spirulina (the first) Spirulina latans () ), Spirulina kuta Spirulina latans () ), and in particular, Spirulina platensis is preferred because it can be easily obtained through artificial cultivation. Spirulina gigantea () ), conjoined spirulina ( ), etc. It should be noted that spirulina ( Also known as arthrospira ( ).

[0043] There are no particular limitations on the method for obtaining spirulina extract; commonly known methods can be used. Examples include the methods for producing extracts described in Non-Patent Document 1 and Japanese Patent Application Publication No. 8-9940. More specifically, the following methods can be listed.

[0044] <<Manufacturing Method of Spirulina Extract>>

[0045] Spirulina extract can be manufactured as follows: Spirulina algae are extracted with hot water at a temperature exceeding 100°C, the pH of the extract is adjusted to a specific acidic condition, and then the insoluble fraction is removed to obtain the spirulina extract.

[0046] When using spirulina extract in liquid form, the spirulina extract obtained as described above can be used directly. However, the spirulina extract can also be concentrated / dried to form a powder before use. The spirulina extract in this invention can be an extract prepared by concentrating or drying the spirulina extract.

[0047] Spirulina can be commercially available or cultivated at home. Alternatively, live spirulina can be used, or it can be dried.

[0048] The cultivation method for spirulina can follow the conventional methods used for culturing cyanobacteria. For example, spirulina can be cultivated outdoors under alkaline conditions to promote its proliferation.

[0049] The cultured spirulina (hereinafter also referred to as spirulina algae) can be used directly, or the cultured spirulina can be recovered using filter cloth or filter paper, washed with water, and then suspended in water to make a suspension. The culture medium or suspension can then be concentrated to produce wet algae, which can be dried using freeze-drying, spray drying, or other methods to produce dried algae, which can then be powdered.

[0050] The spirulina cells used in hot water extraction can be any of the following: wet cells, freeze-dried cells, spray-dried cells, or broken cells. For example, when obtaining broken cells, conventional methods such as high-pressure extrusion methods like the French press can be used to break the cells.

[0051] The hot water extraction process will now be described. For example, the processed spirulina algae, as described above, are pre-suspended in an extraction solvent, such as distilled water, within a pressurized container. There are no particular limitations on the suspension concentration; however, considering extraction efficiency and recovery costs, a concentration of 1-20% by mass relative to the solvent is ideal. Tap water can be used as the extraction solvent, but distilled water is preferred if the extract is intended for use as a food ingredient. The extraction temperature is above 100°C, preferably 105°C to 140°C, and more preferably 110-130°C. The extraction pressure is preferably 1.0 to 2.5 atmospheres. Furthermore, stirring may or may not be performed during extraction; stirring is preferable for thermal efficiency. Longer extraction times result in greater extraction yields; considering efficiency, an extraction time of 0.5 to 4 hours is typically sufficient.

[0052] Then, from the algal residue suspension (pH = 6.8–7.0) after the extraction operation as described above, algal residue and aggregated proteins formed due to heat denaturation are removed. For example, the suspension can be centrifuged or filtered to obtain a supernatant. However, since the supernatant contains a large amount of dissolved protein, it is preferable to further remove the dissolved protein. To further remove the dissolved protein, acid can be added to the suspension to make the pH of the extract below the isoelectric point of the protein. This causes the protein to aggregate, and the aggregated protein is separated by centrifugation or filtration to obtain the spirulina extract. Alternatively, without removing the algal residue, the pH of the algal residue suspension after the extraction operation is adjusted to below the isoelectric point of the protein as described above, and the algal residue and aggregated protein are separated by centrifugation or filtration in the same manner. By removing the aggregated protein, a spirulina extract containing polysaccharides is obtained in high yield.

[0053] Acidic conditions below the isoelectric point of the protein are preferably below pH 4.5. Protein aggregation and precipitation reach their maximum extent at pH 3.75 to 4.25, so pH 4.0 is more preferred.

[0054] Sulfuric acid or hydrochloric acid can be added to adjust the pH. However, if the actual operation process is considered and the product is used as a food ingredient, organic acids such as citric acid and malic acid are preferred over inorganic acids.

[0055] <Promotion of cytotoxic T cell (CTL) activation>

[0056] As shown in the examples below, spirulina extract can induce CTL proliferation in NK cell-insensitive but CTL-sensitive tumors without the injection of exogenous tumor-associated antigens (TAAs). Furthermore, as shown in the examples below, spirulina extract can inhibit tumor proliferation in NK cell-insensitive but CTL-sensitive tumors. When the tumor releases substantially sufficient TAA, spirulina extract can affect CTL activation and induce tumor shrinkage.

[0057] The CTL activation composition of the present invention can be effectively used in pharmaceutical compositions targeting CTL activation.

[0058] <Morphology of the Composition for CTL Activation>

[0059] The CTL activation composition of the present invention can be used in CTL activation pharmaceutical compositions used to promote CTL proliferation.

[0060] The form of the CTL activating composition of the present invention and the CTL activating pharmaceutical composition containing the CTL activating composition (hereinafter collectively referred to as "the CTL activating composition of the present invention") is not particularly limited and can be suitably selected according to the purpose, for example, it can be in solid, liquid, gel or the like. In addition, it can be, for example, tablets (including chewable tablets, etc.), capsules, granules or the like.

[0061] The CTL-activating pharmaceutical composition containing the CTL-activating composition of the present invention can contain various pharmaceutically acceptable conventional formulation ingredients such as carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrants, solubilizers, dissolving agents, isotonic agents, etc. Furthermore, the CTL-activating pharmaceutical composition of the present invention can be for oral or non-oral use, more preferably for oral use. For oral use, it can be in commonly used dosage forms, such as tablets, powders, granules, capsules, syrups, suspensions, etc. For non-oral use, commonly used administration methods can be listed, such as intranasal administration of solutions, emulsions, suspensions, etc., in the form of injections (subcutaneous injection, intravenous injection, intramuscular injection, etc.) or sprays.

[0062] When the composition for CTL activation of the present invention is in the form of, for example, tablets (also known as lozenges), spirulina extract can be appropriately combined with additives such as excipients, binders, disintegrants, lubricants, preservatives, antioxidants, isotonic agents, buffers, coating agents, flavoring agents, solubilizers, base agents, dispersants, stabilizers, and colorants, and prepared according to conventional methods.

[0063] Examples of excipients include starch and its derivatives (dextrin, carboxymethyl starch, etc.), cellulose and its derivatives (methylcellulose, hydroxypropyl methylcellulose, etc.), sugars (lactose, white sugar, glucose, trehalose, etc.), citric acid or its salts, malic acid or its salts, and ethylenediaminetetraacetic acid or its salts.

[0064] Examples of binding agents include starch and its derivatives (α-starch, dextrin, etc.), cellulose and its derivatives (ethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, etc.), gum arabic, astragalus gum, gelatin, sugars (glucose, white sugar, etc.), and ethanol.

[0065] Examples of disintegrants include starch and its derivatives (carboxymethyl starch, hydroxypropyl starch, etc.), cellulose and its derivatives (sodium carboxymethyl cellulose, crystalline cellulose, hydroxypropyl methyl cellulose, etc.), carbonates (calcium carbonate, calcium bicarbonate, etc.), astragalus gum, gelatin, agar, etc.

[0066] Examples of lubricants include stearic acid, calcium stearate, magnesium stearate, talc, titanium dioxide, calcium hydrogen phosphate, dried aluminum hydroxide gel, sucrose fatty acid esters, and edible oils.

[0067] Examples of preservatives include parabens, sulfites (sodium sulfite, sodium metabisulfite, etc.), phosphates (sodium phosphate, calcium polyphosphate, sodium polyphosphate, sodium metaphosphate, etc.), dehydroacetic acid, sodium dehydroacetate, sorbitol glyceride, and sugars.

[0068] As antioxidants, examples include sulfites (sodium sulfite, sodium bisulfite, etc.), isoascorbic acid, L-ascorbic acid, cysteine, thioglycerol, butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate, ascorbate palmitate, dl-α-tocopherol, etc.

[0069] Examples of isotonic agents include sodium chloride, sodium nitrate, potassium nitrate, dextrin, glycerol, and glucose.

[0070] Examples of buffering agents include sodium carbonate, hydrochloric acid, boric acid, and phosphates (such as sodium hydrogen phosphate).

[0071] Examples of coating agents include cellulose derivatives (hydroxypropyl cellulose, cellulose acetate phthalate, hydroxypropyl methyl cellulose phthalate, etc.), shellac, polyvinylpyrrolidone, polyvinylpyridines (poly-2-vinylpyridine, poly-2-vinyl-5-ethylpyridine, etc.), polyvinyl acetyl diethylaminoacetate, polyvinyl alcohol phthalate, methacrylate-methacrylic acid copolymer, etc.

[0072] Examples of flavoring agents include sugars (glucose, white sugar, lactose, etc.), sodium saccharin, and sugar alcohols.

[0073] Examples of solvent aids include ethylenediamine, nicotinamide, sodium saccharin, citric acid, citrates, sodium benzoate, polyvinylpyrrolidone, polysorbates, sorbitan fatty acid esters, glycerin, polypropylene glycol, and benzyl alcohol.

[0074] Examples of base agents include fats (such as lard), vegetable oils (such as olive oil and sesame oil), animal oils, lanolin, petrolatum, paraffin wax, resins, bentonite, glycerin, and glycol oils.

[0075] Examples of dispersants include gum arabic, astragalus gum, cellulose derivatives (such as methylcellulose), sodium alginate, polysorbates, and sorbitan fatty acid esters.

[0076] Examples of stabilizers include sulfites (such as sodium bisulfite), nitrogen, and carbon dioxide.

[0077] The content of spirulina extract in the CTL activation composition of the present invention varies depending on the type, composition, and form of the pharmaceutical or food product, and can be appropriately selected. For example, in liquid pharmaceuticals or beverages, the content of spirulina extract, calculated based on the dry weight of the spirulina extract, is preferably 0.1% by mass or more, more preferably 1% by mass or more. Furthermore, the content of spirulina extract in liquid pharmaceuticals or beverages, calculated based on the dry weight of the spirulina extract, is preferably 20% by mass or less, more preferably 10% by mass or less.

[0078] Furthermore, the content of spirulina extract in pharmaceuticals and food products, calculated based on the dry weight of the spirulina extract, is preferably 0.1% by mass or more, more preferably 1% by mass or more. Additionally, the content of spirulina extract in pharmaceuticals and food products, calculated based on the dry weight of the spirulina extract, is preferably 30% by mass or less, more preferably 20% by mass or less.

[0079] Furthermore, especially when the composition for CTL activation of the present invention is in the form of a tablet, for example, the content of spirulina extract in the tablet, calculated by the dry weight of the spirulina extract, is preferably 20% by mass or more, more preferably 50% by mass or more in its total mass. Additionally, the content of spirulina extract in the tablet, calculated by the dry weight of the spirulina extract, is preferably 95% by mass or less, more preferably 80% by mass or less in its total mass.

[0080] In this invention, the intake of spirulina extract is not particularly limited and can be appropriately selected according to the type and ingredients of the medicine. For example, for an adult, the daily intake is preferably 0.5g or more, more preferably 1g or more, and preferably 6g or less, more preferably 4g or less, based on the dry weight of the spirulina extract.

[0081] <Pharmaceutical Compositions for CTL Activation>

[0082] The CTL activating composition of the present invention is effective against a variety of diseases requiring CTL activity. For example, it can be used as a tumor therapeutic agent. In particular, when the tumor is NK cell insensitive and CTL sensitive, it can inhibit tumor proliferation and can be effectively used as a cancer preventive or therapeutic agent.

[0083] Therefore, the CTL activation composition of the present invention is an effective anticancer treatment for tumors that can shrink in dependence on CTLs, as described below.

[0084] Types of cancer that can be targeted for treatment include, for example, malignant melanoma, non-small cell lung cancer, Hodgkin's lymphoma, head and neck cancer, stomach cancer, kidney cancer, and bladder cancer.

[0085] <Combinations with Immune Checkpoint Inhibitors>

[0086] The CTL activation composition of the present invention, or the CTL activation pharmaceutical composition containing the CTL activation composition, can be further combined with immune checkpoint inhibitors to prepare a composition.

[0087] As an immune checkpoint inhibitor, there are no particular limitations as long as it can exert the effects of the present invention; examples include PD-1 pathway inhibitors.

[0088] PD-1 pathway inhibitors are drugs that inhibit the PD-1 / PD-1 ligand pathway. As a PD-1 pathway inhibitor, there is no particular limitation as long as the drug inhibits the PD-1 / PD-1 ligand pathway; examples include anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-PD-L2 antibodies.

[0089] By using PD-1 pathway inhibitors, the binding process of PD-1 to PD-L1 (PD-1 ligand) and PD-L2 (PD-2 ligand) can be inhibited, improving the suppressed state of immune cell activity. This exerts its anti-cancer effect and inhibits tumor proliferation by activating autoimmunity.

[0090] Examples of anti-PD-1 antibodies include nivolumab (Opdivo (registered trademark)), REGN-2810, pembrolizumab (Keytruda (registered trademark)), PDR-001, BGB-A317, AMP-514 (MEDI0680), BCD-100, IBI-308, JS-001, PF-06801591, TSR-042, etc.

[0091] Examples of anti-PD-L1 antibodies include atezolizumab (RG7446, MPDL3280A), avelumumab (PF-06834635, MSB0010718C), durvalumab (MEDI4736), BMS-936559, CA-170, LY-3300054, and FAZ053.

[0092] Spirulina extract, as shown in the examples below, can be used in combination with anti-PD-L1 antibodies. In experiments using it in combination with anti-PD-L1 antibodies, it inhibited tumor proliferation in tumors that were insensitive to NK cells but sensitive to CTLs. Therefore, anti-PD-L1 antibodies and other PD-1 pathway inhibitors can be used in combination with spirulina extract for the purpose of inhibiting tumor proliferation in CTL-sensitive tumors.

[0093] Example

[0094] The present invention is further described in detail below with reference to specific embodiments, but the scope of the present invention is not limited to these embodiments.

[0095] (Material)

[0096] Spirulina extract

[0097] Spirulina platensis was cultured in an outdoor culture tank under alkaline conditions (pH 11). Proliferation. Then, 50g of the propagated Spirulina platensis powder obtained by spray drying was suspended in 500mL of distilled water in an autoclave, and the pressure was adjusted to extract at 120℃ for 1 hour.

[0098] The extract was adjusted to pH 4.0 with citric acid. Algal residues and proteins (insoluble fractions) were removed by centrifugation to obtain the Spirulina extract, which is used as a hot water extract of Spirulina.

[0099] For mice, 200 μL of spirulina extract was administered each time (relative to 96 mg of spirulina powder).

[0100] <Low endotoxin ovalbumin (OVA)>

[0101] The OVA is manufactured by Fujifilm and Kojun Pharmaceutical Co., Ltd.

[0102] <Durbeco-modified PBS (D-PBS)>

[0103] The Dulbec-modified PBS (D-PBS) used in the matrix solution or for injection into mice is of the endotoxin-free grade (Merck Millipore).

[0104] <Anti-PD-L1 antibody (Ab) (clone: ​​10F.9G2)>

[0105] Purchased from Bio X Cell.

[0106] <Rat IgG2b isotype control antibody (Ab) (LTF-2)>

[0107] Purchased from Bio X Cell. LTF-2 is useful as an isotype control against rat IgG2b antibodies.

[0108] <Pam2CSK4>

[0109] 2,3-Bis(palmitoyloxy)propyl-Cys-Ser-Lys-Lys-Lys-Lys (Pam2CSK4) was synthesized by Biologica Co., Ltd.

[0110] <7AAD>

[0111] 7-Aminoactinomycin D (7AAD, cell survival solution) used for staining dead cells was purchased from BD Biosciences.

[0112] <Mouse>

[0113] Wild-type C57BL / 6 mice were purchased from CLEA Japan, Inc. Mice were maintained under specific pathogen-free conditions using mice aged 7–11 weeks.

[0114] OT-1 mice are mice that are forced to express T-cell receptors that specifically recognize OVA antigens.

[0115] <B16 melanoma cells expressing OVA (MO5)>

[0116] B16 melanoma cells (MO5) expressing OVA were cultured in RPMI 1640 (Thermo Fisher Scientific) supplemented with 10% heat-inactivated fetal bovine serum (FBS, purchased from GE Healthcare Life Sciences), 100 IU of penicillin / 100 μg / mL of streptomycin (Thermo Fisher Scientific), and 100 μg / mL of G418 (Roche).

[0117] <EL4 lymphoma cells expressing OVA (EG7)>

[0118] EL4 lymphoma cells (EG7) expressing OVA (ATCC CRL-2113TM) were purchased from ATCC.

[0119] EL4 lymphoma cells (EG7) expressing OVA were cultured in RPMI 1640 containing 10% heat-inactivated FBS, 10 mM HEPES (Thermo Fisher Scientific), 1 mM sodium pyruvate (Thermo Fisher Scientific), 55 μM 2-mercaptoethanol (Thermo Fisher Scientific), 100 IU penicillin / 100 μg / mL streptomycin, and 500 μg / mL G418.

[0120] (Example 1)

[0121] For C57BL / 6 mice, MO5 tumor cells (1×10⁻⁶) were subcutaneously injected into the shaved back of each mouse. 6 (1 cell / 200μL D-PBS).

[0122] Tumor size was measured using calipers and expressed using the following formula.

[0123] Tumor volume (mm) 3 = 0.52 × (major axis) × (minor axis) 2

[0124] When the tumor volume reaches approximately 200 mm 3 At that time, 200 μL of spirulina extract or H2O was administered orally via a gastric tube.

[0125] H2O or spirulina extract was administered orally to mice every 2 or 3 days from the day following tumor inoculation (transplantation).

[0126] For the group that initially received OVA stimulation, 100 μg of OVA protein was injected subcutaneously around the tumor on days 1 and 8 after tumor inoculation.

[0127] The experimental protocol is shown below. Figure 1 .

[0128] The results are shown in Figure 2 .

[0129] Figure 2 This graph illustrates the increase in MO5 tumors induced by oral administration of H2O or spirulina extract. Regarding the data shown in the graph, the mean values ​​for each administration group are presented for H2O administration, and the results for each mouse are presented for spirulina extract administration.

[0130] MO5 tumors are tumors that are sensitive to both NK cells and CTLs.

[0131] like Figure 2As shown, it can be confirmed that regardless of whether OVA was administered subcutaneously, mice given spirulina extract showed promoted tumor shrinkage.

[0132] (Example 2)

[0133] For C57BL / 6 mice, EG7 lymphoma cells (2 × 10⁻⁶) were subcutaneously injected into the shaved back of each mouse. 6 (1 cell / 200μL D-PBS).

[0134] Tumor size was measured using calipers and expressed using the following formula.

[0135] Tumor volume (mm) 3 = 0.52 × (major axis) × (minor axis) 2

[0136] When the tumor volume reaches approximately 200 mm 3 At that time, 200 μL of spirulina extract or H2O was administered orally via a gastric tube.

[0137] Mice were orally administered H2O or spirulina extract every 2 or 3 days starting from the day after tumor inoculation.

[0138] The results are shown in Figures 3-4 .

[0139] Figure 3 The growth of tumors in mice orally administered with H2O or spirulina extract is shown. Data are the mean for each group (n=5). Figure 4 The results show tumor suppression in mice that were orally administered spirulina extract, with particularly significant tumor shrinkage.

[0140] EG7 tumors are NK cell insensitive and CTL sensitive tumors.

[0141] like Figures 3-4 As shown, it can be confirmed that mice with EG7 tumors that were given spirulina extract exhibited tumor shrinkage that was promoted.

[0142] (Example 3)

[0143] The following experiment was conducted: In mouse experiments conducted under the same conditions as in Example 2, mice that had been orally administered spirulina extract were further injected intraperitoneally with anti-CD8β antibody.

[0144] To deplete CD8β cells, mice were intraperitoneally (ip) injected with 15 μL of hybridoma ascites containing an anti-CD8β monoclonal antibody (clone: ​​H35.17-2) the day before administration of spirulina extract. The ascites typically contained 10–15 mg / mL of IgG.

[0145] The results are shown in Figure 5 .

[0146] In mice given spirulina extract, tumor growth was inhibited in response to the extract. However, in mice given both spirulina extract and anti-CD8β antibody, tumor growth resumed due to CD8β cell depletion. This confirms that CTLs are associated with tumor growth inhibition in this experimental system.

[0147] In other words, it can be confirmed that in mice with EG7 tumors that were given spirulina extract, tumor growth was slowed down by the proliferation of CD8+CTL.

[0148] (Example 4)

[0149] For C57BL / 6 mice, each mouse was orally administered 200 μL of H2O or Spirulina extract three times over 7 days (on days 0, 2 and 4).

[0150] For the group initially receiving OVA stimulation, 100 μg of OVA protein was subcutaneously injected into the shaved back of the mice on day 1.

[0151] The experimental protocol is shown below. Figure 6 .

[0152] Then, spleen cells were isolated from the spleens of mice that had been orally administered H2O or spirulina extract by treatment with collagenase D (prepared by Roche Diagnostics KK).

[0153] While blocking with anti-CD16 / CD32 antibody (Ab), CD11c+ DC cells were isolated from spleen cells using CD11c-microbeads (Miltenyi Biotec).

[0154] OT-1 T cells were isolated from the spleen of OT-1 mice using CD8 microbeads (manufactured by Miltenyi Biotec).

[0155] The isolated OT-1 CD8+ T cells were labeled with 1 μM carboxyfluorescein diacetate succinimide ester (CFSE, Thermo Fisher Scientific) at 37°C for 10 minutes.

[0156] Inoculate 1×10⁻³ times into the wells of a 96-well plate with a U-shaped bottom. 5 CD11c+ DC cells, with or without 2.5 μg / mL OVA, were incubated at 37°C.

[0157] Four hours later, add 5×10 to each hole 4Equal amounts of CFSE-labeled OT-1 CD8+ T cells were cultured at 37°C for 62 hours.

[0158] The steps of co-cultivation are shown below. Figure 7 .

[0159] Cells were stained with anti-CD8α antibody, anti-CD3 antibody, and anti-TCR Vβ5.1 and 5.2 antibodies. Dead cells were excluded by 7AAD staining.

[0160] OT-1 proliferation was evaluated using CFSE attenuation obtained from FACS AriaII (manufactured by BD Biosciences) and Flowjo software (manufactured by Tree Star).

[0161] According to this experimental system, it can be confirmed whether T cells are activated by dendritic cells obtained from mice given spirulina extract by co-culturing dendritic cells (DCs) obtained from mice given spirulina extract with T cells obtained from other mice.

[0162] The results are shown in Figure 8 .

[0163] Figure 8 The figure shows the proportion of cells that have undergone division in CFSE-labeled OT-1 cells.

[0164] Figure 8 The data shown demonstrate the results of co-culturing OVA-restimulated T cells with CD11c+ DCs, with or without OVA.

[0165] Figure 8 The results shown are representative of Exp.1 and Exp.2 for two independent experimental systems.

[0166] Figure 8 The results of samples co-cultured with CD11c+DC stimulated with Pam2CSK4 are also shown in the comparative experiment as a positive control.

[0167] Depend on Figure 8 The results confirmed that splenic DCs in mice given spirulina extract could induce OVA-specific CTLs independently of exogenous OVA administration.

[0168] The experimental results were confirmed in vitro using a mixture of spleen CD11c+ DCs and OT-1 CD8+ T cells.

[0169] As shown in Exp.2, Spirulina treatment of mouse dendritic cells induced CD8+ T cell proliferation after OVA restimulation. This proliferation was independent of the initial OVA stimulation.

[0170] (Example 5)

[0171] As in Example 1, MO5 tumor cells (1 × 10⁻⁶) were subcutaneously injected into the shaved back of each C57BL / 6 mouse. 6 In an experimental system of 1 cell / 200 μL D-PBS, H2O or Spirulina extract was administered orally on days 6 and 8 post-tumor transplantation.

[0172] Mice were sacrificed on day 11 after tumor transplantation.

[0173] To analyze intratumoral immune cells, tumor tissue was minced and treated in 25°C Hanks balanced salt solution (Sigma-Aldrich) with 0.05 mg / mL collagenase I (Sigma-Aldrich), 0.05 mg / mL collagenase IV (Sigma-Aldrich), 0.025 mg / mL hyaluronidase (Sigma-Aldrich), and 0.01 mg / mL DNase I (Roche Diagnostics KK) for 15 minutes.

[0174] After enzyme treatment, the red blood cells were lysed with ACK buffer.

[0175] Tumor-infiltrating CD8+ T cells, CD4+ T cells, and NK cells were stained with antibodies and analyzed using FACS AriaII (BDBiosciences) and Flowjo software (Tree Star).

[0176] The results are shown in Figures 9-10 .

[0177] Figure 9 The results show an increase in tumor cells when H2O and spirulina extract are administered orally.

[0178] Figure 9 In the data, the H2O-treated group is shown as the mean (n=2), and the Spirulina extract-treated group is shown as individual points (n=3).

[0179] As mentioned above, Figure 10 The results of flow cytometry analysis of CD8+ T cells, CD4+ T cells, and NK cells are shown. Figure 10 The relationship between the proportions of CD8+ T cells, CD4+ T cells, and NK cells among CD45+ cells in the tumor and the tumor proliferation rate is shown.

[0180] like Figures 9-10As shown, in MO5 mice with tumor proliferation inhibition after oral administration of spirulina extract, the number of CD8+ T cells in the tumor increased, and the ratio of CD8+ T cells in the tumor increased significantly with tumor shrinkage. On the other hand, the number of CD4+ T cells and NK cells in the tumor was not correlated with tumor growth.

[0181] It can be confirmed that in mice that were given spirulina extract and showed observable antitumor effects, CD8+ T cells were induced within the tumor.

[0182] (Example 6)

[0183] As in Example 1, MO5 tumor cells (1 × 10⁻⁶) were subcutaneously injected into the shaved back of each C57BL / 6 mouse. 6 In an experimental system of 1 cell / 200 μL D-PBS, H2O or Spirulina extract was administered orally on days 6 and 8 post-tumor transplantation.

[0184] Six, eight, and ten days after tumor transplantation, mice were intraperitoneally injected with 200 μg of rat IgG2b isotype control antibody (Ab) (LTF-2) or anti-PD-L1 antibody, respectively.

[0185] The results are shown in Figure 11 .

[0186] The results show tumor growth in mice orally administered H2O + isotype control Ab or Spirulina extract + anti-PD-L1 Ab.

[0187] like Figure 11 As shown, the combination of anti-PD-L1 antibody and spirulina extract showed good results in inhibiting tumor growth in stages, compared with the combination of H2O and isotype control Ab (LTF-2) without anti-PD-L1 antibody.

[0188] This confirms that immune checkpoint inhibitors (especially PD-1 pathway inhibitors such as anti-PD-L1 antibodies) can be used in combination with spirulina extract for the purpose of inhibiting tumor proliferation in CTL-sensitive tumors.

[0189] As shown in the above embodiments, under the action of spirulina extract, cytotoxic T cells (CTLs) are spontaneously activated, and tumor shrinkage is dependent on CTLs.

[0190] That is, the CTL activation composition containing spirulina extract of the present invention can induce CTL proliferation and also inhibit tumor proliferation.

[0191] Therefore, the pharmaceutical composition containing the CTL activating composition of the present invention can be effectively used in CTL activating pharmaceutical compositions for the purpose of CTL activation.

Claims

1. Use of a CTL-activating pharmaceutical composition in the preparation of a medicament for treating Hodgkin's lymphoma, said pharmaceutical composition comprising a spirulina extract, said spirulina extract being obtained by processing Spirulina platensis (… Spirulina extract is prepared by hot water extraction at a temperature of 110-130°C, adjusting the pH of the extract to an acidic condition below pH 4.5, and then removing algal residues and proteins.

2. The use according to claim 1, wherein the pharmaceutical composition is for oral administration.

3. Use of a combination of a CTL-activating pharmaceutical composition and an immune checkpoint inhibitor in the preparation of a medicament for treating Hodgkin's lymphoma by a method comprising the combination of the CTL-activating pharmaceutical composition and the immune checkpoint inhibitor. The CTL activation pharmaceutical composition contains a spirulina extract, which is obtained by processing Spirulina platensis (…). Spirulina extract is prepared by hot water extraction at a temperature of 110-130°C, adjusting the pH of the extract to an acidic condition below pH 4.5, and then removing algal residues and proteins.

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