Novel adhesion molecules

Abstract

To simply utilize a biocompatible material such as chitin or chitosan as a cell culture surface and to provide a fusion protein simply utilizable for purification and proliferation of an objective cell.SOLUTION: The present invention provides a fusion protein comprising a region consisting of at least one peptide capable of adhering to chitins and a region consisting of at least one peptide or protein capable of adhering to cells.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a cell adhesive substance capable of adhering to both chitins and cells. [Background technology]

[0002] It is known that cell culture can be achieved by adding collagen to the cell culture surface to impart cell adhesion, but collagen is a heterologous protein, and there are concerns about its infectivity. Furthermore, many cell adhesion proteins, such as collagen, have low specificity and cannot be used for selective cell culture.

[0003] Chitin, chitosan, or their derivatives are known as biocompatible materials and are widely used in various medical fields. However, chitin and chitosan do not have cell adhesion properties and cannot be used directly as cell culture surfaces. Chitin-binding peptides are known to be used primarily in protein purification (Non-Patent Document 1), but their use as cell adhesion proteins has not been known.

[0004] Patent Document 1 describes a cell culture carrier that includes a substrate containing a polyuronic acid such as alginic acid as a biodegradable polymer having anionic groups, and a coating portion provided on at least a portion of the substrate and containing chitosan as a biodegradable polymer having cationic groups to which cell adhesive groups are bound, the substrate having a crosslinked portion that is ionically crosslinked with the biodegradable polymer having cationic groups (claims 1 to 4 of Patent Document 1).However, there is no description of using chitosan as a cell culture surface without binding cell adhesive groups to chitosan by condensation, or coating chitin or chitosan with a cell adhesive protein to use as a cell culture surface, or selectively culturing cells using a highly specific cell adhesive protein. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2022-126087 [Non-patent literature]

[0006] [Non-Patent Document 1] Methods in Enzymology, Volume 559, 2015, Pages 111-125 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a fusion protein that can be easily used to purify and grow target cells, and to provide a biocompatible material such as chitin or chitosan that can be easily used as a cell culture surface. [Means for solving the problem]

[0008] The present inventors discovered that desired cells can be selectively purified and grown by coating a biocompatible material such as chitin or chitosan with a fusion protein combining a peptide capable of adhering to chitins with a peptide or protein capable of adhering to cells. Based on this finding, the inventors conducted further research and completed the present invention.

[0009] That is, the present invention relates to the following. [1] A fusion protein comprising a region consisting of at least one peptide capable of adhering to chitin and a region consisting of at least one peptide or protein capable of adhering to cells. [2] The fusion protein according to [1], wherein the chitins are chitin, chitosan, or derivatives thereof. [3] The fusion protein according to [1], wherein the peptide capable of adhering to chitins comprises the amino acid sequence represented by TTNPGVSAWQVNTAYTAGQLVXYNGKTYK (SEQ ID NO: 1) (X represents any amino acid). [4] The fusion protein according to [3], wherein the peptide capable of adhering to chitins comprises the amino acid sequence represented by TTNPGVSAWQVNTAYTAGQLVTYNGKTYK (SEQ ID NO: 2) or TTNPGVSAWQVNTAYTAGQLVIYNGKTYK (SEQ ID NO: 3). [5] The fusion protein according to any one of [1] to [4], wherein the protein capable of adhering to cells is an antibody, a DARPins, or another target-binding protein. [6] The peptide capable of adhering to cells is RGD, GRGDS (SEQ ID NO: 4), YIGSR (SEQ ID NO: 5), IKVAV (SEQ ID NO: 6), VTCG (SEQ ID NO: 7), AGTFALRGDNPQG (SEQ ID NO: 8), DYATLQLQEGRLHFMFDLG (SEQ ID NO: 9), SYWYRIEASRTG (SEQ ID NO: 10), RKRLQVQLSIRT (SEQ ID NO: 11), KNSFMALYLSKGRLVFALG (SEQ ID NO: 12), RQVFQVAYIIIKA (SEQ ID NO: 13), KAFDITYVRLKF (SEQ ID NO: 14), or The fusion protein according to any one of [1] to [4], which contains an amino acid sequence represented by SEQ ID NO: 14), EPDIM (SEQ ID NO: 15), NKDIL (SEQ ID NO: 16), GRKRK (SEQ ID NO: 17), KYGAASIKVAVSADR (SEQ ID NO: 18), NGEPRGDTYRAY (SEQ ID NO: 19), PQVTRGDVFTM (SEQ ID NO: 20), AVTGRGDSPASS (SEQ ID NO: 21), TGRGDSPA (SEQ ID NO: 22), or CTGRGDSPAC (SEQ ID NO: 23), or polylysine, polyarginine, or polyornithine. [7] The fusion protein according to any one of [1] to [4], wherein the protein capable of adhering to cells is a cell adhesion molecule, a partial sequence thereof, or a derivative thereof selected from extracellular matrices such as fibronectin, laminin, or vitronectin, protein ligands such as cadherin, Delta / Notch, the immunoglobulin superfamily, cytokines, growth factors, and lectins. [8] A fusion protein according to any one of [1] to [7], which contains a sequence for the purpose of purification or detection, a sequence for improving solubility or hydrophilicity / hydrophobicity, or a sequence for use as a spacer / linker between the region consisting of a peptide capable of adhering to chitins and the region consisting of a peptide or protein capable of adhering to cells. [9] The fusion protein according to any one of [1] to [8], wherein the cell is an animal cell that is purified and cultured.

[10] A method for coating a fusion protein-chitin complex, comprising: The method includes coating a scaffold containing chitins with the fusion protein described in [1] to [9], or coating a scaffold having at least a portion of its surface containing one or more types of polysaccharides containing chitins with the fusion protein described in [1] to [9], or coating a scaffold that is a substance that interacts with chitins with a complex of chitins and the fusion protein described in [1] to [9].

[11] The method according to

[10] , wherein the scaffold material containing chitins is non-cell-adhesive and has a surface coated with at least one type of polysaccharide containing chitins.

[12] The method according to

[10] , wherein the scaffold that interacts with chitin is a gel formed from an anionic polysaccharide such as alginate gel.

[13] The method according to

[10] , wherein the scaffold material has a plate-like, fiber-like, knitted, or microcarrier-like shape.

[14] A method for recovering a target cell, the method comprising: Coating a scaffold containing chitins with the fusion protein according to [8] or [9], or coating a scaffold having at least a portion of its surface made of polysaccharides containing one or more chitins with the fusion protein according to [8] or [9], or coating a scaffold that is a substance that interacts with chitins with a complex of chitins and the fusion protein according to [8] or [9], incubating cells on the fusion protein-coated scaffold; and purifying the cells of interest captured by the fusion protein; It encompasses The method according to [8] or [9], wherein the cell-adhering protein of the fusion protein specifically binds to a target cell.

[15] A method for growing a target cell, comprising: Coating a scaffold containing chitins with the fusion protein according to [8] or [9], or coating a scaffold having at least a portion of its surface made of polysaccharides containing one or more chitins with the fusion protein according to [8] or [9], or coating a scaffold that is a substance that interacts with chitins with a complex of chitins and the fusion protein according to [8] or [9], incubating cells on the fusion protein-coated scaffold; Purifying the cells of interest captured by the fusion protein; and growing cells on the fusion protein-coated scaffold; It encompasses The method according to [8] or [9], wherein the cell-adhering protein of the fusion protein specifically binds to a target cell. [Effects of the Invention]

[0010] By using the fusion protein of the present invention, desired cells can be purified and grown simply and selectively using biocompatible materials such as chitin and chitosan as cell culture surfaces. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows a schematic diagram of the fusion protein of the present invention and its mode of use. The upper diagram of FIG. 1 is a schematic diagram of the fusion protein of the present invention, which contains a "peptide or protein capable of adhering to cells" at the N-terminus and a "peptide capable of adhering to chitin" at the C-terminus. The left diagram of FIG. 1 shows a scaffold material that is a spherical alginate gel or microcarrier, the surface of which is coated with chitin, and the fusion protein of the present invention is bound to the scaffold material. The right diagram of FIG. 1 shows a non-cell-adhesive scaffold material that is coated with chitin, and the fusion protein of the present invention is bound to the scaffold material. [Figure 2]FIG. 2 shows a specific embodiment of a fusion protein of the present invention and its three-dimensional structure. The upper diagram of FIG. 2 shows a specific example of a fusion protein of the present invention, which contains an RGD sequence at the N-terminus as a "peptide or protein capable of adhering to cells" and a chitin-binding domain at the C-terminus as a "peptide capable of adhering to chitins." The middle diagram of FIG. 2 shows another specific example of a fusion protein of the present invention, which contains, from the N-terminus, an anti-HER2 antibody as a "peptide or protein capable of adhering to cells," followed by a FLAG® tag for fusion protein purification and a 6×His tag for fusion protein detection, and finally a chitin-binding domain at the C-terminus as a "peptide capable of adhering to chitins." The lower left diagram of FIG. 2 shows the three-dimensional structure of the fusion protein of the present invention shown in the upper diagram of FIG. 2. The lower right diagram of FIG. 2 shows the three-dimensional structure of the fusion protein of the present invention shown in the middle diagram of FIG. 2. [Figure 3] Figure 3 shows the results of chitosan coating with a fusion protein of the present invention (peptide 1 or peptide 2). The fusion protein of the present invention (peptide 2 (also referred to as sequence 2 in the figure)) is N-terminally labeled with the fluorescent dye FAM, contains an RGD sequence at the N-terminus as a "peptide or protein capable of adhering to cells," contains a chitin-binding domain at the C-terminus as a "peptide capable of adhering to chitins," and contains a linker between the "peptide or protein capable of adhering to cells" and the "peptide capable of adhering to chitins." Figure 3 also shows the presence or absence of fluorescent dye emission in the presence or absence of the fusion protein of the present invention (photograph). The scaffold is non-cell-adhesive and has a surface coated with chitin, and the fusion protein of the present invention binds to the scaffold and emits light. [Figure 4A] FIG. 4A shows human cell adhesion on chitosan in the presence or absence of the fusion protein of the present invention (peptide 2) (photograph). [Figure 4B] FIG. 4B shows the number of human cells attached on chitosan in the presence or absence of the fusion protein of the present invention (peptide 2). [Figure 5A] FIG. 5A shows human cell adhesion on chitosan in the presence or absence of the fusion protein of the present invention (peptide 3) (photograph). [Figure 5B] FIG. 5B shows the number of human cells attached on chitosan in the presence or absence of the fusion protein of the present invention (peptide 3). [Figure 6] Figure 6 shows the adhesion of the fusion protein of the present invention (peptide 2) to a scaffold made of calcium alginate beads coated with chitosan. From left to right, the results are shown for a group coated with chitosan solution only, a group coated with chitosan solution and a chitosan-free solution (3:1), a group coated with chitosan solution and a chitosan-free solution (1:1), a group coated with chitosan solution and a chitosan-free solution (1:3), and a group coated with a chitosan-free solution only. The upper panel of Figure 6 shows the phase contrast results, and the lower panel of Figure 6 shows the fluorescence results (photographs). [Figure 7] Figure 7 shows the results of culturing on a scaffold made of calcium alginate beads coated with chitosan, without and with the fusion protein of the present invention (peptide 2). [Figure 8] Figure 8 shows the specific binding between the fusion protein of the present invention (peptide 3) using an antibody as the "peptide capable of adhering to cells" and target cells. The scaffold is calcium alginate beads, the surface of which is coated with chitosan. The fusion protein of the present invention binds to the scaffold, and cells also bind to it. Figure 8 shows the results of culturing with and without the fusion protein of the present invention (peptide 3). [Figure 9A] FIG. 9A shows the results of human cell adhesion after coating chitosan with the fusion proteins of the present invention (peptide 3 and peptide 2) (also referred to as sequence 3 and sequence 2, respectively) (photograph). [Figure 9B] FIG. 9B shows the results of human cell proliferation after coating chitosan with the fusion proteins of the invention (peptide 3 and peptide 2). [Figure 9C] FIG. 9C shows the percentage of human cells after coating chitosan with the fusion proteins of the invention (peptide 3 and peptide 2). [Figure 10A]FIG. 10A shows the results of bovine cell adhesion after coating of chitosan with the fusion protein of the invention (peptide 2) (photograph). [Figure 10B] FIG. 10B shows the results of bovine cell proliferation after coating of chitosan with the fusion protein of the invention (peptide 2) (photograph). DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in detail below.

[0013] Fusion proteins of the present invention The present invention relates to a fusion protein (also referred to as an adhesion factor of the present invention) comprising at least one domain consisting of a peptide capable of adhering to chitins and at least one domain consisting of a peptide or protein capable of adhering to cells. The fusion protein of the present invention can comprise one or more "domains consisting of a peptide capable of adhering to chitins" and one or more "domains consisting of a peptide or protein capable of adhering to cells." In the present invention, chitins are chitin, chitosan, or derivatives thereof. Chitin is a type of mucopolysaccharide, a natural material found in organisms such as shrimp, crabs, insects, mushrooms, and shellfish. Chitin is a polymer of N-acetylglucosamine, consisting of 90% N-acetylglucosamine and 10% glucosamine. Chitosan can be obtained by hydrolyzing and deacetylating chitin. Chitosan sugar chains are also known to have intercellular recognition ability and can be used to immobilize the fusion protein of the present invention. Chitin derivatives are not particularly limited as long as they are derivatives of aminopolysaccharides in which N-acetyl-D-glucosamine is linked in a long chain, but specifically refer to chitin in which the hydroxyl groups have been esterified or the N-acetyl groups have been removed. Chitosan derivatives include phosphorylated chitosan, glycol chitosan, hydroxyethyl chitosan, hydroxypropyl chitosan, trimethyl chitosan, carboxymethyl chitosan, chitosan dimer dihydrochloride, glycochitosan methacrylate, chitosan oligosaccharide lactate, and the like.

[0014] In one embodiment of the present invention, the "peptide capable of adhering to chitins" comprises the amino acid sequence of a chitin-binding domain represented by TTNPGVSAWQVNTAYTAGQLVXYNGKTYK (SEQ ID NO: 1), where X represents any amino acid, and is preferably T or I. Threonine (T) has a hydroxyethyl group in its side chain and is classified as a polar, uncharged amino acid. Threonine residues are susceptible to numerous post-translational modifications, such as phosphorylation. Isoleucine (I) has a sec-butyl group in its side chain and is an amino acid with a hydrophobic side chain. Alanine, like isoleucine, is a hydrophobic amino acid, and is used in the alanine scanning method, in which each side chain residue in a protein is mutated to alanine to create mutants, thereby site-specifically identifying residues important for protein structure and function. Since the X portion is replaced with isoleucine, which has a significantly different function from threonine, it is thought to have a low contribution to binding to chitin, and therefore can be any amino acid sequence. Therefore, in one embodiment of the present invention, the "peptide capable of adhering to chitins" comprises the amino acid sequence of a chitin-binding domain represented by TTNPGVSAWQVNTAYTAGQLVTYNGKTYK (SEQ ID NO: 2) or TTNPGVSAWQVNTAYTAGQLVIYNGKTYK (SEQ ID NO: 3). In another embodiment of the present invention, a sequence known to those skilled in the art as a chitin binding protein or a part thereof can be used as a "peptide capable of adhering to chitins."

[0015] In one embodiment of the present invention, a "peptide or protein capable of adhering to cells" is a peptide or protein capable of specifically binding to a cell of interest, and an antibody can be used, for example. The target site of the "peptide or protein capable of adhering to cells" can be of any type as long as it is expressed on the surface of the cells to be purified and grown, but for example, a protein or sugar chain expressed on the cell surface can be used as the target site. More specifically, target-binding proteins such as antibodies against receptors specifically expressed on the surface of cells, DARPins, and ligands or portions of ligands that bind to receptors can be used as "proteins capable of adhering to cells." In the present invention, antibodies that can be used include full-length polyclonal or monoclonal antibodies, antigen-binding fragments thereof (Fab, Fab', F(ab')2, Fv, etc.), single-chain (scFv) antibodies, VHHs, heavy-chain antibodies, diabodies, etc., but scFVs and VHHs with small molecular weights are preferred so that they can be expressed in E. coli, etc. In the fusion protein of the present invention, by using the above-mentioned antibody as the "protein capable of adhering to cells," the specificity of the fusion protein of the present invention to the target cells can be increased, and as a result, the fusion protein can specifically bind to the target cells, which can be selected, purified, and cultured. In one embodiment of the present invention, the "protein capable of adhering to cells" is, for example, an antibody against human epidermal growth factor receptor type 2 (HER2). For example, a VHH antibody against HER2 (selected from the VHH antibody library owned by RePHAGEN Inc.) can be used to select and culture only HER2-positive cells.

[0016] In another embodiment of the present invention, the "peptide capable of adhering to cells" may be, for example, a sequence commonly used as a peptide capable of adhering to cells, such as RGD, GRGDS (SEQ ID NO: 4), YIGSR (SEQ ID NO: 5), IKVAV (SEQ ID NO: 6), VTCG (SEQ ID NO: 7), AGTFALRGDNPQG (SEQ ID NO: 8), DYATLQLQEGRLHFMFDLG (SEQ ID NO: 9), SYWYRIEASRTG (SEQ ID NO: 10), RKRLQVQLSIRT (SEQ ID NO: 11), KNSFMALYLSKGRLVFALG (SEQ ID NO: 12), RQVFQVAYIIIK Amino acid sequences represented by A (SEQ ID NO: 13), KAFDITYVRLKF (SEQ ID NO: 14), EPDIM (SEQ ID NO: 15), NKDIL (SEQ ID NO: 16), GRKRK (SEQ ID NO: 17), KYGAASIKVAVSADR (SEQ ID NO: 18), NGEPRGDTYRAY (SEQ ID NO: 19), PQVTRGDVFTM (SEQ ID NO: 20), AVTGRGDSPASS (SEQ ID NO: 21), TGRGDSPA (SEQ ID NO: 22), CTGRGDSPAC (SEQ ID NO: 23), or polylysine, polyarginine, polyornithine, etc. can be used.

[0017] In yet another embodiment of the present invention, the "peptide or protein capable of adhering to cells" may be a cell adhesion molecule, a partial sequence thereof, or a derivative thereof selected from extracellular matrices such as fibronectin, laminin, or vitronectin, cadherin, Delta / Notch, the immunoglobulin superfamily, protein ligands such as cytokines and growth factors, and lectins. Fibronectin is a major glycoprotein that constitutes the extracellular matrix and is involved in cell adhesion, spreading, migration, proliferation, and differentiation. Laminin is the main component of the basement membrane of most tissues and is involved in cell differentiation, proliferation, and cancer metastasis. Vitronectin is a type of glycoprotein present in serum and the extracellular matrix that promotes cell adhesion and spreading and is also used as a cell culture substrate. In the fusion protein of the present invention, by using fibronectin, laminin, or vitronectin as the "cell-adhesive peptide," the fusion protein can adhere to cells that have fibronectin, laminin, or vitronectin receptors, respectively. Although these cell adhesion molecules have low cell specificity, they are preferred because they are not derived from heterologous species.

[0018] In one embodiment of the present invention, the fusion protein of the present invention comprises a "peptide capable of adhering to chitins" and a "peptide or protein capable of adhering to cells" linked together by a linker such as a peptide. In the present invention, the peptide linker is 5 to 100 amino acids long, and the sequence of the peptide linker is represented, for example, by GGGGSGGGSGGGGS (SEQ ID NO: 24).

[0019] In the present invention, the term "cells" refers to cells that can specifically or nonspecifically bind to the "peptide or protein capable of adhering to cells" in the fusion protein of the present invention and are grown by culturing on a scaffold. More specifically, cells have on their surface binding sites, receptors, etc. that are recognized and bound by the "peptide or protein capable of adhering to cells." The fusion protein of the present invention can bind to cells via a "peptide or protein capable of adhering to cells." The cells are, for example, target cells that require purification, target cells that require cell culture, or target cells that require cell culture after purification. If the "protein capable of adhering to cells" is, for example, a highly selective sequence such as an antibody, it will specifically bind to the target cells, resulting in the purification of the target cells and their selective cultivation. In the present invention, proliferation of target cells refers to the adhesion of the fusion protein of the present invention to the scaffold material or the adhesion of the fusion protein of the present invention to the target cells, followed by proliferation of the target cells.

[0020] In the present invention, the type of cells to be purified and expanded is not particularly limited, and examples include stem cells (e.g., pluripotent stem cells, differentiated cells derived from pluripotent stem cells, mesenchymal stem cells, myoblasts, hematopoietic stem cells, etc.), somatic cells (e.g., muscle cells, blood cells (T cells, B cells, etc.), fibroblasts, nervous system cells, epidermal cells, epithelial cells, endothelial cells, bone cells, chondrocytes, adipocytes, etc.). In the present invention, the cells to be purified and expanded may be cells derived from any organism. Examples of such cells include cells derived from animals such as mammals, insects, yeast, and E. coli. Preferably, in the present invention, the mammalian cells are cells derived from humans or cows. More preferably, the cells to be purified and expanded in the present invention are human cells differentiated from human mesenchymal stem cells, such as human mesenchymal stem cells, human myoblasts, human T cells, or human muscle cells.

[0021] Method for coating the fusion protein-chitin complex of the present invention Methods for coating the fusion protein-chitin complex of the present invention include coating a scaffold containing chitin with the fusion protein of the present invention described above, or coating a scaffold having at least a portion of its surface made of a polysaccharide containing one or more types of chitin with the fusion protein of the present invention described above, or coating a scaffold that is a substance that interacts with chitin with a complex of chitin and the fusion protein of the present invention described above.

[0022] By coating a scaffold with the fusion protein of the present invention, it becomes possible for both chitin and cells to adhere, and the target cells can grow on the scaffold.

[0023] In another embodiment of the method for coating a fusion protein-chitins complex of the present invention, the scaffold is non-cell adhesive and has a surface coated with at least one type of polysaccharide containing chitins. Specifically, coating a non-cell-adhesive scaffold with chitosan can be carried out by contacting the culture substrate with a chitosan solution by painting, spraying, immersion, or other procedures. Thereafter, by further coating the scaffold with the fusion protein of the present invention, it becomes possible for both chitin and cells to adhere, and the target cells can be grown on the scaffold.

[0024] In the method for coating a fusion protein-chitin complex of the present invention, the scaffold may be a substance that interacts with chitin, such as a gel formed from anionic polysaccharides such as alginate gel, a beaded alginate gel, or a microcarrier, or a conventional flat scaffold. These scaffolds are not limited as long as they contain chitin on their surface or can be coated with chitin. Alginate gel can be made by using sodium alginate as a gelling agent and reacting it with calcium ions. When sodium alginate aqueous solution is reacted with calcium ions, ionic cross-linking occurs quickly, resulting in gelation. The gelation time can be controlled by suppressing calcium ionization. In the present invention, the alginate gel is, for example, a beaded alginate gel. For example, a kit for easily preparing beaded alginate gel can be used for three-dimensional culture of cells with anchorage-independent growth ability. Alginate gels in the presence of calcium and becomes a solution upon addition of a chelating agent, allowing for easy cultivation and recovery of target cells after purification. The shape of the alginate gel may be, for example, a dish, a fiber, a knit, or a microcarrier.

[0025] Microcarriers can be used as scaffolds to support cells in the medium when cells are suspended and cultured in the medium in three-dimensional culture methods. Adding microcarriers to the medium can promote cell culture. The shape of the microcarrier may be any of spherical, polygonal, conical, pyramidal, fractured, needle-like, etc., but spherical particles are preferred from the viewpoint of providing a wide surface area for adsorbing cells and suppressing cell damage. The average particle size of the microcarriers is preferably 10 to 500 μm, more preferably 50 to 300 μm, and even more preferably 100 to 250 μm.

[0026] In one embodiment of the method for coating the fusion protein-chitin complex of the present invention, alginate gel or beaded alginate gel may be used as a scaffold, but the process of coating the surface with chitin and then the fusion protein of the present invention can be carried out without using a condensing agent.

[0027] The method for recovering the target cells of the present invention comprises the following steps: Coating a scaffold containing chitins with the fusion protein of the present invention, or coating a scaffold having at least a portion of its surface made of polysaccharides containing one or more chitins with the fusion protein of the present invention, or coating a scaffold that is a substance that interacts with chitins with a complex of chitins and the fusion protein of the present invention, incubating cells on the scaffold; and purifying the cells of interest captured by the fusion protein; It encompasses The cell-adherent protein of the fusion protein of the present invention specifically binds to target cells.

[0028] Therefore, in such an embodiment, an antibody sequence, for example, can be used so that the "protein capable of adhering to cells" specifically binds to the target cells. Furthermore, in order to purify the target cells, the fusion protein of the present invention may contain, between the peptide capable of adhering to chitins and the peptide or protein capable of adhering to cells, a sequence for the purpose of purification or detection, a sequence for improving solubility or hydrophilicity / hydrophobicity, or a sequence for the purpose of spacer / linker; more specifically, it may contain a purification tag such as a FLAG (registered trademark) tag or a 6xHis tag.

[0029] In the method of the present invention for recovering target cells, the step of incubating the cells on the scaffold material can be carried out at 1°C or higher for 1 minute or more, for example, at 3 to 60°C for 5 minutes to 1000 hours, at 4 to 45°C for 10 minutes to 720 hours, or at 37°C for 1 to 700 hours.

[0030] In the method of the present invention for recovering target cells, the step of purifying the target cells can be carried out, for example, by coating the surface of chitosan or a derivative thereof with an adhesion factor having an amino acid sequence that specifically binds to an antigen present on the surface of the target cells and SEQ ID NO: 2, incubating with the cells, washing away non-adherent cells, and then performing an enzymatic treatment to recover the adherent cells.

[0031] Other steps are as described in the section on the method for coating the scaffold of the present invention.

[0032] The method for growing the cells of interest of the present invention comprises the steps of: Coating a scaffold containing chitins with the fusion protein of the present invention, or coating a scaffold having at least a portion of its surface made of polysaccharides containing one or more chitins with the fusion protein of the present invention, or coating a scaffold that is a substance that interacts with chitins with a complex of chitins and the fusion protein of the present invention, incubating cells on the scaffold; Purifying the cells of interest captured by the fusion protein; and Growing cells on the scaffold; It encompasses The cell-adherent peptide of the fusion protein of the present invention specifically binds to target cells.

[0033] Therefore, in such an embodiment, for example, an antibody sequence can be used so that the "protein capable of adhering to cells" specifically binds to the target cells, and in order to purify the target cells, the fusion protein of the present invention may contain a purification tag such as a FLAG (registered trademark) tag or a 6xHis tag between the peptide capable of adhering to chitins and the peptide or protein capable of adhering to cells.

[0034] In the method of the present invention for growing cells of interest, the step of growing cells on a scaffold material can be carried out at 1°C or higher for 1 minute or more, for example, at 3 to 60°C for 5 minutes to 1000 hours, at 18 to 45°C for 10 minutes to 720 hours, or at 37°C for 1 to 700 hours.

[0035] The method for growing cells of interest of the present invention may include a step of purifying the cells of interest after growing the cells on the scaffold. The step of purifying the cells of interest can be carried out as described in the section on the method for recovering cells of interest of the present invention.

[0036] Other steps are as described in the section on the method for coating a scaffold material of the present invention or the method for recovering target cells of the present invention. [Example]

[0037] The present invention will be described in more detail below based on examples, but it goes without saying that the present invention is not limited to these examples.

[0038] Example 1 (Coating of Chitosan Surfaces with Fusion Proteins of the Invention) 20 μL of 10 mg / mL chitosan solution was added to a portion of a 24-well plate and allowed to stand for at least 1 hour, followed by the addition of a solution of fluorescein-labeled peptides (Peptide 1 and Peptide 2 below) and allowing to stand for at least 1 hour. Peptide 1: TTNPGVSAWQVNTAYTAGQLVIYNGKTYK (SEQ ID NO: 3) Peptide 2: GRGDSGGGGSGGGGSGGGGSTTNPGVSAWQVNTAYTAGQLVTYNGKTYK (SEQ ID NO: 25) (a fusion protein of the present invention in which a "peptide capable of adhering to cells" is linked to a "peptide capable of adhering to chitins" via a peptide linker sequence) A control was also prepared without adding peptide. After washing with PBS, the area around the border between the plate coated with chitosan solution and the plate without was observed under a fluorescence microscope (Figure 3). Fluorescence was observed under the conditions where peptide 1 and peptide 2 were added, confirming that the peptides had been successfully coated onto the 24-well plate via chitosan.

[0039] Example 2 (Human cell adhesion after coating chitosan surfaces with the fusion protein of the present invention) A 10 mg / mL chitosan solution was added to a 24-well plate and allowed to stand for at least 1 hour. In condition 1, peptide 2 was added and allowed to stand for at least 1 hour. In condition 2, no peptide was added. In condition 3, a 24-well tissue culture plate was used without coating. Adipose-derived cells were seeded at 20,000 cells and cultured until the next day (37°C, 5% CO2). After incubation, the supernatant was discarded, and 4% paraformaldehyde was added and allowed to stand for 10 minutes. After washing with PBS, 0.2% Triton X-100 / PBS was added and allowed to stand for 10 minutes. After washing with PBS, nuclear staining was performed with DAPI (2 μg / mL), and the cells were counted. Under condition 1, with the addition of peptide 2, cells spread and many cells adhered. However, under the condition without peptide 2, cells did not spread and sufficient cell adhesion was not observed (Figures 4A and 4B).

[0040] Example 3 (Human cell adhesion after coating chitosan surfaces with the fusion protein of the present invention) A 10 mg / mL chitosan solution was added to a 24-well plate and allowed to stand for at least 1 hour. Subsequently, in condition 1, peptide 3 (see below) was added and allowed to stand for at least 1 hour. In condition 2, no peptide was added. In condition 3, a 24-well tissue culture plate was used without coating. BT474 cells (HER2-positive human breast cancer cells) were seeded at 200,000 cells and cultured until the next day (37°C, 5% CO2). After incubation, the supernatant was discarded, and 4% paraformaldehyde was added and allowed to stand for 10 minutes. After washing with PBS, 0.2% Triton X-100 / PBS was added and allowed to stand for 10 minutes. After washing with PBS, nuclear staining was performed with DAPI (2 μg / mL), and the cell numbers were counted. Many cells adhered when peptide 3 was added, but not enough cell adhesion was observed without peptide 3 (Figures 5A and 5B). Peptide 3: Anti-HER2 antibody sequence +DYKDHDGDYKDHDIDYKDDDDKLEHHHHHHGGGGS TTNPGVSAWQVNTAYTAGQLVTYNGKTYK (SEQ ID NO: 26) (a sequence in which a FLAG (registered trademark) tag and a 6xHis tag are added to the N-terminal side of SEQ ID NO: 2)

[0041] Example 4 (Coating of calcium alginate beads with a fusion protein of the invention) Calcium alginate beads were left to stand for at least one hour under either Condition 1 (chitosan solution added) or Condition 2 (no chitosan solution added). A solution of fluorescein-labeled peptide 2 was then added and left to stand for at least one hour. After washing, the bead solution under Condition 1 and the bead solution under Condition 2 were mixed in ratios of 1:0, 3:1, 1:1, 1:3, and 0:1, and images were taken using a fluorescence microscope (Figure 6). Fluorescence was only observed in the bead solution under Condition 1, which contained chitosan, confirming that the calcium alginate had been coated with the peptide.

[0042] Example 5 (Human cell proliferation after coating calcium alginate beads with a fusion protein of the invention) Chitosan solution was added to calcium alginate beads and allowed to stand for at least one hour. Then, either Condition 1 (Peptide 2 solution was added and allowed to stand for at least one hour) or Condition 2 (No peptide added) were prepared. Then, 50,000 adipose-derived stem cells were seeded in a 24-well plate and cultured for four days (37°C, 5% CO2). Cell proliferation was superior when Peptide 2 was added (Figure 7).

[0043] Example 6 (Human cell proliferation after coating calcium alginate beads with a fusion protein of the invention) Chitosan solution was added to calcium alginate beads and allowed to stand for at least one hour. Then, either Condition 1 (peptide 3 solution was added and allowed to stand for at least one hour) or Condition 2 (no peptide added) were prepared. BT474 cells were then seeded at 50,000 cells in a 24-well plate and cultured for 7 days (37°C, 5% CO2). Cell proliferation was superior under the condition with peptide 3 added (Figure 8).

[0044] Example 7 (Human cell proliferation after coating chitosan surfaces with the fusion protein of the present invention) A 10 mg / mL chitosan solution was added to a 24-well plate and allowed to stand for at least 1 hour. Subsequently, peptide 2 or peptide 3 solution was added and allowed to stand for at least 1 hour. Then, 120,000 adipose-derived stem cells and BT474 cells were seeded. Similarly, cells were seeded into an uncoated 24-well plate for tissue culture. The plates were cultured until the next day (37°C, 5% CO2). The culture supernatant was removed, 4% paraformaldehyde was added, and discarded after 10 minutes. The plates were washed with 1% BSA / PBS, and 0.2% Triton X-100 / PBS was added and discarded after 10 minutes. A rabbit anti-HER2 antibody was added as the primary antibody and allowed to stand for at least 1 hour. After washing with 1% BSA / PBS, an Alexa488-labeled anti-rabbit antibody and DAPI were added as the secondary antibody and allowed to stand for at least 1 hour. The plates were then washed and observed (Figure 9A). Coating with chitosan and peptide 3 resulted in a higher rate of HER2-positive cell adhesion compared to other conditions (FIGS. 9B and 9C).

[0045] Example 8 (Bovine cell growth after coating chitosan surfaces with fusion proteins of the present invention) A 10 mg / mL chitosan solution was added to a 24-well plate and allowed to stand for at least 1 hour. Next, 10 μL of peptide 2 solution was added and allowed to stand for at least 1 hour. After washing with PBS, 150,000 bovine ES cells were seeded and cultured until the next day (37°C, 5% CO2). After washing with PBS, microscopic observation of the border between coated and uncoated plates confirmed that cells had adhered to the coated area (Figure 10A). Culture medium was added and culture was continued for another 6 days, and the adhered cells were observed to be proliferating (Figure 10B).

Claims

1. A fusion protein comprising a region consisting of at least one peptide capable of adhering to chitins, and a region consisting of at least one peptide or protein capable of adhering to cells.

2. The fusion protein according to claim 1, wherein the chitins are chitin, chitosan or derivatives thereof.

3. The fusion protein according to claim 1, wherein the peptide capable of adhering to chitins comprises the amino acid sequence represented by TTNPGVSAWQVNTAYTAGQLVXYNGKTYK (SEQ ID NO: 1) (X represents any amino acid).

4. The fusion protein according to claim 3, wherein the peptide capable of adhering to chitins comprises the amino acid sequence represented by TTNPGVSAWQVNTAYTAGQLVTYNGKTYK (SEQ ID NO: 2) or TTNPGVSAWQVNTAYTAGQLVIYNGKTYK (SEQ ID NO: 3).

5. The fusion protein of claim 1, wherein the protein capable of adhering to cells is an antibody, a DARPin, or other target-binding protein.

6. Peptides capable of adhering to cells include RGD, GRGDS (SEQ ID NO: 4), YIGSR (SEQ ID NO: 5), IKVAV (SEQ ID NO: 6), VTCG (SEQ ID NO: 7), AGTFALRGDNPQG (SEQ ID NO: 8), DYATLQLQEGRLHFMFDLG (SEQ ID NO: 9), SYWYRIEASRTG (SEQ ID NO: 10), RKRLQVQLSIRT (SEQ ID NO: 11), KNSFMALYLSKGRLVFALG (SEQ ID NO: 12), RQVFQVAYIIIKA (SEQ ID NO: 13), and KAFDITYVRLKF (SEQ ID NO: 14). 14), EPDIM (SEQ ID NO: 15), NKDIL (SEQ ID NO: 16), GRKRK (SEQ ID NO: 17), KYGAASIKVAVSADR (SEQ ID NO: 18), NGEPRGDTYRAY (SEQ ID NO: 19), PQVTRGDVFTM (SEQ ID NO: 20), AVTGRGDSPASS (SEQ ID NO: 21), TGRGDSPA (SEQ ID NO: 22), or CTGRGDSPAC (SEQ ID NO: 23), or polylysine, polyarginine, or polyornithine.

7. The fusion protein of claim 1, wherein the protein capable of adhering to cells is a cell adhesion molecule, a partial sequence thereof, or a derivative thereof selected from extracellular matrices such as fibronectin, laminin, or vitronectin, protein ligands such as cadherin, Delta / Notch, the immunoglobulin superfamily, cytokines, and growth factors, and lectins.

8. The fusion protein of claim 1, which contains a sequence for the purpose of purification or detection, a sequence for improving solubility or hydrophilicity / hydrophobicity, or a sequence for spacer / linker between the region consisting of a peptide capable of adhering to chitins and the region consisting of a peptide or protein capable of adhering to cells.

9. The fusion protein of claim 1, wherein the cell is an animal cell that is purified and cultured.

10. A method for coating a fusion protein-chitin complex, comprising the steps of: The method includes coating a scaffold containing chitins with the fusion protein described in claim 1, or coating a scaffold having at least a portion of its surface made of a polysaccharide containing one or more types of chitins with the fusion protein described in claim 1, or coating a scaffold that is a substance that interacts with chitins with a complex of chitins and the fusion protein described in claim 1.

11. The method according to claim 10, wherein the scaffold material containing chitins is non-cell-adhesive and has a surface coated with at least one type of polysaccharide containing chitins.

12. The method according to claim 10, wherein the scaffold that interacts with chitin is a gel formed from an anionic polysaccharide, such as an alginate gel.

13. The method of claim 10, wherein the scaffold is in the form of a dish, fiber, knit, or microcarrier.

14. 1. A method for recovering a cell of interest, the method comprising: Coating a scaffold containing chitins with the fusion protein of claim 8, or coating a scaffold having at least a portion of its surface made of polysaccharides containing one or more chitins with the fusion protein of claim 8, or coating a scaffold that is a substance that interacts with chitins with a complex of chitins and the fusion protein of claim 8, incubating cells on the fusion protein-coated scaffold; and purifying the cells of interest captured by the fusion protein; It encompasses The method according to claim 8, wherein the cell-adherent protein of the fusion protein specifically binds to a target cell.

15. 1. A method for expanding a cell of interest, comprising: Coating a scaffold containing chitins with the fusion protein of claim 8, or coating a scaffold having at least a portion of its surface made of polysaccharides containing one or more chitins with the fusion protein of claim 8, or coating a scaffold that is a substance that interacts with chitins with a complex of chitins and the fusion protein of claim 8, incubating cells on the fusion protein-coated scaffold; Purifying the cells of interest captured by the fusion protein; and growing cells on the fusion protein-coated scaffold; It encompasses The method according to claim 8, wherein the cell-adherent protein of the fusion protein specifically binds to a target cell.

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