Fluorescent protein, composition for identifying polarization of macrophage, and method for identifying polarization of macrophage

Abstract

Provided are a novel fluorescent protein, a composition for identifying polarization of macrophages, and a method for identifying polarization of macrophages.　A fluorescent protein is chemically modified with a sugar that binds to a C-type lectin receptor, the fluorescent protein being characterized in that: the sugar that binds to the C-type lectin receptor can bind to C-type lectin receptors expressed in macrophages; and in the fluorescent protein chemically modified with the sugar, the fluorescent protein and the sugar that binds to the C-type lectin receptor are bound directly or via a linker.

Description

Fluorescent proteins, compositions for identifying macrophage polarization, and methods for identifying macrophage polarization

[0001] The present invention relates to a fluorescent protein, a composition for identifying the polarization of macrophages, and a method for identifying the polarization of macrophages.

[0002] Macrophages are a type of white blood cell that are ubiquitously present in all organ tissues, exist in various forms, and are intrinsically involved in the innate immune system. Their primary function is to provide host defense against potential pathogens, such as microorganisms, cancer cells, and foreign bodies, and to suppress infection and wound healing. This process involves the phagocytic pathway, in which macrophages inject and directly remove harmful substances, present antigens on their cell surface, secrete cytokines, and stimulate the inflammatory and adaptive immune systems. When inflammation and the immune system are overstimulated, macrophages also perform other important functions, such as releasing anti-inflammatory cytokines to attenuate the immune response and induce an anti-inflammatory state. They also mediate tissue homeostasis. Macrophages exhibit high plasticity, undergoing polarization into different phenotypes to effectively function across a wide spectrum of functions. The two main classifications of polarized macrophage phenotypes are the classical activation state (M1) and the alternative activation state (M2). M1 macrophages exist in pro-inflammatory states while simultaneously maintaining anti-tumor immunity. M2 macrophages exhibit opposing functions by dominating in an anti-inflammatory state, providing functions such as wound healing and tissue repair, while simultaneously promoting tumor growth and proliferation. Undifferentiated macrophages, referred to as M0 macrophages, have the ability to polarize into either M1 or M2 macrophages. These distinct macrophage phenotypes are characterized by distinct metabolic pathways, cytokine secretion profiles, and diverse expression patterns of cell surface receptors.

[0003] Failure to maintain the necessary balance between M1 and M2 macrophage populations can lead to a variety of disease states, including autoimmune diseases, infectious diseases, and non-infectious diseases such as cancer. Rapid identification of macrophage polarization states is important for diagnosing disease states, preventing further infection, and administering therapeutic agents. Therefore, research and development of methods for identifying macrophage polarization is underway.

[0004] For example, Non-Patent Document 1 discloses that a fluorescently labeled anti-CD206 antibody was used to detect CD206, which is expressed at a high level in macrophages.

[0005] However, antibodies are large proteins with two binding sites per molecule. However, due to steric hindrance, antibodies lack multivalent function when binding to antigens on cell surfaces, such as receptors, rather than antigens in solution. This requires higher affinity. Furthermore, antibodies require careful handling during storage and manipulation. Furthermore, antibody production often involves the use of animals, presenting various challenges. Furthermore, because antibodies themselves lack spectroscopic characteristics, fluorescent labeling is required, making fluorescently labeled antibodies very expensive reagents. For these reasons, fluorescently labeled antibodies have been insufficient as biomarkers for identifying macrophage polarization.

[0006] Oncol Lett. 2019 Sep; 18(3):3218-3226 Biomolecules 2021, 11(2), 180FASEB J. 2012 Oct;26(10):4210-7Biochem Biophys Res Commun. 2012 Oct 19;427(2):280-4

[0007] The present invention has been made in view of the above-mentioned problems of the prior art, and aims to provide a novel fluorescent protein, a composition for identifying macrophage polarization, and a method for identifying macrophage polarization.

[0008] As a result of extensive research to achieve the above object, the present inventors discovered that the polarization of macrophages can be identified with high accuracy by using a fluorescent protein chemically modified with sugar that binds to a C-type lectin receptor (hereinafter simply referred to as a "fluorescent protein chemically modified with sugar"). This led to the completion of the present invention.

[0009] Technologies using fluorescent proteins often have additional functions added through genetic engineering. For example, known technologies for visualizing intracellular glycation using fluorescent proteins include a technology for visualizing glycated proteins using a fusion protein of a lectin and a fluorescent protein obtained through genetic engineering (Non-Patent Document 2), and a technology for visualizing glycation by inserting a glycosyltransferase recognition sequence into a fluorescent protein through genetic engineering (Non-Patent Documents 3 and 4). On the other hand, the fluorescent protein chemically modified with sugar used in the present invention is obtained through chemical techniques. The ability to identify macrophage polarization by using a fluorescent protein chemically modified with sugar has not been described or suggested in the prior art.

[0010] That is, the present invention includes the following inventions: [1] A fluorescent protein chemically modified with a sugar that binds to a C-type lectin receptor, wherein the sugar that binds to the C-type lectin receptor is capable of binding to a C-type lectin receptor expressed in macrophages, and the fluorescent protein chemically modified with the sugar is formed by binding the fluorescent protein to the sugar that binds to the C-type lectin receptor directly or via a linker. [2] The fluorescent protein according to [1], wherein the C-type lectin receptor is CD206, CD205, CD209, CD301, Mincle, Lox, CLEC, MGL, DC-IR, DC-SIGN, dectin, selectin, CD23, CD69, CD93, C161, or CD302. [3] The fluorescent protein according to [1], wherein the sugar that binds to the C-type lectin receptor is a monosaccharide selected from the group consisting of glucose, galactose, mannose, xylose, fructose, rhamnose, arabinose, allose, altrose, idose, N-acetylglucosamine, N-acetylgalactosamine, talose, glucuronic acid, glucosamine, galactosamine, N-acetylneuraminic acid, and fucose, or two or more of these monosaccharides, the same or different, bound together. [4] The fluorescent protein according to [1], wherein the C-type lectin receptor has a Ca2+-binding motif, and when the Ca2+-binding motif is an EPN (Glu-Pro-Asn) motif, the sugar that binds to the C-type lectin receptor is mannose, and when the Ca2+-binding motif is a QPD (Gln-Pro-Asp) motif, the sugar that binds to the C-type lectin receptor is galactose. [5] The fluorescent protein according to [1], wherein the sugar that binds to the C-type lectin receptor is mannose, and mannose is capable of binding to CD206 expressed in macrophages. [6] The fluorescent protein according to any one of [1] to [5], wherein the fluorescent protein is green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), or orange fluorescent protein (OFP).[7] The fluorescent protein according to any one of [1] to [6], wherein the fluorescent protein is a green fluorescent protein (GFP) that consists of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and emits fluorescence, or a red fluorescent protein (RFP) that consists of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 and emits fluorescence. [8] The fluorescent protein according to any one of [1] to [7], wherein the fluorescent protein chemically modified with sugars has an average number of sugar bonds per fluorescent protein molecule of 1 to 25. [9] The fluorescent protein according to any one of [1] to [8], wherein the fluorescent protein chemically modified with sugars forms a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer.

[10] The fluorescent protein according to any one of [1] to [9], wherein the fluorescent protein chemically modified with a sugar is formed by binding an amino group, a thiol group, or a carboxyl group contained in the fluorescent protein to the sugar that binds to the C-type lectin receptor, either directly or via a linker.

[11] The fluorescent protein according to any one of [1] to

[10] , wherein the fluorescent protein chemically modified with a sugar is formed by binding an amino group contained in the fluorescent protein to the sugar that binds to the C-type lectin receptor, either directly or via a linker, wherein the bond is an amide bond, a thiourea bond, a urea bond, a sulfonamide bond, an amino bond, an imine bond, an amidine bond, a phosphonamide bond, a thioether bond, or a urethane bond.

[12] A composition for identifying macrophage polarization, comprising the fluorescent protein according to [1] to

[11] .

[13] The composition according to

[12] , wherein the concentration of the fluorescent protein chemically modified with a sugar in the composition is at least 2 μM.

[14] A method for identifying the polarization of macrophages, comprising the steps of contacting a fluorescent protein chemically modified with a sugar that binds to a C-type lectin receptor with macrophages, and measuring the fluorescence of the fluorescent protein chemically modified with the sugar, wherein the sugar that binds to a C-type lectin receptor is capable of binding to a C-type lectin receptor expressed in macrophages, and the fluorescent protein chemically modified with the sugar is formed by a reaction between a fluorescent protein and a sugar that binds to a C-type lectin receptor or a modifying agent containing a sugar that binds to a C-type lectin receptor.

[0011] According to the present invention, high-affinity binding to C-type lectin receptors expressed on macrophages is possible, resulting in high fluorescence intensity. This allows for highly accurate identification of macrophage polarization. Furthermore, according to the present invention, the antibody can be easily handled in analysis, the use of animals in production can be reduced, and production can be performed more inexpensively than conventional fluorescently labeled antibodies.

[0012] This figure shows a comparison between mannose-modified FP, one embodiment of a fluorescent protein chemically modified with sugars used in the present invention, and a fluorescently labeled antibody. FP stands for fluorescent protein. SDS-PAGE results for mannose-modified FP are shown. (a) GFP, (b) RFP. Arrow (1) indicates the possible location of FP monomers or oligomers. Arrow (2) indicates the band shift from unmodified to maximum modification. The dotted line indicates the position of the unmodified FP band as the baseline. Fluorescence spectra of unmodified FP and mannose-modified FP are shown. (a) GFP excited at 488 nm. (b) RFP excited at 565 nm. Fluorescence recovery patterns of unmodified FP, mannose-modified FP, and FPs with different degrees of modification upon Con A agarose binding are shown. (a) GFP, (b) RFP. Flow cytometry analysis of macrophages with different treatments is shown. (a) Dot plot analysis of FSC vs. SSC. (b) Histogram derived from dot plot analysis of FSC. (c) Histogram derived from dot plot analysis of SSC. Comparison of the binding profiles of mannose-modified FP to M0 macrophages, M1 macrophages, and M2 macrophages obtained by flow cytometry. (a) Results for mannose-modified GFP. (b) Results for mannose-modified RFP. Comparison of the binding profiles of mannose-modified FP to M0 macrophages, M1 macrophages, and M2 macrophages obtained by flow cytometry. (a) Results for mannose-modified GFP. (b) Results for mannose-modified RFP. (c) Results for antibody-labeled antibodies. By utilizing the difference in fluorescence intensity due to differences in the binding mode and amount of mannose-modified FP when contacted with M0 macrophages, M1 macrophages, or M2 macrophages, it was possible to distinguish M0 macrophages, M1 macrophages, or M2 macrophages with high accuracy (see Figures 7a and 7b, in particular Figure 7b) ii and iv). On the other hand, when antibody-labeled antibodies were used, it was not possible to distinguish M0 macrophages, M1 macrophages, or M2 macrophages with high accuracy (see Figure 7c).

[0013] A preferred embodiment for carrying out the present invention will be described below. Note that the embodiment described below is an example of a typical embodiment of the present invention, and the scope of the present invention should not be construed as being narrow.

[0014] 1. Fluorescent Protein Chemically Modified with a Sugar that Binds to a C-Type Lectin Receptor The present invention uses a fluorescent protein chemically modified with a sugar that binds to a C-type lectin receptor. The sugar that binds to a C-type lectin receptor can bind to a C-type lectin receptor expressed in macrophages.

[0015] In the present invention, "macrophages" refer to cells differentiated from monocytes (mononuclear leukocytes), a type of white blood cell, in response to the influence of various environmental and inductive factors in each tissue. In the present invention, "macrophages" include macrophages present in various tissues and peripheral blood, and include, but are not limited to, macrophages present in peripheral blood.

[0016] As described in the Background Art section, macrophages are broadly classified into M0 macrophages, M1 macrophages, and M2 macrophages depending on their polarization state. In the present invention, macrophages may be M0 macrophages, M1 macrophages, or M2 macrophages. On the other hand, macrophages present in cancer tissues are in an activation state different from normal and are therefore called tumor-associated macrophages (TAM). Tumor-associated macrophages do not meet the classification criteria for M1 macrophages or M2 macrophages. In the present invention, macrophages may be tumor-associated macrophages (TAM).

[0017] C-type lectin receptors are expressed on the surface of immune cells such as dendritic cells and macrophages. They recognize characteristic glycans and glycolipids derived from pathogens and damaged autologous cells in the immune system, inducing immune responses such as cytokine production and phagocytosis. C-type lectin receptors form a protein family that specifically recognizes ligands such as glycans and glycolipids via small C-type lectin domains. C-type lectin receptors in this family share high homology in the three-dimensional structure of their C-type lectin domains. Although the types of saccharides recognized by individual receptors and the motifs that determine their recognition differ, they are believed to share a fundamental binding mode that primarily binds to glycans via calcium ions. However, there are also receptors whose ligands are still unknown, and interactions based on recognition of molecules other than glycans have also been reported. For example, CD209 has been reported to bind to IgE, CD69 to lectins and oxidized LDL, CD161 to LLT-1, and CLEC to hemin and podoplanin.

[0018] In one aspect of the present invention, the C-type lectin receptor is preferably CD206, CD205, CD209, CD301, Mincle, Lox, CLEC, MGL, DC-IR, DC-SIGN, dectin, selectin, CD23, CD69, CD93, C161, or CD302, and more preferably CD206.

[0019] In one aspect of the present invention, the sugar that binds to a C-type lectin receptor is a monosaccharide selected from the group consisting of glucose, galactose, mannose, xylose, fructose, rhamnose, arabinose, allose, altrose, idose, N-acetylglucosamine, N-acetylgalactosamine, talose, glucuronic acid, glucosamine, galactosamine, N-acetylneuraminic acid, and fucose, or a monosaccharide in which two or more of these monosaccharides, the same or different, are bound. The monosaccharide may be a derivative. The monosaccharide is preferably mannose, fucose, or N-acetylglucosamine, and more preferably mannose. Examples of a sugar chain in which two or more identical or different monosaccharides are linked together (hereinafter also referred to as a "sugar chain") include a sugar chain in which two or more monosaccharides are linked together in a linear or branched chain via glycosidic bonds, but preferably a sugar chain containing mannose, fucose, or N-acetylglucosamine, more preferably a sugar chain containing terminal mannose, terminal fucose, or terminal N-acetylglucosamine.

[0020] In one aspect of the present invention, the saccharide that binds to a C-type lectin receptor is preferably a saccharide that binds to CD206 expressed on macrophages. More preferably, the saccharide that binds to a C-type lectin receptor is mannose, fucose, or N-acetylglucosamine, and mannose, fucose, or N-acetylglucosamine is capable of binding to CD206 expressed on macrophages. Even more preferably, the saccharide that binds to a C-type lectin receptor is mannose, and mannose is capable of binding to CD206 expressed on macrophages.

[0021] In one aspect of the present invention, specific combinations of C-type lectin receptors and saccharides that bind to C-type lectin receptors include, for example, CD205 and mannose; CD209 and sialic acid; CD301 and galactose; Mincle and trehalose; CD23 and mannose or galactosamine; Lox and sialic acid; CLEC and fucoidan (fucose, galactose, mannose, xylose, glucuronic acid); MGL and galactose or galactosamine; DC-SIGN and mannose or galactosamine; Dectin and mannose or β-glucan; Selectin and sialyl Lewis acid.

[0022] In one aspect of the present invention, the C-type lectin receptor is Ca ２＋ It has a Ca binding motif ２＋ When the binding motif is an EPN (Glu-Pro-Asn) motif (for example, when the C-type lectin receptor is CD206), the sugar that binds to the C-type lectin receptor is mannose, and Ca ２＋ When the binding motif is a QPD (Gln-Pro-Asp) motif, the sugar that binds to the C-type lectin receptor is galactose.

[0023] The fluorescent protein is not particularly limited and can be appropriately selected depending on the purpose. The fluorescent protein may be, for example, green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), or orange fluorescent protein (OFP). In one aspect of the present invention, the fluorescent protein may be red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), or orange fluorescent protein (OFP). In one aspect of the present invention, the fluorescent protein may be one other than green fluorescent protein (GFP).

[0024] The green fluorescent protein (GFP) is not particularly limited and can be appropriately selected depending on the purpose. For example, it may be a wild-type GFP or a mutant GFP such as eGFP_A206K (F64L, S65T, A206K, H231L) or sfGFP (F64L, S65T, H231L, S30R, Y39N, N105T, Y145F, M153T, V163A, I171V, A206V). Similarly, red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), and orange fluorescent protein (OFP) may be wild-type or mutant.

[0025] A specific example of a green fluorescent protein (GFP) is a green fluorescent protein (GFP) that consists of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and that emits fluorescence. A specific example of a red fluorescent protein (RFP) is a red fluorescent protein (RFP) that consists of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 and that emits fluorescence. The green fluorescent protein (GFP) is preferably a green fluorescent protein (GFP) consisting of the amino acid sequence set forth in SEQ ID NO: 1. The red fluorescent protein (RFP) is preferably a red fluorescent protein (RFP) consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[0026] The number of sugars bound to one fluorescent protein molecule can be specified as the average number of sugars bound to one fluorescent protein molecule. For example, the average number of sugars bound to one fluorescent protein molecule can be 1 to 25, preferably 5 to 25, and more preferably 10 to 25.

[0027] The average number of sugar bonds per fluorescent protein molecule can be controlled. For example, let us consider the case where amino groups contained in a fluorescent protein are reacted with a modifying agent containing a sugar that binds to a C-type lectin receptor. The amino groups exposed on the surface of the fluorescent protein and the amino group at the N-terminus vary depending on the fluorescent protein. For the green fluorescent protein (GFP) used in the examples described below, the total number of amino groups exposed on the fluorescent protein surface and the amino group at the N-terminus can be predicted to be 21. Therefore, those skilled in the art can obtain a green fluorescent protein (GFP) with an average number of sugar bonds of 21 per fluorescent protein molecule. Furthermore, for the red fluorescent protein (RFP) used in the examples described below, the total number of amino groups exposed on the fluorescent protein surface and the amino group at the N-terminus can be predicted to be 22. Therefore, those skilled in the art can obtain a red fluorescent protein (RFP) with an average number of sugar bonds of 22 per fluorescent protein molecule.

[0028] Furthermore, those skilled in the art can control the average number of sugar bonds per fluorescent protein molecule, for example, by increasing or decreasing the number of amino acids having an amino group through genetic engineering. The average number of sugar bonds per fluorescent protein molecule can also be controlled by using a modifying agent that reacts with groups other than amino groups (e.g., carboxyl groups, thiol groups) contained in the fluorescent protein. In these cases, it is possible to increase the average number of sugar bonds per fluorescent protein molecule beyond the 25 exemplified above.

[0029] In one embodiment of the present invention, a fluorescent protein chemically modified with a sugar that binds to a C-type lectin receptor has a relative fluorescence intensity equivalent to that of an unmodified fluorescent protein when excited at an excitation wavelength appropriate for that fluorescent protein and the relative fluorescence intensity is measured at a fluorescence wavelength appropriate for that fluorescent protein. The fluorescent protein chemically modified with a sugar that binds to a C-type lectin receptor preferably has a relative fluorescence intensity of at least 70, 75, 80, 85, 90, or 95% of that of the unmodified fluorescent protein.

[0030] In a fluorescent protein chemically modified with a sugar that binds to a C-type lectin receptor, the fluorescent protein can be made into a multimer by modifying the terminal sequence (Nat Commun 6, 7134 (2015)). In one aspect of the present invention, in a fluorescent protein chemically modified with a sugar that binds to a C-type lectin receptor, the fluorescent protein forms a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer. In one aspect of the present invention, the fluorescent protein forms a trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer. In one aspect of the present invention, the fluorescent protein forms a tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer. In one aspect of the present invention, the fluorescent protein is a multimer excluding a dimer. In one aspect of the present invention, the fluorescent protein is a multimer excluding a dimer or trimer. When the fluorescent protein is a red fluorescent protein (RFP), the red fluorescent protein (RFP) preferably forms a tetramer.

[0031] The fluorescent protein chemically modified with a sugar may be one in which the fluorescent protein and the sugar that binds to the C-type lectin receptor are bound directly or via a linker. Preferably, the fluorescent protein chemically modified with a sugar may be one in which an amino group, a thiol group, or a carboxyl group contained in the fluorescent protein is bound directly or via a linker to the sugar that binds to the C-type lectin receptor. More preferably, the fluorescent protein chemically modified with a sugar may be one in which an amino group contained in the fluorescent protein is bound directly or via a linker to the sugar that binds to the C-type lectin receptor.

[0032] The bond may be, for example, an amide bond, a thiourea bond, a urea bond, a sulfonamide bond, an amino bond, an imine bond, an amidine bond, a phosphonamide bond, a thioether bond or a urethane bond.

[0033] Next, the fluorescent protein chemically modified with a sugar that binds to a C-type lectin receptor is produced by reacting a fluorescent protein with a sugar that binds to a C-type lectin receptor or a modifying agent containing a sugar that binds to a C-type lectin receptor.

[0034] (1) The reaction between a fluorescent protein and a modifying agent containing a sugar that binds to a C-type lectin receptor will be described below.

[0035] The "fluorescent protein" is as defined above.

[0036] A "modifying agent comprising a sugar that binds to a C-type lectin receptor" refers to a modifying agent that can chemically modify a fluorescent protein with a sugar that binds to a C-type lectin receptor. A "modifying agent comprising a sugar that binds to a C-type lectin receptor" is, for example, a modifying agent that comprises a sugar that binds to a C-type lectin receptor and a functional group that reacts with a functional group (e.g., an amino group, a carboxyl group, or a thiol group) contained in an amino acid that constitutes the fluorescent protein. In the modifying agent, the sugar that binds to a C-type lectin receptor and the functional group that reacts with a specific functional group contained in an amino acid that constitutes the fluorescent protein may be bonded directly or via a linker. That is, the modifying agent may further comprise a linker.

[0037] The linker is a group that connects the fluorescent protein and the sugar that binds to the C-type lectin receptor, and may have a group that binds to the fluorescent protein or a group that binds to a functional group that reacts with the fluorescent protein, and a group that binds to the sugar that binds to the C-type lectin receptor. The bond may be a chemical bond or a physical bond. The linker is not particularly limited as long as it is a known linker, but is preferably a divalent hydrocarbon group. When the linker is a divalent hydrocarbon group, it is preferably a hydrocarbon group having 2 or more carbon atoms. More preferably, it is a hydrocarbon group having 2 to 20 carbon atoms, and even more preferably, it is a hydrocarbon group having 2 to 10 carbon atoms. Examples of divalent hydrocarbon groups include alkylene groups having 2 to 20 carbon atoms, arylene groups having 2 to 20 carbon atoms, and groups formed by combining these. Other examples of divalent hydrocarbon groups include methylene groups, ethylene groups, propylene groups, butylene groups, phenylene groups, and groups formed by combining these.

[0038] By reacting (or contacting) a fluorescent protein with a modifying agent containing a sugar that binds to a C-type lectin receptor, a functional group (e.g., an amino group, a carboxyl group, or a thiol group) contained in the fluorescent protein reacts with a functional group of the modifying agent containing a sugar that binds to a C-type lectin receptor, thereby obtaining a fluorescent protein chemically modified with a sugar that binds to a C-type lectin receptor.

[0039] When reacting with an amino group contained in a fluorescent protein, the modifying agent containing a sugar that binds to a C-type lectin receptor is preferably a modifying agent containing a sugar that binds to a C-type lectin receptor and a functional group that reacts with an amino group contained in an amino acid that constitutes the fluorescent protein. More preferably, the modifying agent contains a sugar that binds to a C-type lectin receptor and a functional group selected from the group consisting of an N-hydroxysuccinimide ester group, an isothiocyanate group, an isocyanate group, an acyl azide group, a sulfonyl group, an aldehyde group, an epoxy group, an aryl group, a carboxy group, a glyoxal group, an anhydride group, an imido ester group, a fluoroaryl group, a fluorophenyl ester group, a pentafluorophenyl ester group, a hydroxymethylphosphine group, a carbonate group, and a carbodiimide group. The amino group contained in the fluorescent protein can bond to the functional group of the modifying agent, for example, by forming an —NH— bond. A specific example of the modifying agent is 4-isothiocyanatephenyl alpha-D-mannopyranoside (used in the Examples described below).

[0040] When reacting with an amino group contained in a fluorescent protein, an alkyne compound prepared from a sugar may be used as a modifying agent containing a sugar that azidotates the amino group of the fluorescent protein and binds to a C-type lectin receptor. Other click chemistry methods that minimize denaturation of the fluorescent protein during the binding of the sugar to the fluorescent protein may also be used.

[0041] When reacting with a carboxy group contained in a fluorescent protein, the modifying agent containing a sugar that binds to a C-type lectin receptor is preferably a modifying agent containing a sugar that binds to a C-type lectin receptor and a functional group that reacts with a carboxy group contained in an amino acid that constitutes the fluorescent protein. More preferably, the modifying agent contains a sugar that binds to a C-type lectin receptor and a functional group selected from the group consisting of an amino group, a carboxy group, a hydroxy group, an epoxy group, an isocyanate group, and a vinyl group. The carboxy group contained in the fluorescent protein can bond to the functional group of the modifying agent, for example, by forming -O-.

[0042] When reacting with a thiol group contained in a fluorescent protein, the modifying agent containing a sugar that binds to a C-type lectin receptor is preferably a modifying agent containing a sugar that binds to a C-type lectin receptor and a functional group that reacts with a thiol group contained in an amino acid that constitutes the fluorescent protein. More preferably, the modifying agent contains a sugar that binds to a C-type lectin receptor and a functional group selected from the group consisting of a halogen group, an epoxy group, a hydroxy group, an isocyanate group, a methylol group, a carboxy group, and a carbonyl group. The thiol group contained in the fluorescent protein can bond to the functional group of the modifying agent, for example, by forming -S-.

[0043] For example, 1 to 150 moles, preferably 25 to 150 moles, more preferably 75 to 150 moles, and even more preferably 100 to 150 moles of a modifying agent containing a sugar that binds to a C-type lectin receptor are reacted with 1 mole of the fluorescent protein. If the number of moles of the modifying agent containing a sugar that binds to a C-type lectin receptor is equal to or greater than the above-mentioned lower limit, the average number of sugars bound per fluorescent protein molecule is increased, thereby further increasing the affinity of the fluorescent protein chemically modified with sugars for the C-type lectin receptor, thereby enabling more sensitive identification of macrophage polarization. On the other hand, if the number of moles of sugar that binds to a C-type lectin receptor is equal to or less than the above-mentioned upper limit, a state in which M2 macrophages are present in excess, such as a state of cancer proliferation or angiogenesis, or a state of accelerated wound healing, can be determined.

[0044] In one embodiment of the present invention, a modifying agent containing a sugar that binds to a C-type lectin receptor is a compound represented by the formula R-L-A. In the formula, R is a sugar that binds to a C-type lectin receptor (or a monovalent group derived from a sugar that binds to a C-type lectin receptor), L is a linker, and A is a functional group that reacts with a functional group contained in an amino acid that constitutes the fluorescent protein. The sugar, the linker, and the functional group that reacts with a functional group contained in an amino acid that constitutes the fluorescent protein are as described above.

[0045] (2) The reaction between a fluorescent protein and a sugar that binds to a C-type lectin receptor will be explained below.

[0046] Methods for reacting a protein with a sugar to obtain a protein chemically modified with a sugar are known. For example, a protein in which an arbitrary amino acid residue is Cys is produced according to a known protein synthesis method, and then a sugar chain is added to the Cys by a chemical reaction to produce a protein chemically modified with a sugar. In the present invention, for example, a fluorescent protein chemically modified with a sugar that binds to a C-type lectin receptor can be obtained based on such a known method by reacting a fluorescent protein with a sugar that binds to a C-type lectin receptor.

[0047] Mannose-modified FP, one embodiment of a fluorescent protein chemically modified with sugars that bind to C-type lectin receptors, will be described with reference to Figure 1. Figure 1 shows a comparison between mannose-modified FP and a fluorescently labeled antibody. FP is an acronym for fluorescent protein.

[0048] As shown in Figure 1(a), the fluorescently labeled antibody has only two binding sites and, due to its large size, cannot avoid steric hindrance. On the other hand, the mannose-modified FP has many binding sites and, due to its smaller size compared to the fluorescently labeled antibody, can avoid steric hindrance. As shown in Figure 1(b), the fluorescently labeled antibody does not exhibit multivalent binding and cannot bind in any direction. On the other hand, the mannose-modified FP exhibits multivalent binding and can bind in any direction.

[0049] The fluorescent proteins chemically modified with sugars used in the present invention have a molecular weight approximately one-fifth that of antibodies. Many fluorescent proteins form dimers or tetramers, but even these aggregates have a smaller molecular weight than antibodies. Furthermore, the surface of a single fluorescent protein typically contains approximately 20 sugar-modifiable sites, including the termini, allowing for multivalent binding and optimization of the degree of modification. These features enable fluorescent proteins chemically modified with sugars to bind with high affinity to C-type lectin receptors expressed in macrophages, resulting in high fluorescence intensity. This allows for highly accurate identification of macrophage polarization. Furthermore, the fluorescent proteins chemically modified with sugars used in the present invention often have a stable tertiary structure and are easy to handle. Furthermore, unlike antibodies, fluorescent proteins can be produced using bacterial transformation systems. Therefore, the fluorescent proteins chemically modified with sugars used in the present invention can reduce the use of animals in antibody production. Furthermore, the fluorescent proteins chemically modified with sugars used in the present invention do not require expensive fluorescent reagents and do not require the use of animals for antibody production, and therefore can be produced more inexpensively than conventionally used fluorescently labeled antibodies.

[0050] 2. Composition for identifying macrophage polarization The composition for identifying macrophage polarization of the present invention (hereinafter also simply referred to as "composition") contains a fluorescent protein chemically modified with a sugar that binds to a C-type lectin receptor. The composition of the present invention may optionally contain other additives, etc.

[0051] In the composition of the present invention, the concentration of the fluorescent protein chemically modified with a sugar that binds to a C-type lectin receptor in the composition is, for example, at least 0.2 μM, preferably 1 μM, more preferably at least 2 μM, and even more preferably 2 to 100 μM. When the concentration of the chemically modified fluorescent protein is equal to or greater than the above-mentioned lower limit, the affinity of the chemically modified fluorescent protein for the C-type lectin receptor can be further increased, thereby enabling more sensitive identification of macrophage polarization. The concentration of the chemically modified fluorescent protein can be measured using known methods. For example, the ultraviolet absorption method, which is easy to operate, can be applied (see * below). This method quantifies the amount of tryptophan and tyrosine in a protein and estimates the total protein concentration. The concentration (mg / mL) can be calculated from the absorbance at 280 nm and the molar extinction coefficient of the protein to be quantified. The molar extinction coefficient of each protein can be determined by entering the amino acid sequence of the protein to be quantified into the software provided at the following website: https: / / web.expasy.org / protparam / *Pace CN, Vajdos F, Fee L, Grimsley G, Gray T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 1995 Nov;4(11):2411-23. doi: 10.1002 / pro.5560041120. PMID: 8563639; PMCID: PMC2143013

[0052] The composition of the present invention can distinguish macrophage polarization with high accuracy. The composition of the present invention can preferably distinguish between M0 macrophages, M1 macrophages, and / or M2 macrophages. Furthermore, the composition of the present invention can preferably distinguish between tumor-associated macrophages (TAM).

[0053] 3. Method for identifying macrophage polarization

[0054] The method of the present invention for identifying macrophage polarization comprises the steps of contacting a fluorescent protein chemically modified with a sugar that binds to a C-type lectin receptor with a macrophage and measuring the fluorescence of the fluorescent protein chemically modified with the sugar. The sugar that binds to a C-type lectin receptor can bind to a C-type lectin receptor expressed in macrophages. The fluorescent protein chemically modified with a sugar that binds to a C-type lectin receptor is produced by reacting a fluorescent protein with a sugar that binds to a C-type lectin receptor or a modifying agent containing a sugar that binds to a C-type lectin receptor.

[0055] The step of contacting a fluorescent protein chemically modified with a sugar that binds to a C-type lectin receptor with a macrophage can be carried out by a method known in the art, for example, by adding the fluorescent protein chemically modified with a sugar to a macrophage or a sample containing a macrophage.

[0056] Macrophages can be prepared by methods known in the art. Macrophages are not particularly limited, but may be isolated, unestablished macrophages (hereinafter referred to as "isolated macrophages"), established macrophage cell lines, or macrophages differentiated from monocytes in vitro. Sources of isolated macrophages include, for example, human peripheral blood, human bone marrow, human tissue, mouse peripheral blood, mouse bone marrow, mouse tissue, etc. Examples of isolated macrophages include isolated human macrophages and isolated mouse macrophages. Isolation methods for isolated macrophages have been established for each source. Examples of established macrophage cell lines include, but are not particularly limited to, RAW-264.7, J774, MV-4-11, KG-1, etc. A method for differentiating monocytes into macrophages in vitro can be carried out, for example, by culturing monocytes in a monocyte medium containing GM-CSF and / or M-CSF under culture conditions suitable for maintaining the physiological functions of monocytes.

[0057] The step of measuring the fluorescence of the fluorescent protein chemically modified with sugar can be carried out using, for example, a flow cytometer.

[0058] A flow cytometer is an instrument that performs flow cytometry. Specifically, flow cytometry is a technique in which a suspension of a measurement object is made into a high-speed fluid, and the fluid is irradiated with laser light, mercury light, or the like to measure and quantify the scattered light and fluorescence generated by the fluid, thereby accurately measuring the size, quantity, etc. of the measurement object or separating the particles of the measurement object. The measurement object is introduced into a very fine tube (flow cell), and the irradiation is performed while the measurement object flows through the tube in an aligned state.

[0059] The flow cytometer used in the present invention can automatically measure the size, number, etc. of foreign matter by irradiating the foreign matter with excitation light and measuring the reflected light and / or scattered light from the foreign matter and / or the fluorescence from the excited state of the foreign matter. Flow cytometers are widely used, particularly in the field of biotechnology, and the devices themselves are commercially available, so those skilled in the art can use them as appropriate.

[0060] In the present invention, preferably, M0 macrophages, M1 macrophages, and M2 macrophages can be distinguished in flow cytometry by utilizing the difference in fluorescence intensity due to the difference in the binding mode and amount of a fluorescent protein chemically modified with sugar when contacted with M0 macrophages, M1 macrophages, and M2 macrophages. For example, in the Examples described below, M0 macrophages, M1 macrophages, and M2 macrophages can be distinguished with high accuracy by utilizing the difference in fluorescence intensity due to the difference in the binding mode and amount of a mannose-modified FP when contacted with M0 macrophages, M1 macrophages, and M2 macrophages when contacted with M0 macrophages, M1 macrophages, and M2 macrophages. (See Figures 7a and 7b, particularly Figure 7b) ii and iv.)

[0061] The method for identifying macrophage polarization of the present invention can identify macrophage polarization with high accuracy. The method for identifying macrophage polarization of the present invention can preferably identify M0 macrophages, M1 macrophages, and / or M2 macrophages. Furthermore, the method for identifying macrophage polarization of the present invention can preferably identify tumor-associated macrophages (TAM). Furthermore, the method for identifying macrophage polarization of the present invention can be used to elucidate the relationship between changes in the pathology of inflammatory diseases and subtypes. Furthermore, the method for identifying macrophage polarization of the present invention can also be used to determine or diagnose the pathology of inflammatory diseases in the research or clinical fields.

[0062] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples, and these examples should not be construed as limiting in any sense.

[0063] Experimental Section (Isolation and Purification of FP) The previously prepared plasmids encoding the GFP mutant (pUV5trypS1tag) and RFP mutant (pT3castag) were transformed into E. coli cells (BL21(DE3) Merck Biosciences, Darmstadt, Germany). The transformed bacteria were then grown overnight at 37°C in Luria-Bertani (LB) medium supplemented with 75 μg / mL ampicillin. An aliquot of the culture medium was diluted with fresh LB medium containing 75 μg / mL ampicillin and incubated at 28°C for 24 hours, while 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) (Takara Bio. Inc. Japan) was used to promote FP expression. After centrifugation, the collected bacterial cells were treated with an appropriate amount of bacterial lysis reagent B-PER II (Thermo Scientific, USA) and rotated overnight at room temperature on a rotary shaker. The lysate was centrifuged at 12,000 rpm for 10 minutes at room temperature to separate the supernatant and cell debris pellet. The supernatant was collected and applied to Ni2+-NTA resin (Qiagen, Düsseldorf, Germany) to isolate the histidine-tagged FP using standard procedures for histidine-tagged protein purification. The purified FP was reconstituted in phosphate-buffered saline (PBS) by exchanging the elution buffer for affinity chromatography through a gel filtration column. The properties of the FP were then checked using a spectrophotometer (DU 640, Beckman Coulter Inc., Japan). The amino acid sequences of the GFP variant and the RFP variant are represented by SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0064] (Dose-dependent modification of FP by mannose derivatives) One hundred microliters (100 μL) of a 25 μM FP (GFP or RFP) solution containing PBS (pH 9) was mixed with 2.5 μL of 25, 75, and 100 mM mannose derivative (4-isothiocyanatophenyl alpha-D-mannopyranoside, Sigma-Aldrich Japan K.K., Tokyo, Japan) containing DMSO, resulting in FP:mannose derivative molar ratios of 1:25, 1:75, and 1:100. Under alkaline conditions (pH 9-11), the isothiocyanate functional group (R １ -N-C=S) and an amine group (R ２ -NH ２ ) is reactive with thiourea (R １ -N-C=S-NR ２ ), we determined the mixing ratio based on the number of lysines present on the FP surface and N-terminus, the number of lysines present on the FP surface and N-terminus, and the number of lysines present on the GFP (PDB DOI: https: / / doi.org / 10.2210 / pdb2b3q / pdb) variants used by us in this study (according to [reference] 21), and the number of lysines present on the RFP (PDB DOI: https: / / doi.org / 10.2210 / pdb2vae / pdb) variants used by us in this study (according to [reference] 22), to estimate the sufficiency of the reaction. A control reaction of 1:0 FPs:mannose derivative was performed by mixing the corresponding FP solution with 2.5 μL of DMSO. The mixture was incubated at 37°C for 4 hours, followed by further incubation at 4°C overnight. To remove excess mannose derivatives, the reaction mixture was then applied to an ultrafiltration column (Amicon Ultra-0.5 10 K column, Merck Millipore Ltd, Tullagreen, Carrigtwohill, Co. Cork IRL, Darmstadt, Germany) and centrifuged at 14,000 rpm. Centrifugation was repeated by adding an appropriate amount of PBS to reconstitute the mixture.

[0065] (SDS-PAGE of unmodified and mannose-modified FP) Aliquots of unmodified control FP and mannose-modified FP were subjected to SDS-PAGE under non-denaturing conditions using 4% stacking gels and 15% running gels, along with protein standard markers (Precision Plus Dual Xtra Standards, Bio-Rad, USA). All FP samples were prepared by simply mixing with loading dye (Takara Bio Inc., Tokyo, Japan) without heating, which could maintain fluorescence and FP association. Electrophoresis was performed for 4 hours at a constant voltage of 100 V, with a current of 10 mA for stacking gels and 20 mA for running gels. After electrophoresis, the gels were visualized using a fluorescent imager (Typhoon FLA 9500, GE Healthcare, Ltd.), and the gel images were further analyzed using Quantity one software.

[0066] (Acquisition of Fluorescence Spectra of Unmodified and Mannose-Modified FP) 0.5 μM unmodified and modified FP solutions were prepared in PBS solution, and fluorescence spectra were acquired using a fluorescence spectrophotometer (Shimadzu RF-5300PC, Shimadzu, Kyoto, Japan) with excitation at 488 nm and 565 nm for GFP and RFP, respectively. As described in the "FP Separation and Purification" section, protein concentrations were determined by UV-Vis spectroscopy through estimation of the molar extinction coefficient of the protein based on its amino acid composition. Modification of FP with mannose via a lysine residue does not affect protein quantification. To examine the fluorescence properties after modification, unmodified and modified protein solutions were prepared at exactly the same concentrations. These comparisons allow for interchangeable examination of their quantum yields.

[0067] (Verification of Mannose-Specific and Mannose-Modification Level-Dependent Binding to Concanavalin A (Con A)) The affinity binding of unmodified and mannose-modified FP to concanavalin A (Con A agarose-bound, AL-1003, Vector Laboratories, Inc., 6737 Mowry Avenue, Newark, CA 94560 USA), a type of mannose-recognizing lectin, was verified. An aliquot of the Con A solution was diluted with 10 mM HEPES, 150 mM NaCl, 0.1 mM CaCl and 0.1 mM ATP. ２ , and 0.01 mM MnCl ２ The FP was thoroughly washed with 100 μL of mannose-modified FP (3 μM) in the binding buffer (pH 7.5) and resuspended in the same buffer to obtain a 50% Con A slurry. The elution buffer also contained 25 mM, 50 mM, and 75 mM α-methyl-D-mannoside (methyl α-D-mannopyranoside, Tokyo Chemical Industry Co., Ltd., 6-15-9 Toshima, Kita-ku, Tokyo). After initial fluorescence intensity determination, 100 μL of 3 μM unmodified and mannose-modified FP solutions resuspended in the binding buffer were mixed with 200 μL of Con A slurry and incubated at room temperature for 1 hour with continuous vortexing. The supernatant, containing the FP fraction not bound to Con A, was collected by centrifugation at 1,000×g for 15 minutes. The remaining Con A slurry was washed with 200 μL of binding buffer by vortexing for 15 minutes, followed by centrifugation at 1000 g for 15 minutes and then washed twice with 400 μL of the same buffer to obtain combined supernatants. These washing procedures were repeated with buffers containing progressively more concentrated methyl α-D-mannopyranoside. The entire supernatant was individually probed for fluorescence intensity. The resulting fluorescence intensity was calculated as a percentage of the initial fluorescence intensity.

[0068] J774A.1 mouse macrophage cells were provided by RIKEN BRC through the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology of Japan. Cells were subcultured in T25 cell culture flasks in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin at 37°C under 5% carbon dioxide.

[0069] Macrophage polarization. J774A.1 cells were grown in 24-well plates until they reached confluence. To polarize M0 macrophages to M1 or M2, cells were treated with 100 ng / mL lipopolysaccharide (LPS) (SIGMA-ALDRICH, Co., 3050 Spruce Street, St. Louis, USA) or 20 ng / mL interleukin-4 (IL-4) (recombinant mouse IL-4, PEPROTECH, Thermo Fisher Scientific), respectively, and incubated at 37°C for 24 hours according to the passaging procedure described above. Untreated cells were analyzed as M0. M1 and M2 polarization was confirmed using bright-field microscopy with a Nikon Eclipse TE 2000-U inverted fluorescent microscope (Nikon Solutions Co. Ltd., Tokyo, Japan) equipped with a ×40 water-immersion objective and forward scatter (FSC) histograms obtained by flow cytometry analysis using a Cell Sorter (SH800S SONY Corp., Tokyo, Japan) and FlowJo software (Becton, Dickinson and Company, San Jose, CA, USA).

[0070] Flow cytometry analysis of polarized macrophages treated with mannose-modified FP and anti-CD206 antibody. Polarization was induced to M1, M2, and M0. M0 macrophages remained in their original state and were scraped from the cell culture plate by adding fresh culture medium, DMEM supplemented with 10% FBS. The collected cell suspension was centrifuged at 300 g for 5 minutes at 4°C, and a cell pellet was obtained after removing the supernatant. The cell pellet was washed twice with PBS and resuspended in ice-cold flow cytometry staining buffer (R&D systems, USA) to a cell concentration of 1 x 10 ６The cell suspension was adjusted to a concentration of 1 x 10 cells / 100 μL. Four combinations of FP solutions with two different mannose modification levels (Fp:mannose derivative 1:25 and 1:100), 0.2 μM and 2 μM, were incubated with aliquots of the cell suspension for 1 hour on ice in the dark. After incubation, the resulting cell solution was washed twice with ice-cold flow cytometry staining buffer by repeated centrifugation at 300 g for 5 minutes at 4°C and resuspended in 200 μL of ice-cold flow cytometry staining buffer for flow cytometry analysis. For antibody-based detection of individual conditioned macrophages, 1 x 10 cells were used. ６ Cells collected at a concentration of 100 μL were treated with 0.2 μM phycoerythrin (PE)-labeled anti-mouse CD206 antibody (excitation: 565 nm, emission: 575 nm) (BioLegends Inc., San Diego, CA, USA) for 30 minutes on ice under dark conditions. Treated cells were washed and resuspended in ice-cold FACS buffer (flow cytometry staining buffer), compatible with FP processing, for flow cytometry analysis. All data acquired in this study were processed using FlowJo software.

[0071] Results and Discussion (Confirmation of Dose-Dependent Mannose Modification of FP by SDS-PAGE) In this study, we attempted to develop an FP-based molecular sensor for sensitive and quantitative detection of mannose receptors expressed on the cell surface of macrophages. To this end, we aimed to achieve multivalent binding of mannose to the CD206 receptor on the cell surface of macrophages as multiple ligands. Therefore, we performed dose-dependent modification of FP using mannose derivatives, conjugating mannose moieties to FP at various levels through conjugation reactions with several molar ratios. We then performed SDS-PAGE under non-denaturing conditions to confirm the dose-dependent modification of FP by the mannose derivatives (Figure 2). As shown in Figure 2(a), an unmodified GFP variant with an approximate molecular weight of 27 kDa, based on molecular markers of 25 kD and 75 kD, demonstrated several bands with various mobilities (indicated by arrows (1)), which are believed to represent monomeric, dimeric, trimeric, tetrameric, and higher-associated GFP variants. While GFP can exist essentially in a monomeric state, GFP-like FPs have been reported to possess an unusual concentration-dependent ability for self-association in vitro. Therefore, our band assignments were consistent under these conditions. Therefore, we focused on four bands highlighted by arrows (1), which demonstrated a ratio-dependent gradual shift toward the bottom of the gel, highlighted by arrows (2) and dotted lines, with increasing amounts of conjugated mannose. Furthermore, the shifted bands gradually expanded and became blurred. This observation indicated that the increase in molecular weight of proteins due to glycosylation, unlike simple proteins, can promote reverse migration, and the amount of increase may be distributed depending on the reaction efficiency. The mannose dose-dependence of specific migrations was enhanced, suggesting that mannose modification may progress as the mixing ratio increased. In addition, modified GFP variants, regardless of the degree of mannose modification, may spontaneously associate with each other as unmodified GFP variants. This could be expected to function as a multivalent molecular sensor binding to its target receptor (Figure 1(b)).We also performed corresponding SDS-PAGE analysis of the RFP variants (Figure 2(b)). In most detection methods based on binding to specific cell surface receptors, color changes are expected to facilitate multiplex monitoring. Because our RFP variants are DsRed derivatives, their molecular weights are approximately 28 kDa, comparable to other FPs. However, DsRed derivatives are notably tetrameric in nature. The distinct band appearing around 75 kDa could be expected to represent the tetrameric form formed by the RFP variants. Therefore, we anticipated that the use of GFP and RFP variants would provide not only color options but also exemplary associations between them. We obtained comparable band patterns for the RFP variants, demonstrating a ratio-dependent band shift toward the bottom of the gel, as highlighted by the arrow (2) and dotted line, suggesting that the emission and association properties of FPs may be robust to mannose modification. Overall, we managed to modulate the approximate degree of mannose modification of FP in a dose-dependent manner.

[0072] (Fluorescence Properties of Mannose-Modified FP) Although the inventors were able to make a rough estimate of the fluorescence emission of mannose-modified FP from the band intensity detected on SDS-PAGE, they checked the fluorescence spectra of the modified FP along with the unmodified group of FP to identify any changes in the emission profile. According to the obtained spectra shown in Figure 3, the inventors could not find any significant changes in the emission profile. Therefore, the inventors could try to apply FP with any modification level for further experiments.

[0073] (Mannose-specific and Mannose-Modification Level-Dependent Binding of Unmodified and Mannose-Modified FP to Con A) Since we obtained consistent results for the mannose-modification level-dependent band shift of FP using SDS-PAGE and demonstrated no significant changes in the fluorescence properties of FP, we attempted to investigate the mannose-specific and modification level-dependent binding properties using concanavalin A resin. Aiming for target binding to the CD206 receptor, a mannose-recognizing C-type lectin, we utilized concanavalin A as a lectin with similar recognition properties. As shown in Figures 4(a) and (b), both unmodified FPs were recovered in the unbound and first supernatant fractions, whereas the corresponding recovery rates of mannose-modified FPs decreased in a modification level-dependent manner. Furthermore, modified RFPs appeared to have a higher affinity for Con A agarose compared to comparable GFP profiles. We further observed an inverse correlation between fluorescence recovery rates in the latter fractions. These results demonstrated that the mannose-modification approach successfully generated FPs with different mannose-modification conditions that exhibited various affinities for Con A depending on the mannose-modification level, suggesting the possibility of multivalent binding between mannose-modified FPs and Con A.

[0074] (Verification of the polarization state of M0 macrophages into M1 and M2 populations) Having verified distinct mannose modification levels of FP, we next attempted to evaluate the performance of mannose-modified FP using receptors. For these studies, we first verified macrophage polarization through a previously established approach. For this purpose, we used the J774A.1 murine reticulosarcoma cell line. As described in the experimental section, we treated J774A.1 with LPS (100 ng / mL) or IL-4 (20 ng / mL) toward M1 or M2 polarization, respectively. Furthermore, it has previously been shown that these polarizations can cause changes in cell size and organelle interiors (mitochondria and lysosomes), which are suitable for microscopic observation and flow cytometry analysis. First, we examined the morphological changes of the three macrophage populations using a microscope. We were able to observe typical cell morphologies (shape and size) for the three differently treated macrophages, as previously reported (not shown). Untreated macrophages (designated as M0) exhibited small, spherical appearances. The LPS-treated cell population (designated as M1) exhibited a type of spindle-shaped cell, while the IL-4-treated cells (designated as M2) exhibited more elongated cells. Furthermore, somewhat more enlarged cell types were also observed in the IL-4-treated group. Additionally, compared with the untreated cell population, both treated cell populations exhibited a scattered appearance. Next, we attempted to more quantitatively demonstrate macrophage polarization using flow cytometry (Fig. 5). In all macrophage populations, there were three distinct cell distributions, circled in black, which were likely a consequence of their cell cycle (Fig. 5(a)). We were able to clearly reveal the differences in their cell population distribution through histograms processed from forward scatter (FSC; an index of cell size) and side scatter (SSC; an index of internal cell complexity). A significant shift in cell size expansion in the case of M2 and a smaller but significant shift in M1 (Fig. 5(b)) and similar changes in internal cell complexity were observed in both M1 and M2 (Fig. 5(c)).We also used anti-CD206 antibodies to check polarization using a representative polarization marker such as CD206 (FIG. 7(c)).

[0075] (Binding Profile Examination of FP Conjugates Developed for Polarized Macrophages by Flow Cytometry) To evaluate the appropriate mannose modification level for monitoring macrophage polarization, we selected two distinct conjugation conditions, FP:mannose 1:25 and 1:100, to treat each of the following cell populations: M0 (untreated), M1 (LPS-treated), and M2 (I1-4-treated). In addition, we set two concentrations for the binding reaction, 0.2 μM and 2 μM, according to the standard antibody binding assay for each molecular sensor type. Briefly, we used four combination treatments for each FP: i) 1:25, 0.2 μM (low modification, low dose); ii) 1:25, 2 μM (low modification, high dose); iii) 1:100, 0.2 μM (high modification, low dose); and iv) 1:100, 2 μM (high modification, high dose). To avoid the stimulation of signal transduction inside cells upon mannose-modified FP binding and the internalization of these ligand-like compounds and receptor complexes, we treated all types of cell suspensions (M0, M1, and M2) with the conjugation mixtures described in the Experimental Section under ice-cold and dark conditions. The binding profiles of individual cell populations were compared as histograms of the fluorescence intensity of each FP source. Autofluorescence was also assessed (Figure 6). As shown in Figure 6(a), mannose-modified GFP conjugates appeared to achieve mannose receptor binding at higher treatment doses, regardless of the modification level. On the other hand, RFP-derived molecular sensors were able to function similarly on each cell type (M0, M1, and M2) shown in Figure 6(b). In both GFP and RFP, a shift toward higher fluorescence intensity (to the right of the x-axis) of the bound population was observed in the order M1 < M0 << M2. We then used all combinations of FP-based molecular sensors to check the identity of each polarized cell type. We considered further evaluation using anti-CD206 antibodies in contrast to conventional approaches.

[0076] (Discrimination of M0, M1, and M2 Macrophages Using FP-Based Molecular Sensors) We compared the performance of four combinations of FP-based molecular sensors among three macrophage types: M0, M1, and M2. For further objective validation, we treated the same cell populations with PE-labeled anti-CD206 antibody. Due to the excitation and emission maxima of the PE-labeled anti-CD206 antibody, we were only able to evaluate the function of the RFP-based molecular sensor on the performance of the antibody ( Figure 7(c) ). We obtained distinct FP-dependent patterns among the three treated cell populations. In the case of the GFP-based molecular sensor, the GFP-based molecular sensor failed to provide substantial discrimination except at high doses ( Figure 7(a)(ii)(iv) ). As shown in the inset of Figure 7(a) containing the processed data on a logarithmic scale, we observed a population shift toward higher intensities. In these cases, a small but significant population of M2 macrophages was identified as targeting the molecular sensor. However, nonspecific binding could still prevail in both cases. On the other hand, RFP variant-based molecular sensor treatment (Figure 7(b)) demonstrated a major peak among all macrophages, similar to the anti-CD206 antibody treatment experiment (Figure 7(c)). Interestingly, a common shoulder peak with low fluorescence intensity was present in the M2 macrophage population, highlighted by the black arrow. Histograms from macrophages treated with a low dose of the molecular sensor overlapped with each other, demonstrating nearly identical profiles among the three cell populations. Fortunately, we were able to observe three distinct patterns under high-dose molecular sensor application (Figure 7(b)(ii)(iv)). We detected more differences among the three cell populations compared to the patterns from the anti-CD206 antibody experiment. These pattern changes depending on the macrophage cell population resulted in a peak shift to higher fluorescence intensity in the order M1 < M0 << M2. Such pattern changes simply indicated that more binding could occur for the molecular sensor.Notably, we found a much clearer peak shift, M1 < M0 << M2, under high-modification and high-dose conditions (Figure 7(b)(iv)). Previous studies have already shown that M2 and the remaining two macrophage populations can be distinguished through high-throughput analysis using antibodies, but further differentiation can be achieved through additional approaches such as expression profiling. Therefore, we speculated that RFP variants in the tetrameric state achieve multivalent mannose binding, thereby reflecting the amount of receptor present on the cell surface and more sensitively detecting subtle differences. We attempted to introduce a dual indicator for the three polarization states of detailed macrophage differentiation. As shown in Figure 5, we considered that FSC may be a more promising marker. We combined FSC and the fluorescence intensity from each channel to create a dot plot graph for further validation (not shown). We were able to recognize three distinct populations, highlighted by P1, P2, and P3, for each FP variant-based molecular sensor treatment. Obvious population changes were detected, as highlighted by arrows or quartile bars. We calculated the percentages and abundance ratios among the three populations for each treatment, demonstrating that these ratios, while somewhat complex for interpretation of macrophage cellular characteristics, provide useful information due to the wide variety of ratios obtained (not shown).

[0077] Conclusion: Macrophages exhibit polarization from the M0 state to M1 and M2 states when confronted with different inflammatory conditions. Rapid identification of macrophage polarization states is important for identifying inflammatory conditions or diagnosing disease states. Common macrophage identification methods utilize fluorescent dyes to stain cells or fluorescently labeled antibodies to bind to specific receptors expressed on the cell surface, but these antibodies are expensive and require special care when handled. In cases where these methods are still insufficient, additional approaches are used to monitor the expression profile of polarization markers. In this study, we presented a simple approach to overcome these challenges by developing molecular sensors containing FPs. Since CD206 is one of the major cell surface markers of polarized M2 macrophages that recognizes mannose as part of the ligand molecule, we achieved mannose modification of conventional FPs to obtain molecular sensors based on GFP and RFP variants. We utilized the reaction of mannose derivatives with amine groups on the FP surface for conjugation. The many modifiable sites on the FP surface allowed us to assemble FPs with varying degrees of mannose loading. We expected that the optimized conjugates would perform molecular sensors targeting the CD206 receptor on polarized macrophages with efficient multivalent binding, demonstrating highly sensitive detection of CD206 expression. Initial investigations of the binding properties of each FP conjugate using Con A agarose revealed, as expected, diverse results. More modified FPs exhibited stronger binding to Con A agarose. Comparing GFP and RFP, RFP may have a higher affinity for Con A than GFP. As a result, we found that high-dose treatment of the mannosylated RFP variant-based molecular sensor enabled highly sensitive discrimination between M2 macrophages and M0 and M1 macrophages, but only marginally between GFP variants.We observed that the binding profiles of each FP to Con A differed, potentially due to the association preference of each FP—as a dimer for the GFP variant and as a tetramer for the RFP variant. Because GFP is known to have dose-dependent association properties in vitro, as previously described, we will establish the appropriate concentration of mannose-modified GFP in advanced association conditions applied to macrophages. However, at this stage, we have evaluated the significant mannose content of each FP variant, as well as the availability of detectable but distinct multivalent binding to CD206, which enhances binding in different ways. Furthermore, previous studies on delivery systems employed to target the mannose receptor were based on the assumption that not only mannose but also mannose moieties contained in glycans can be recognized by the receptor. We propose that our approach is useful for identifying macrophage types because it is a simple, cost-effective, time-saving, and sensitive throughput method. Our approach could be more applicable if we could utilize FP variants modified with aptamers or potential ligands specific to proteins expressed on the surface of target cells. We could also utilize various FPs depending on the expression profile of the target protein. Our approach could also be considered as one of the strategies for the Sustainable Development Goals (SDGs). In this case, FPs can be produced economically using a bacterial transformation system, reducing the invasive use of animals (in antibody production) and increasing the use of FPs, which are an underutilized resource.

Claims

1. A fluorescent protein chemically modified with a sugar that binds to a C-type lectin receptor, wherein the sugar that binds to the C-type lectin receptor is capable of binding to a C-type lectin receptor expressed in macrophages, and the fluorescent protein chemically modified with the sugar is formed by binding the fluorescent protein to the sugar that binds to the C-type lectin receptor directly or via a linker.

2. The fluorescent protein of claim 1, wherein the C-type lectin receptor is CD206, CD205, CD209, CD301, Mincle, Lox, CLEC, MGL, DC-IR, DC-SIGN, dectin, selectin, CD23, CD69, CD93, C161, or CD302.

3. The fluorescent protein according to claim 1, wherein the sugar that binds to the C-type lectin receptor is a monosaccharide selected from the group consisting of glucose, galactose, mannose, xylose, fructose, rhamnose, arabinose, allose, altrose, idose, N-acetylglucosamine, N-acetylgalactosamine, talose, glucuronic acid, glucosamine, galactosamine, N-acetylneuraminic acid, and fucose, or two or more of these monosaccharides, whether identical or different, bound together.

4. The C-type lectin receptor is a Ca ２＋ It has a Ca binding motif ２＋ When the binding motif is an EPN (Glu-Pro-Asn) motif, the sugar that binds to the C-type lectin receptor is mannose, and Ca ２＋ 2. The fluorescent protein according to claim 1, wherein when the binding motif is a QPD (Gln-Pro-Asp) motif, the sugar that binds to the C-type lectin receptor is galactose.

5. The fluorescent protein of claim 1, wherein the sugar that binds to the C-type lectin receptor is mannose, and mannose can bind to CD206 expressed in macrophages.

6. The fluorescent protein according to any one of claims 1 to 5, wherein the fluorescent protein is a green fluorescent protein (GFP), a red fluorescent protein (RFP), a blue fluorescent protein (BFP), a yellow fluorescent protein (YFP), or an orange fluorescent protein (OFP).

7. The fluorescent protein according to any one of claims 1 to 6, wherein the fluorescent protein is a green fluorescent protein (GFP) that has an amino acid sequence that is 90% or more identical to the amino acid sequence set forth in SEQ ID NO: 1 and emits fluorescence, or a red fluorescent protein (RFP) that has an amino acid sequence that is 90% or more identical to the amino acid sequence set forth in SEQ ID NO: 2 and emits fluorescence.

8. A fluorescent protein according to any one of claims 1 to 7, wherein the fluorescent protein chemically modified with sugars has an average of 1 to 25 sugar bonds per fluorescent protein molecule.

9. The fluorescent protein according to any one of claims 1 to 8, wherein the fluorescent protein chemically modified with sugar forms a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer.

10. The fluorescent protein according to any one of claims 1 to 9, wherein the fluorescent protein chemically modified with a sugar is formed by binding an amino group, a thiol group, or a carboxyl group contained in the fluorescent protein to the sugar that binds to the C-type lectin receptor, either directly or via a linker.

11. The fluorescent protein chemically modified with a sugar is characterized in that an amino group contained in the fluorescent protein is bound to the sugar that binds to the C-type lectin receptor either directly or via a linker, and the bond is an amide bond, thiourea bond, urea bond, sulfonamide bond, amino bond, imine bond, amidine bond, phosphonamide bond, thioether bond, or urethane bond, the fluorescent protein according to any one of claims 1 to 10.

12. A composition for identifying the polarization of macrophages, comprising the fluorescent protein according to any one of claims 1 to 11.

13. The composition according to claim 12, wherein the concentration of the fluorescent protein chemically modified with the sugar in the composition is at least 2 μM.

14. A method for identifying the polarization of macrophages, comprising the steps of contacting macrophages with a fluorescent protein chemically modified with a sugar that binds to a C-type lectin receptor, and measuring the fluorescence of the fluorescent protein chemically modified with the sugar, wherein the sugar that binds to a C-type lectin receptor is capable of binding to a C-type lectin receptor expressed in macrophages, and the fluorescent protein chemically modified with the sugar is formed by reacting a fluorescent protein with a sugar that binds to a C-type lectin receptor or a modifying agent containing a sugar that binds to a C-type lectin receptor.

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