Macrophage activators

Abstract

The present invention provides a macrophage activator comprising Gc protein by a simple process. This macrophage activator comprises Gc protein to which no N-acetylgalactosamine is attached.

Description

Technical Field

[0001] The present invention relates to a macrophage activator.

Background Art

[0002] Macrophages are a type of white blood cell and are present in all tissues of the body, such as the skin, lungs, intestines, and brain. Macrophages are known to have important functions in both innate and acquired immunity. In innate immunity, together with neutrophils, they phagocytize foreign substances that have invaded the living body and digest them with proteolytic enzymes and lipases compartmentalized within the macrophages. In acquired immunity, they present molecules derived from phagocytized foreign substances as antigens on the cell surface and activate helper T cells. Furthermore, macrophages are known to be involved in the formation of various pathological conditions such as inflammatory diseases, arteriosclerosis, obesity, and cancer.

[0003] Gc protein is a protein present in plasma and is also called vitamin D-binding protein because it binds to vitamin D. Gc protein has the ability to activate macrophages, but conventionally, it has been considered that the sugar chain structure added after the translation of Gc protein is important for the activation of macrophages. Gc protein is expressed in a state in which an O-type sugar chain consisting of a trisaccharide in which sialic acid and galactose are bound to GalNAc (N-acetylgalactosamine) is bound to threonine (Thr) at position 418 or 420. It has been considered that macrophages cannot be activated when sialic acid or galactose is bound to GalNAc, or when GalNAc is not bound to threonine at position 418 or 420. Therefore, Patent Documents 1 and 2 disclose methods of allowing β-galactosidase or sialidase to act on Gc protein to remove sialic acid and galactose.

[0004] Recombinant expression of Gc protein in specific cells can produce Gc protein in which only GalNAc is bound to the threonine (Thr) at position 418 or 420 (Patent Document 3). However, the cell types that could be used were limited. Furthermore, the relationship between the glycosylation structure of Gc protein and its macrophage activation ability had not been sufficiently clarified. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Special Publication No. 6-510908 [Patent Document 2] Special Publication No. 11-511962 [Patent Document 3] International Publication No. WO2019 / 117295 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The present invention aims to provide a macrophage activator consisting of Gc protein through a simple process. [Means for solving the problem]

[0007] The inventors investigated the relationship between the glycosylation structure of Gc protein and its ability to activate macrophages. As a result, they found that N-acetylgalactosamine at the 418th or 420th Thr of Gc protein is not essential for macrophage activation, leading to the completion of the present invention.

[0008] In other words, the present invention relates to a macrophage activator comprising a Gc protein that does not have N-acetylgalactosamine attached.

[0009] Preferably, the Gc protein is a purified product derived from human, bovine, goat, or mouse serum or milk.

[0010] It is preferable that the amino acid without N-acetylgalactosamine attached is the threonine at position 418 or 420 in SEQ ID NO: 1.

[0011] Furthermore, the present invention relates to a pharmaceutical composition comprising the macrophage activator for anti-cancer, anti-infective, anti-autoimmune disease, anti-autism, anti-inflammatory disease, anti-brain / neurodegenerative disease, skin improvement, or heart disease treatment.

[0012] Furthermore, the present invention relates to a method for producing a macrophage activator, comprising the step of contacting Gc protein with N-acetylgalactosaminidase.

[0013] The above-mentioned manufacturing method preferably includes a step of contacting the Gc protein with neuraminidase before the step of contacting it with N-acetylgalactosaminidase. [Effects of the Invention]

[0014] The Gc protein contained in the macrophage activator of the present invention does not have N-acetylgalactosamine attached and can be produced by simple enzymatic treatment. [Brief explanation of the drawing]

[0015] [Figure 1] The results of the macrophage phagocytic activity test are shown. [Figure 2A] The results of the NO production test are shown. [Figure 2B] The results of the NO suppression test are shown. [Figure 3] The results of the TNF-α suppression test are shown. [Figure 4] The results of the M2 differentiation ability evaluation test are shown. [Modes for carrying out the invention]

[0016] <<Macrophage activator>> Gc protein is a protein present in body fluids such as mammalian plasma and milk, and is also referred to as vitamin D binding protein because it binds to vitamin D. Gc protein exists in body fluids such as plasma and milk in a form in which an O-glycan consisting of a trisaccharide in which sialic acid and galactose are bound to GalNAc (N-acetylgalactosamine) is bound to threonine (Thr) at position 418 or 420, and this form is said to have no ability to activate macrophages. On the other hand, it is known that a form in which only GalNAc is bound to threonine at position 418 or 420 has the ability to activate macrophages, and it is called GcMAF (Gc protein-derived macrophage activating factor). In contrast, the macrophage activator of the present invention is characterized by containing a Gc protein to which N-acetylgalactosamine is not added.

[0017] <Gc protein> The Gc protein is not particularly limited as long as it is derived from mammals, and examples include those derived from humans, cows, goats, and mice. However, a human-derived Gc protein is preferred because it does not cause an immune reaction when administered to humans.

[0018] Specific examples of the Gc protein include a polypeptide having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, or a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, inserted, substituted, and / or added in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing.

[0019] The sequence identity with the amino acid sequence shown in SEQ ID NO: 1 is preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and particularly preferably 99% or more.

[0020] In the amino acid sequence shown in Sequence ID No. 1, the number of deleted, inserted, substituted and / or added amino acids is preferably 68 or less, more preferably 45 or less, even more preferably 22 or less, even more preferably 9 or less, and particularly preferably 4, 3, or 2 or less.

[0021] The position of the amino acid to which N-acetylgalactosamine is not attached is not particularly limited, but it is preferably a threonine in the amino acid sequence of the Gc protein, and preferably the threonine at position 418 or 420 in SEQ ID NO: 1.

[0022] <Serum, milk> Gc protein can be obtained from bodily fluids such as the milk or serum of the aforementioned mammals. The milk or serum containing Gc protein can be used as is, or the Gc protein can be purified from the milk or serum before use.

[0023] The serum is not particularly limited as long as it is prepared from blood collected from a mammal. The serum can be prepared by conventional methods. Furthermore, in order to reduce the risk of immune response and infection that may arise from using serum from another person, serum prepared from the patient's own blood to be administered the macrophage activator of the present invention may be used. Alternatively, serum prepared from the blood of a healthy other person may also be used.

[0024] One method for purifying Gc protein from serum is to remove albumin and other components from the serum, adsorb and elute the Gc protein using a vitamin D-binding column, and then perform dialysis.

[0025] The milk is preferably colostrum, which is secreted only during a predetermined number of days after childbirth. When purifying Gc protein from milk, for example, it can be purified by reducing the water content and removing casein and fat.

[0026] <Genetically modified> Gc protein may be a protein expressed by genetic engineering. Methods for expressing Gc protein by genetic engineering include culturing host cells into which the gene encoding Gc protein has been introduced, or using a cell-free expression system containing the gene encoding Gc protein. The host cells are not particularly limited and include microorganisms such as Streptomyces, Rhodococcus, Escherichia, Bacillus, Pseudomonas, Brevibacterium, Streptococcus, Lactobacillus, Saccharomyces, and Kluiveromyces, as well as cells of higher eukaryotes. Examples of higher eukaryote cells include cells derived from humans, hamsters, chickens, and insects, and specific cell lines include CHO cells, HeLa cells, HEK293 cells, sf9 cells, and DT40 cells. The cell culture method may be suspension culture or adhesion culture, but suspension culture is preferred. It is also preferable to use serum-free medium to avoid contamination with impurities.

[0027] <Enzyme treatment> If N-acetylgalactosamine is attached to the Gc protein, the Gc protein according to the present invention can be obtained by removing the N-acetylgalactosamine. The method for removing N-acetylgalactosamine is not particularly limited, but it is preferably carried out by enzymatic treatment. Examples of enzymes that can be used include endo-α-N-acetylgalactosaminidase and exo-α-N-acetylgalactosaminidase.

[0028] Examples of endo-α-N-acetylgalactosaminidases include those derived from Escherichia coli, Bifidobacterium longum, Streptococcus pneumoniae, and bovine liver. A commercially available example is MERCK catalog #324716. Endo-α-N-acetylgalactosaminidases may be used alone or in combination of two or more types.

[0029] Furthermore, if sialic acid is bound to N-acetylgalactosamine, the cleavage effect of endo-α-N-acetylgalactosaminidase on N-acetylgalactosamine is reduced; therefore, it is preferable to cleave the sialic acid beforehand. Sialic acid cleavage can be performed by treating Gc protein with sialidase.

[0030] Examples of sialidases include those derived from Clostridium perfringenes, Streptococcus 6646K, Vibrio cholerae, and Arthrobacter ureafaciens. Commercially available examples include SIGMA-ALDRICH product numbers N2876, N2133, N2904, N3001, and N5631; Biochemical Biobusiness code number 120052; and BioLabs catalog numbers P0720L and P0720S. Sialidases may be used alone or in combination of two or more types.

[0031] The contact between Gc protein and endo-α-N-acetylgalactosaminidase or sialidase is preferably carried out using a sufficient amount of enzyme for a sufficient amount of time until substantially no further enzymatic reaction proceeds. The amount of endo-α-N-acetylgalactosaminidase used is preferably 1 to 500 mU, more preferably 100 to 200 mU, per 1 μg of Gc protein. The amount of sialidase used is preferably 1 to 500 mU, more preferably 10 to 200 mU, per 1 μg of Gc protein. In either case, the enzyme treatment temperature is preferably 35 to 39°C. The enzyme treatment time is preferably 60 to 200 minutes.

[0032] Enzyme treatment may optionally be carried out in the presence of a buffer solution. Examples of such buffer solutions include physiological saline and phosphate-buffered saline (PBS).

[0033] After enzyme treatment, the enzyme may be inactivated by heat treatment. The heat treatment conditions are not particularly limited as long as the enzyme can be inactivated, but for example, heating at a temperature of around 60°C for about 10 minutes is used. Alternatively, after enzyme treatment, the enzyme may be removed from the Gc protein by combining a vitamin D-binding column, gel filtration column, ultrafiltration, etc.

[0034] Enzyme treatment can also be carried out using enzymes immobilized on a solid phase (immobilized enzymes). Methods for immobilizing enzymes on a solid phase are known to those skilled in the art. For example, endo-α-N-acetylgalactosaminidase or sialidase can be immobilized on agarose beads using a coupling agent such as cyanide bromide. Enzyme reactions can be carried out by applying Gc protein to the solid phase on which the enzyme is immobilized. By using immobilized enzymes, the enzymes can be recovered after enzymatic treatment without being inactivated by heat treatment, and impurities (such as proteins including enzymes inactivated by heat treatment) can be removed.

[0035] The Gc protein obtained by the above method may be further freeze-dried to obtain a solid or powder form. Furthermore, the removal of N-acetylgalactosamine from the Gc protein can be confirmed by the presence or absence of binding to lectins. Examples of lectins include those derived from Wisteria floribunda and Helix pomatia. If the Gc protein does not bind to the lectin, it can be determined that N-acetylgalactosamine has been removed.

[0036] <Macrophage activation ability> Macrophage activation ability can be evaluated by measuring the phagocytic activity of macrophages. This is done by adding Gc protein to the macrophage culture medium, allowing the macrophages to phagocytose test substances such as fluorescent latex beads or zymozan, and then calculating the percentage of macrophages that phagocytose and the number of phagocytic particles of the test substance. Macrophage activation ability can be determined when the percentage of phagocytic macrophages and the number of phagocytic particles are higher than those of the control group. Examples of macrophage cell lines that can be used include RAW264.7, NR8383, and J774.1.

[0037] The macrophage activator of the present invention preferably does not increase NO production by macrophages. NO production capacity can be evaluated by adding the macrophage activator to macrophages and measuring the amount of NO produced after standing for about 24 hours. The amount of NO produced can be measured, for example, by the Griess method described in the examples.

[0038] The macrophage activator of the present invention preferably suppresses NO production by macrophages. The ability to suppress NO production can be evaluated by adding the macrophage activator and an NO production stimulant such as lipopolysaccharide (LPS) to macrophages and measuring the amount of NO produced after standing for about 24 hours. Suppressing NO production by macrophages means having an anti-inflammatory effect.

[0039] The macrophage activator of the present invention preferably does not increase TNF-α production by macrophages. TNF-α production capacity can be evaluated by adding the macrophage activator, an NO production stimulant such as lipopolysaccharide (LPS), and IFN-γ to macrophages, and measuring the amount of TNF-α produced after standing for approximately 24 hours. TNF-α production can be measured, for example, by the ELISA method described in the examples. Not increasing TNF-α production by macrophages implies an anti-inflammatory effect.

[0040] The macrophage activator of the present invention may have the ability to differentiate into M2 type macrophages. The ability to differentiate into M2 type macrophages can be evaluated by adding the macrophage activator to macrophages and measuring the expression level of a marker gene in the macrophages after they have been allowed to stand for about 24 hours. For example, ARG-1 can be used as the marker gene. The expression level of the marker gene can be evaluated, for example, by observing fixed cells with a fluorescence microscope as described in the examples. Generally, inflammatory M1 type and anti-inflammatory M2 type are known as differentiation types of macrophages. It is thought that the anti-inflammatory response can be promoted by administering the macrophage activator of the present invention.

[0041] <<Pharmaceutical Composition>> The pharmaceutical composition of the present invention is characterized by comprising the macrophage activator and being a pharmaceutical composition for anticancer, anti-infective, anti-autoimmune disease, anti-autism, anti-inflammatory disease, anti-brain / neurodegenerative disease, skin improvement, or heart disease treatment.

[0042] <Composition> The pharmaceutical composition may contain, in addition to the macrophage activator, a pharmaceutically acceptable carrier as appropriate. Examples of pharmaceutically acceptable carriers include diluents, stabilizers, preservatives, and buffers.

[0043] The form of the pharmaceutical composition is not particularly limited and includes injectable compositions, oral compositions, eye drop compositions, intravenous solutions, nasal drops, ear drops, suppositories, and enteral nutrition compositions. Among these, injectable compositions are preferred. Forms of injection include intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and intraperitoneal injection, with intramuscular injection being preferred. Forms of oral compositions include powders, granules, tablets (including sublingual tablets), capsules, pills, enteric-coated tablets, oral solutions (including suspensions, emulsions, syrups, etc.), and inhalants.

[0044] The dosage of the pharmaceutical composition varies depending on the patient's age, sex, weight, symptoms, and method of administration. However, as a typical example, the total amount of protein contained in the pharmaceutical composition is preferably 0.1 mg to 4.0 mg per kg of body weight per single dose, more preferably 0.2 mg to 2.0 mg, and even more preferably 0.3 mg to 1.3 mg.

[0045] When administering the pharmaceutical composition at the above-mentioned single dose, the administration interval and number of doses may be 1 to 2 times per week for a total of 12 to 24 doses. Alternatively, it may be administered twice a week during the initial period (e.g., 1 to 2 months), followed by once a week thereafter.

[0046] <Indications> The pharmaceutical composition can be suitably used for anti-cancer, anti-infective, anti-autoimmune disease, anti-autism, anti-inflammatory disease, anti-brain and neurodegenerative disease, skin improvement, and heart disease treatment.

[0047] Cancer includes carcinomas, sarcomas, and other malignant tumors, such as skin cancer, bronchial cancer, lung cancer, non-small cell lung cancer, breast cancer, ovarian cancer, tongue cancer, pharyngeal cancer, esophageal cancer, stomach cancer, small intestine cancer, colorectal cancer, rectal cancer, colon cancer, liver cancer, pancreatic cancer, kidney cancer, renal cell carcinoma, bladder cancer, prostate cancer, uterine cancer, cervical cancer, Wilms' tumor, malignant melanoma, meningioma, neuroblastoma, osteosarcoma, Kaposi's sarcoma, lymphoma, and leukemia. Furthermore, cancer also includes metastases of these malignant tumors.

[0048] Infectious diseases include, for example, viral infections and bacterial infections. Specifically, these include COVID-19, HIV infection, AIDS, as well as hepatitis B, hepatitis C, herpes, influenza, pneumonia, tuberculosis, and EB virus infection.

[0049] Autism is a behavioral disorder characterized by difficulties in forming social relationships with others and delays in language development.

[0050] Inflammatory diseases are diseases caused by inflammation, which is one of the body's defense responses to physical stimuli, chemical stimuli, and microbial infections. Furthermore, the pharmaceutical composition of the present invention is also effective against autoinflammatory diseases in which inflammatory responses occur spontaneously due to abnormalities in innate immunity, leading to organ damage.

[0051] Examples of brain and neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, spinocerebellar degeneration, amyotrophic lateral sclerosis, and Lewy body dementia.

[0052] Skin improvement includes skin whitening, suppression or improvement of pigmentation, exfoliation or promotion of keratin turnover, anti-aging, suppression or improvement of wrinkles, moisturizing, regeneration, and treatment or prevention of alopecia.

[0053] Heart diseases include congestive heart failure, arrhythmias, angina pectoris, and myocardial infarction.

[0054] <<Composition for Quasi-Drug Use>> Macrophage activators can be formulated into quasi-drug compositions by adding auxiliary agents as needed. These quasi-drug compositions can take various forms, such as solutions, suspensions, syrups, granules, creams, pastes, and jellies, and can be molded into desired shapes as needed. All quasi-drug compositions can be manufactured by conventional methods.

[0055] The amount of Gc protein used in the quasi-drug composition is not particularly limited, but the same amount as the dosage used in the above-mentioned pharmaceutical composition, or an amount appropriately set based on the above, can be adopted.

[0056] <<Food Composition>> Macrophage activators can be made into food compositions by appropriately blending them with various additives commonly used in food and beverages, such as auxiliary agents, sweeteners, spices, seasonings, preservatives, antibacterial agents, and antioxidants, as needed. These food compositions can take various forms, such as solutions, suspensions, syrups, granules, creams, pastes, and jellies.

[0057] The amount of Gc protein used in the food composition is not particularly limited, but it can be the same as the dosage used in the pharmaceutical composition described above, or an amount appropriately determined based on the above.

[0058] Food compositions can include so-called health foods, health drinks, functional foods, nutritional functional foods, health supplements, nutritional supplements, foods for special dietary uses, foods for specified health uses, etc.

[0059] <<Method for producing macrophage activators>> The present invention relates to a method for producing a macrophage activator, characterized by comprising the step of contacting Gc protein with N-acetylgalactosaminidase. The N-acetylgalactosaminidase and contact conditions are as described above with respect to the macrophage activator.

[0060] The method for producing the macrophage activator of the present invention may include a step of contacting Gc protein with neuraminidase before the step of contacting with N-acetylgalactosaminidase. The neuraminidase and contact conditions are as described above with respect to the macrophage activator. [Examples]

[0061] (1) Purification of Gc protein (Manufacturing example 1) Serum components were obtained by centrifuging human blood collected in an SSTII (new serum separator) blood collection tube (13mm x 100mm) (#367528, BD) at 4°C, 3,000 rpm, and 10 min.

[0062] Subsequently, Gc protein was separated and purified from serum using the BioLogic LP Core system (#731-8300, BIO RAD) by passing it through the following three columns. Specifically, albumin and other components were removed from the serum using Blue Sepharose 6 Fast Flow (#17094801, GE Healthcare). Next, the recovered albumin-removed serum was passed through a vitamin D-binding column to adsorb the Gc protein onto the column. This was then eluted and recovered using guanidine hydrochloride [6M] (#077-02435, Wako).

[0063] The recovered solution was desalted by dialysis (4°C, 15h) in SPB [2mM, 5L] using a Snakeskin Dialysis Tubing, 3.5 K MWCO, 35mm ID (#88244, Thermo Fisher SCIENTIFIC). Subsequently, it was purified by passing it through a Bio-Scale Mini CHT Type II Cartridge (#7324332, BIO RAD).

[0064] The recovered solution was concentrated using a Vivaspin Turbo 4,10kDa,PES,25pc (#VS04T01,sartorius) (4℃, 7,500rpm, 10min) to obtain the final recovered product, Gc protein. The recovery of Gc protein was confirmed by antigen-antibody reaction with polyclonal rabbit anti-human Gc globulin (#A002102-2,Dako) and electrophoresis.

[0065] (2) Preparation of Gc protein (GcMMF) without N-acetylgalactosamine (Manufacturing Example 2) Two enzymes, neuraminidase (#N2876-6U,MeRCK) and endo-α-N-acetylgalactosaminidase (#324716,MERCK), were used to cleave the sugar chains attached to the 418th or 420th threonine residue of the Gc protein.

[0066] Sialic acid bound to GalNAc was removed by incubation (37°C, 180 min) with neuraminidase [200 mU] in Gc protein [10 μg]. Subsequently, the bond between the threonine residue and GalNAc was cleaved by incubation (37°C, 120 min) in the Gc protein from which the sialic acid had been cleaved with endo-α-N-acetylgalactosaminidase [1600 mU]. Antigen-antibody reactions using Wisteria floribunda Lectin, Biotin (#B-1355, Funakoshi) and Lectin from Helix pomatia (#L6512-1MG, SAJ) were used to confirm whether the enzymatic cleavage was complete. The Gc protein obtained in this way, without the addition of N-acetylgalactosamine, is called GcMMF.

[0067] Next, the enzyme used for cleavage was removed from the GcMMF. Enzyme removal was performed using low-pressure chromatography, similar to the Gc protein recovery process, by passing the solution through a vitamin D-binding column and a Bio-Scale Mini CHT Type II Cartridge (#7324332, BIO RAD). The resulting solution was then ultrafiltered (4°C, 7,500 rpm, 5-10 min) using a Vivaspin Turbo 4, 10kDa, PES, 25pc (#VS04T01, sartorius) to obtain the final product.

[0068] In this case, confirmation was also performed using antigen-antibody reactions with Wisteria floribunda lectin, biotin and lectin from Helix pomatia, polyclonal rabbit anti-human Gc-globulin. Furthermore, the gel after electrophoresis was stained with CBB Stain One Super (Ready To Use) (#11642-31, nacalai tesque) to confirm whether enzyme removal had been achieved.

[0069] (3) Macrophage phagocytic activity test (Example 1, Comparative Examples 1-4) In this study, all dilutions were performed using D-MEM(-)in 1% PS. [Day 1] RAW264.7 cells (#EC91062702-F0,KAC) that had been subcultured up to the second generation using D-MEM in 10%FBS, 1%PS were recovered using D-MEM(-) in 1%PS and 1 × 10⁻¹⁶ cells. 6 The cells were diluted to a concentration of cells / ml. 100 μl of each solution was seeded into 96-well black clear-bottom flat-bottom TC-treated plates (#353219, FALCON), and the plates were incubated (37°C, 5% CO2, 15 hours).

[0070] [Day 2] 10 μl of various activators (GcMMF [10 ng, 100 ng] (Example 1), Gc protein [10 ng] (Comparative Example 2), GcMAF [10 ng] (Comparative Example 3), LPS [100 ng] (Comparative Example 4)) were added and gently suspended by pipetting. For Gc protein, the purified protein from Production Example 1 was used. For GcMAF, the protein obtained by treating Gc protein purified from human serum with β-galactosidase and sialidase, as described in International Publication WO2013 / 038997, was used. Comparative Example 1 was a sample without added activators. Macrophages were then activated by incubation (37°C, 5% CO2, 180 min). After incubation, 10 μl of Latex-Beads IgG-FITC Solution was added and gently suspended by pipetting. After the procedure was completed, the macrophages were allowed to phagocytose the Latex-Beads by shielding them from light with aluminum foil and maintaining a warm temperature (37°C, 5% CO2, 24h).

[0071] [Day 3] All experimental procedures on the third day were performed under light-shielding conditions. After incubation, the culture medium was removed, 100 μl of primary methanol (#136-01837, Wako) was added, and it was allowed to stand for 10 minutes. The methanol was then removed, and the plate was air-dried for 5 minutes. After air-drying, the plate was washed twice with 100 μl of Dulbecco's phosphate-buffered saline (Ca, Mg-free, liquid) (#14249-95, nacalai tesque). The 96-well black plate was then placed on a fluorescence microscope (#BZ-X710, KEYENCE) and images were taken.

[0072] On the third day, during fluorescence microscopy observation and imaging, a total of three images were captured using a 20x lens: a phase contrast image, a fluorescence image (filter cube: BZ-X filter GFP), and a merged image. The imaging locations were predetermined to be 3-4 locations. However, since RAW264.7 cells are semi-adherent cells, they may detach or aggregate during experimental procedures (methanol fixation, etc.). In such cases, imaging was performed while excluding abnormal points from the predetermined imaging locations.

[0073] Of the captured phase-contrast, fluorescence, and merge images, the fluorescence images were used for data processing with a BZ-X Analyzer. First, Hybrid Cell Count was performed on the fluorescence images of the Control group, and the fluorescence count and fluorescence intensity (integrated) were measured. At this time, a threshold value for fluorescence labeling was set, and the data processing conditions were saved. Macro Cell Count was performed on all captured fluorescence images using these saved processing conditions.

[0074] The fluorescence intensity is shown in Figure 1. GcMMF from Example 1 showed macrophage activation ability equivalent to that of GcMAF from Comparative Example 3. From these results, it became clear that GalNAc glycans are not essential for macrophage activation by GcMAF.

[0075] (4) NO production / inhibition test (Examples 2-3, Comparative Examples 5-13) NO production in this experiment was measured using the Griess method. [Day 1] RAW264.7cell (#EC91062702-F0,KAC) that had been passed down through two generations using D-MEM in 10%FBS, 1%PS was recovered and processed into 0.5 × 10⁻¹⁴ cells. 6 The solution was diluted to a concentration of cells / ml. 1 ml of this solution was then seeded into 24-well cell culture plates (#VTC-P24, AS ONE) using D-MEM in 10% FBS and 1% PS, and the plates were incubated at 37°C, 5% CO2, for 15 hours.

[0076] [Day 2] (In the case of NO production tests) Each well was washed with 1 mL of Dulbecco's phosphate-buffered saline (Ca, Mg-free, liquid) (#14249-95, nacalai tesque), and 1 mL of D-MEM(-)in 1% PS was applied. Then, 100 μl of various activators (GcMMF [10 ng, 100 ng] (Example 2), Gc protein [10 ng] (Comparative Example 6), GcMAF [10 ng] (Comparative Example 7), LPS [100 ng] (Comparative Example 8)) were added, and the mixture was incubated at 37°C, 5% CO2, for 15 hours. Comparative Example 5 was prepared without the addition of any activators.

[0077] (In the case of NO suppression tests) Each well was washed with 1 mL of Dulbecco's phosphate-buffered saline (Ca, Mg-free, liquid) (#14249-95, nacalai tesque), and 1 mL of D-MEM(-)in 1% PS was applied. Then, 100 μl of various activators (GcMMF [10 ng, 100 ng] (Example 3), LPS [100 ng] (Comparative Example 10), Gc protein [10 ng] (Comparative Example 11), GcMAF [10 ng] (Comparative Example 12), glucosamine [10 mM] (Comparative Example 13)) were added, and the mixtures were incubated at 37°C, 5% CO2, for 15 hours. Comparative Example 9 was created by adding no activators. Here, LPS [100 ng] was simultaneously added to all groups except the control group (Comparative Example 9), and glucosamine was added to a final concentration of 10 mM as a positive control for NO suppression.

[0078] [Day 3] The culture supernatant from each well was collected in an Eppendorf tube and thoroughly mixed using a vortex mixer. Then, 100 μl of the culture supernatant collected from each Eppendorf tube was applied to a 96-well cell culture plate (#TR5003, True Line). 100 μl of Griess reagent was added to each well, and the plates were incubated under light-shielding conditions (rt, 5-10 min). The absorbance (550 nm) was then measured using a microplate reader (#infinite M200, TECAN). Sodium nitrite was used as the calibration curve.

[0079] The results of the NO production test are shown in Figure 2A, and the results of the NO inhibition test are shown in Figure 2B. The GcMMF of Examples 2-3 showed no NO production ability, similar to the GcMAF of Comparative Examples 7 and 12, and only showed NO inhibition ability.

[0080] (5) TNF-α inhibition test (Example 4, Comparative Examples 14-18) The tests were performed using the TNF alpha Mouse Uncoated ELISA Kit with plates (#88-7324-22, eBioscience). The following protocols were also prepared according to the kit's instructions.

[0081] [Day 1] RAW264.7cell (#EC91062702-F0,KAC) that had been passed down through two generations using D-MEM in 10%FBS, 1%PS was recovered and processed into 0.4 × 10⁻¹⁴ cells. 6 The solution was diluted to a concentration of cells / ml. 1 ml of this solution was then seeded into 24-well cell culture plates (#VTC-P24, AS ONE) using D-MEM in 10% FBS and 1% PS, and the plates were incubated at 37°C, 5% CO2, for 15 hours.

[0082] [Day 2] Each well was washed with 1 mL of Dulbecco's phosphate-buffered saline (Ca, Mg-free, liquid) (#14249-95, nacalai tesque), and 1 mL of D-MEM(-)in 1% PS was applied. Then, 100 μl of various activators (GcMMF [10 ng, 100 ng] (Example 4), curcumin [20 mM] in DMSO (Comparative Example 15), Gc protein [10 ng] (Comparative Example 16), GcMAF [10 ng, 100 ng] (Comparative Example 17), LPS [1 μg] + IFN-γ [10 ng] (Comparative Example 18)) were added, and the mixtures were incubated at 37°C, 5% CO2, for 24 hours. Comparative Example 14 was prepared without the addition of any activators. Here, LPS [1 μg] + IFN-γ [10 ng] was added to all sample groups, and 2 μl of curcumin [10 mM] in DMSO was added to each well as a positive control for TNF-α inhibition.

[0083] In addition to the above procedure, capture antibody-in-coating buffer was prepared and 100 μl was added to each of the provided 96-well plates. The plates were then sealed and incubated (4°C, shaking, 15 hours).

[0084] [Day 3] The solution in the 96-well plate was discarded, and the plates were washed three times with wash buffer [250 μl]. After washing, 200 μl of the prepared 1× ELISA / ELISPOT was added to each well, the plate was sealed, and it was incubated (rt, 60 min). (During incubation, the standard (1000 pg / ml) was prepared and the culture supernatant was collected after 24 hours of stimulation.)

[0085] After incubation, the 1×ELISA / ELISPOT was removed and the plates were washed once with wash buffer [250 μl]. Then, the Standard was diluted on the plate, and 100 μl of the culture supernatant after 24 hours of stimulation was added to each plate and incubated (4°C, 15 hours). (100 μl of 1×ELISA / ELISPOT was added to the blank.)

[0086] [Day 4] The solution in each well was removed, and the plates were washed five times with wash buffer [250 μl]. Then, 100 μl of TNF-α 1×DetAb was added to each well, the plates were sealed, and the plates were incubated (rt, 60 min). (Avidin-HRP was prepared during incubation.)

[0087] After incubation, the plates were washed three times with wash buffer [250 μl], then 100 μl of Avidin-HRP was added at a time, the plates were sealed, and incubated for 30 minutes. After incubation, the solution was discarded and the plates were washed seven times with wash buffer [250 μl]. Subsequently, 100 μl of 1×TMB was added at a time, and incubation was continued for another 15 minutes. (During this time, the stop solution was prepared.)

[0088] After incubation, 100 μl of STOP Solution was added to each plate, and the absorbance (450 nm (reference 570 nm)) was measured using a microplate reader (#infinite M200, TECAN).

[0089] The results of the TNF-α inhibition test are shown in Figure 3. GcMMF in Example 4 did not show TNF-α inhibitory ability, similar to GcMAF in Comparative Example 17.

[0090] (6) Evaluation of M2 differentiation ability (Example 5, Comparative Examples 19-22) [Day 1] RAW264.7 cells (#EC91062702-F0,KAC) that had been subcultured up to the second generation using D-MEM in 10%FBS, 1%PS were recovered using D-MEM (#044-29765,Wako) in 1%PS and 1 × 10 6 The cells were diluted to a concentration of cells / ml. 100 μl of each solution was seeded into 96-well black clear-bottom flat-bottom TC-treated plates (#353219, FALCON), and the plates were incubated (37°C, 5% CO2, 15 hours).

[0091] [Day 2] Various activators (GcMMF [10 ng, 100 ng] (Example 5), Gc protein [10 ng] (Comparative Example 20), GcMAF [10 ng] (Comparative Example 21), IL-4 [50 ng] + IL-13 [50 ng] (Comparative Example 22)) were added in 10 μl and gently suspended by pipetting. A sample without activator was designated as Comparative Example 19. The samples were then incubated at 37°C, 5% CO2, for 24 hours. (The positive control was IL-4 [50 ng] + IL-13 [50 ng] (Comparative Example 22)).

[0092] [Day 3] After incubation, the culture medium was removed and 100 μl of primary methanol (#136-01837, Wako) was added, and the mixture was allowed to stand for 10 minutes. The methanol was then removed, and the mixture was air-dried for 5 minutes. After air-drying, the mixture was washed twice with 100 μl of Dulbecco's phosphate-buffered saline (Ca, Mg-free, liquid) (#14249-95, nacalai tesque). Then, 150 μl of 0.1% BSA in PBS was added to each portion, and the mixture was incubated (rt, 15h).

[0093] [Day 4] The plates were washed three times with PBS [100 μl], and 30 μl of anti-h / m Arginase 1 Antibody was added to each plate, followed by incubation (rt, 30 min). Afterward, the plates were washed three more times with PBS [100 μl], and the fluorescence intensity (ex: 488 nm, em: 530 nm) was measured using a microplate reader.

[0094] The fluorescence intensity is shown in Figure 4. The GcMMF of Example 5 showed weak M2 type culture activity, similar to that of the GcMAF of Comparative Example 21.

Claims

1. Contains Gc protein without N-acetylgalactosamine added. The Gc protein is a macrophage activator having more than 90% sequence identity with the amino acid sequence shown in Sequence ID No.

1.

2. The macrophage activator according to claim 1, wherein the Gc protein is a purified product from human, bovine, goat, or mouse serum or milk.

3. The macrophage activator according to claim 1 or 2, wherein the amino acid not to which N-acetylgalactosamine is attached is threonine at position 418 or 420 in SEQ ID NO:

1.

4. A pharmaceutical composition comprising a macrophage activator according to any one of claims 1 to 3, for use in anticancer, anti-infective, anti-autoimmune disease, anti-autism, anti-inflammatory disease, anti-brain / neurodegenerative disease, skin improvement, or treatment of heart disease.

5. A step of contacting Gc protein with N-acetylgalactosaminidase, A method for producing a macrophage activator, comprising the step of contacting a Gc protein with neuraminidase before contacting it with N-acetylgalactosaminidase, The method for producing a macrophage activator, wherein the Gc protein has 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 1.

Images