Novel adhesion molecule

Abstract

The present invention addresses the problem of easily using biocompatible materials such as chitin and chitosan as cell culture surfaces, and the problem of providing a fusion protein that can be easily used for the purification and proliferation of target cells. The present invention provides a fusion protein that includes a region comprising a peptide that can be adhered to at least one or more chitins, and a region comprising a peptide or protein that can be adhered to at least one or more cells.

Description

Novel adhesion molecule

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

[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 lack cell adhesion properties and cannot be used directly as cell culture surfaces. Chitin-binding peptides are known to be primarily used 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 comprising 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 ionically crosslinked with the biodegradable polymer having cationic groups (claims 1 to 4 of Patent Document 1). However, Patent Document 1 does not describe the use of chitosan as a cell culture surface without binding cell adhesive groups to the chitosan by condensation, the use of chitin or chitosan as a cell culture surface by coating with a cell adhesive protein, or the selective culturing of cells using a highly specific cell adhesive protein.

[0005] Paragraph 0036 of Patent Document 2 describes a fusion protein of a chitin-binding protein and a single-chain antibody, and describes as the chitin-binding protein the amino acid sequences of SEQ ID NOs: 22 and 24, which contain the same sequence as the amino acid sequence of SEQ ID NO: 2 of the present application. However, the fusion protein described in Patent Document 2 is used to use a single-chain antibody that has binding ability to a subject to which cosmetics are applied (see paragraphs 0007 and 0008 of Patent Document 2), and binds to a human subject, such as skin, to which cosmetics are applied. Therefore, its structure and use are different from those of the fusion protein of the present invention, which, for example, determines the binding ability to a scaffold when cells adhere to the scaffold for cell culture.

[0006] Claims 26 and 30 of Patent Document 3 describe a cell targeting molecule comprising SEQ ID NO: 363, and paragraph 0741 of Patent Document 3 describes a chitin-binding domain, including exemplary cell targeting molecule 76 represented by SEQ ID NO: 363, which contains the chitin-binding domain represented by SEQ ID NO: 2 of the present application. However, its structure and use differ from those of the fusion protein of the present invention, which determines the binding ability of cells to a scaffold when they adhere to the scaffold for cell culture.

[0007] Japanese Patent Application Laid-Open No. 2022-126087 Japanese Patent Application Laid-Open No. 2002-363026 Special Publication No. 2020-513779

[0008] Methods in Enzymology, Volume 559, 2015, Pages 111-125

[0009] 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.

[0010] 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.

[0011] That is, the present invention relates to the following: [1] A fusion protein comprising a region consisting of at least one or more peptides capable of adhering to chitins, and a region consisting of at least one or more peptides or proteins 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] or [2], 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 peptide or protein capable of adhering to cells is an antibody, DARPins, or other target-binding protein. [6] The peptide or protein 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), E The fusion protein according to any one of [1] to [4], comprising PDIM (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), KGGPQVTRGDVFTMP (SEQ ID NO: 28), polylysine, polyarginine, or polyornithine.[7] The fusion protein according to any one of [1] to [4], wherein the cell-adhesive peptide or protein 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] The fusion protein according to any one of [1] to [7], wherein the fusion protein contains, between the region consisting of the peptide capable of adhering to chitins and the region consisting of 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 use as a spacer / linker. [9] The fusion protein according to any one of [1] to [8], wherein the cell is an animal cell to be purified and cultured.

[0012]

[10] A method for coating a fusion protein-chitin complex, comprising coating a scaffold containing chitins with the fusion protein of any one of [1] to [9], or coating a scaffold at least a portion of whose surface contains one or more chitin-containing polysaccharides with the fusion protein of any one of [1] to [9], or coating a scaffold that is a substance that interacts with chitins with a complex of chitins and the fusion protein of any one of [1] to [9].

[11] The method according to

[10] , wherein the chitin-containing scaffold is non-cell-adhesive and has a surface coated with at least one or more chitin-containing polysaccharides.

[12] The method according to

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

[13] The method according to

[10] , wherein the shape of the scaffold is dish-shaped, fibrous, knitted, or microcarrier-shaped.

[0013]

[14] A method for recovering target cells, 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 scaffold coated with the fusion protein; and purifying the target cells captured by the fusion protein, wherein the cell-adherent protein of the fusion protein according to [8] or [9] specifically binds to the target cells.

[0014]

[15] A method for growing cells of interest, comprising: coating a scaffold containing chitins with the fusion protein of [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 of [8] or [9]; or coating a scaffold that is a substance that interacts with chitins with a complex of chitins and the fusion protein of [8] or [9]; incubating cells on the scaffold coated with the fusion protein; purifying the cells of interest captured by the fusion protein; and growing cells on the scaffold coated with the fusion protein, wherein the cell-adherent protein of the fusion protein of [8] or [9] specifically binds to the cells of interest.

[0015]

[16] A composition for adhering cells on a scaffold material, comprising a fusion protein comprising a region consisting of at least one or more peptides capable of adhering to chitins and a region consisting of at least one or more peptides capable of adhering to cells, wherein the peptide capable of adhering to chitins is a peptide consisting of the amino acid sequence represented by TTNPGVSAWQVNTAYTAGQLVTYNGKTYK (SEQ ID NO: 2) or TTNPGVSAWQVNTAYTAGQLVIYNGKTYK (SEQ ID NO: 3).

[0016]

[17] A composition for adhering cells on a scaffold material, comprising: a fusion protein comprising a region consisting of at least one or more peptides capable of adhering to chitins; a region consisting of at least one or more proteins capable of adhering to cells; and a purification / detection tag or a spacer or linker sequence present between the region consisting of the peptides capable of adhering to chitins and the region consisting of the protein capable of adhering to cells, wherein the peptides capable of adhering to chitins are peptides consisting of the amino acid sequence represented by TTNPGVSAWQVNTAYTAGQLVTYNGKTYK (SEQ ID NO: 2) or TTNPGVSAWQVNTAYTAGQLVIYNGKTYK (SEQ ID NO: 3).

[0017] 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.

[0018] 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 chitins" 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 chitins, 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 chitins, and the fusion protein of the present invention is bound to the scaffold material. FIG. 2 shows a specific embodiment of the fusion protein of the present invention and its three-dimensional structure. The upper diagram of FIG. 2 is a specific example of the fusion protein of the present invention, which contains an RGD sequence as a "peptide or protein capable of adhering to cells" at the N-terminus and a chitin-binding domain as a "peptide capable of adhering to chitins" at the C-terminus. The middle diagram in Figure 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," a FLAG® tag for fusion protein purification, a 6xHis tag for fusion protein detection, and a chitin-binding domain at the C-terminus as a "peptide capable of adhering to chitins." The lower left diagram in Figure 2 shows the three-dimensional structure of the fusion protein of the present invention shown in the upper diagram in Figure 2. The lower right diagram in Figure 2 shows the three-dimensional structure of the fusion protein of the present invention shown in the middle diagram in Figure 2. 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 as a "peptide or protein capable of adhering to cells" at the N-terminus, contains a chitin-binding domain as a "peptide capable of adhering to chitins" at the C-terminus, and contains a linker between the "peptide or protein capable of adhering to cells" and the "peptide capable of adhering to chitins." Figure 3 shows (photographs) whether or not the fluorescent dye emits light in the presence or absence of the fusion protein of the present invention. 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 shows human cell adhesion on chitosan in the presence or absence of the fusion protein of the present invention (peptide 2) (photograph). Figure 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 shows human cell adhesion on chitosan in the presence or absence of the fusion protein of the present invention (peptide 3) (photograph). Figure 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 shows the adhesion of the fusion protein of the present invention (peptide 2) on 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 shows the results of culturing on a scaffold made of calcium alginate beads coated with chitosan. Figure 7 shows the results of culturing with and without the fusion protein of the present invention (peptide 2). Figure 8 shows the specific binding between the fusion protein of the present invention (peptide 3) and target cells, using an antibody as the "cell-adhesive peptide." The scaffold is made of 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 thereto. Figure 8 shows the results of culturing with and without the fusion protein of the present invention (peptide 3). Figure 9A shows the results of human cell adhesion after chitosan coating with the fusion proteins of the present invention (peptide 3 and peptide 2) (also referred to as sequence 3 and sequence 2, respectively) (photographs). Figure 9B shows the results of human cell growth after chitosan coating with the fusion proteins of the present invention (peptide 3 and peptide 2). Figure 9C shows the percentage of human cells after coating chitosan with the fusion proteins of the present invention (peptide 3 and peptide 2), and Figure 10A shows the results of bovine cell adhesion after coating chitosan with the fusion protein of the present invention (peptide 2) (photograph).FIG. 10B shows the results of bovine cell proliferation after coating chitosan with the fusion protein of the present invention (peptide 2) (photograph).

[0019] The present invention will be described in detail below.

[0020] The present invention relates to a fusion protein (also referred to as an adhesion factor of the present invention) comprising at least one region consisting of a peptide capable of adhering to chitins and at least one region consisting of a peptide or protein capable of adhering to cells. The fusion protein of the present invention can comprise one or more "regions consisting of a peptide capable of adhering to chitins" and one or more "regions consisting of a peptide or protein capable of adhering to cells." In the present invention, chitins refer to 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 composed of 90% N-acetylglucosamine and 10% glucosamine. Chitosan can be obtained by hydrolyzing and deacetylating chitin. Chitosan sugar chains are also known to have cell-cell 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 derivatives 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, glycol chitosan methacrylate, and chitosan oligosaccharide lactate.

[0021] In one embodiment of the present invention, a "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, preferably T or I. Threonine (T) has a hydroxyethyl group in its side chain and is classified as a polar, uncharged side chain 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, a hydrophobic amino acid like isoleucine, is used in the alanine scanning method, in which each side chain residue in a protein is mutated to alanine to create a mutant, thereby site-specifically identifying residues important for the structure and function of the protein. Since the X portion is replaced with isoleucine, which has a significantly different function from threonine, it is believed to have a low contribution to binding to chitins, 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 portion thereof, can be used as the "peptide capable of adhering to chitins."

[0022] As described in The Journal of Biological Chemistry, Vol. 275, No. 18, Issue of May 5, pp. 13654-13661, 2000, the term "chitin-binding domain" is known to those skilled in the art, and its structure is as shown in Figure 5. For example, the third paragraph in the left column on page 13660 suggests that threonine, proline, and aromatic residues are conserved in chitin-binding domains, and that these are important for interaction. SEQ ID NOS: 2 and 3 of the present application correspond to the first half of the sequence of Bacillus circulans WL-12 Chitinase A1 shown in the top row of the alignment shown in Figure 5. Although the N-terminal sequences of SEQ ID NOS: 2 and 3 of the present application are not shown in Figure 5, their C-terminal sequences correspond to the 676th amino acid residue, K, in the alignment shown in Figure 5. The explanation for Figure 5 on page 13659 also mentions ChBDs proposed as candidates for interaction with chitin. chiA1The amino acids marked with diamonds correspond to the 681st, 682nd, 687th, and 689th amino acids. Even though the chitin-binding domains of the present invention (SEQ ID NOS: 2 and 3) lack these amino acid residues that are candidates for chitin interaction and thus exhibit reduced chitin-binding ability, they still possess sufficient chitosan-binding ability, as described in the Examples, and can be used for cell purification or cultivation. Furthermore, as described in the first paragraph of the right column on page 13654 of The Journal of Biological Chemistry, Vol. 275, No. 18, Issue of May 5, pp. 13654-13661, 2000, it is described that chitinase A1 (ChiA1) has a high affinity for insoluble chitin, and the second paragraph states that ChBDChiA1 binds to insoluble chitin but not to soluble derivatives of chitin. Furthermore, the abstract in the Journal of Bacteriology, June 2000, pp. 3045-3054, states that ChBDChiA1 does not bind to chitosan. Therefore, since it was known that chitinase and chitin-binding domains bind to insoluble chitin but not soluble chitin, those skilled in the art believed that chitinase and chitin-binding domains would not bind to soluble chitosan.

[0023] Furthermore, Insect Molecular Biology (2017) 00(00), 00-00 doi: 10.1111 / imb.12308 describes BmCPAP3s (BmCPAP3-A1, BmCPAP3-A2, BmCPAP3-B, BmCPAP3-C, BmCPAP3-D1, and BmCPAP3-D2) as chitin-binding proteins derived from silkworms, and describes that BmCPAP3-D1 has the highest binding activity to deacetylated chitin (i.e., chitosan) (see Abstract and Figure 2B). In yet another aspect of the present invention, the amino acid sequence of the chitin-binding protein BmCPAP3-D1 (SEQ ID NO: 27) described in the document, or an amino acid sequence having 90% or more homology thereto and maintaining chitin-binding activity, or a fragment thereof that maintains said activity, can be used as a "peptide capable of adhering to chitins" of the present invention.

[0024] In one embodiment of the present invention, the "peptide or protein capable of adhering to cells" is a peptide or protein capable of specifically binding to a target cell, 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. 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, ligands that bind to receptors, or parts of ligands can be used as the "protein capable of adhering to cells." In the present invention, the antibody may be a full-length polyclonal or monoclonal antibody, or an antigen-binding fragment thereof (Fab, Fab', F(ab')). ２Although antibodies such as scFv (e.g., Fv), single-chain (scFv) antibodies, VHHs, heavy-chain antibodies, and diabodies can be used, those with small molecular weights such as scFv and VHHs are preferred so that they can be expressed in E. coli and the like. By using the above-mentioned antibodies as the "protein capable of adhering to cells" in the fusion proteins of the present invention, the specificity of the fusion proteins for target cells can be enhanced, resulting in specific binding to target cells, which can then be selected, purified, and cultured. In the fusion proteins of the present invention, the "protein capable of adhering to cells" refers to, for example, a protein having a length of 50 amino acids or more. 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 Co., Ltd.) can be used to select and culture only HER2-positive cells.

[0025] In another embodiment of the present invention, the "peptide capable of adhering to cells" is, 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), RQVFQVAYIIIKA (SEQ ID NO: 13), or the like. 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), KGGPQVTRGDVFTMP (SEQ ID NO: 28), polylysine, polyarginine, or polyornithine, etc., can be used. These peptides are known to those skilled in the art and are readily available, as described on the Fujifilm Wako Pure Chemical Corporation website (https: / / labchem-wako.fujifilm.com / jp / product / detail / W01W0118-0153.html), the Peptide Institute, Inc. website (https: / / www.peptide.co.jp / support / useful / rgd-peptides), the Funakoshi Co., Ltd. website (https: / / www.funakoshi.co.jp / contents / 70313), and the Sigma-Aldrich Co., Ltd. website (https: / / www.sigmaaldrich.com / JP / ja / substance / lamininmimeticanimalfreeecmpeptides1234598765). In the fusion protein of the present invention, a "peptide capable of adhering to cells" refers to a peptide that is, for example, 3 to 19 amino acids in length, less than 30 amino acids in length, less than 40 amino acids in length, or less than 50 amino acids in length.

[0026] In yet another embodiment of the present invention, the "cell-adhesive peptide or protein" can be a cell adhesion molecule, a partial sequence thereof, or a derivative thereof selected from extracellular matrix molecules such as fibronectin, laminin, or vitronectin; protein ligands such as cadherin, delta, notch, the immunoglobulin superfamily, cytokines, and growth factors; and lectins. Fibronectin is a major glycoprotein constituting the extracellular matrix and is involved in cell adhesion, spreading, migration, proliferation, and differentiation. Laminin is a major component of the basement membrane of most tissues and is involved in cell differentiation, proliferation, and cancer metastasis. Vitronectin is a glycoprotein present in serum and the extracellular matrix that promotes cell adhesion and spreading and is also used as a cell culture substrate. By using fibronectin, laminin, or vitronectin as the "cell-adhesive peptide" in the fusion protein of the present invention, adhesion to cells bearing fibronectin, laminin, or vitronectin receptors, respectively, is possible. Although these cell adhesion molecules have low cell specificity, they are preferred because they are not derived from heterologous species.

[0027] 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 GGGGSGGGGSGGGGS (SEQ ID NO: 24).

[0028] In the present invention, the term "cells" refers to cells that can specifically or nonspecifically bind to the "cell-adhesive peptide or protein" 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 the "cell-adhesive peptide or protein" recognizes and binds to. The fusion protein of the present invention can bind to cells via the "cell-adhesive peptide or protein." The cells are, for example, target cells that require purification, cell culture, or target cells that require cell culture after purification. When the "cell-adhesive protein" has a highly selective sequence, such as an antibody, it specifically binds to the target cells, resulting in the purification of the target cells and their selective culturing. In the present invention, the growth of target cells refers to the adhesion of the fusion protein of the present invention to the scaffold or simultaneously with the adhesion, followed by the growth of the target cells. The fusion protein of the present invention can be used as an agent or coating agent for adhering cells on a scaffold material, which contains the fusion protein, or as a composition or coating composition for adhering cells on a scaffold material, which contains the fusion protein.

[0029] In the present invention, the type of cells to be purified and expanded is not particularly limited, and examples thereof include stem cells (e.g., pluripotent stem cells, pluripotent stem cell-derived differentiated 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 Escherichia coli. Preferably, in the present invention, the mammalian cells are cells derived from humans or bovines. 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.

[0030] 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 chitins with the fusion protein of the present invention described above, or coating a scaffold having at least a portion of its surface made of polysaccharides containing one or more types of chitins with the fusion protein of the present invention described above, 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 described above.

[0031] 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.

[0032] In another embodiment of the method for coating a fusion protein-chitin complex of the present invention, a scaffold is non-cell-adhesive and its surface is coated with at least one type of chitin-containing polysaccharide. Specifically, coating a non-cell-adhesive scaffold with chitosan can be carried out by contacting a chitosan solution with a culture substrate by a process such as painting, spraying, or immersion. The scaffold is then further coated with the fusion protein of the present invention, making it adhesive to both chitins and cells, allowing target cells to grow on the scaffold.

[0033] 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. Alternatively, a conventional flat scaffold may be used. These scaffolds are not limited as long as they contain chitin on their surface or can be coated with chitin. Alginate gel can be prepared by using sodium alginate as a gelling agent and reacting it with calcium ions. The interaction of aqueous sodium alginate with calcium ions rapidly causes ionic crosslinking and 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 potential. Alginate gels in the presence of calcium and becomes a solution upon the addition of a chelating agent, allowing the target cells to be easily cultured and recovered after purification. The shape of the alginate gel may be, for example, a dish, a fiber, a knit, or a microcarrier.

[0034] Microcarriers can be used as scaffolds to adhere and support cells in a medium when cells are suspended and cultured in the medium in a three-dimensional culture method. Adding microcarriers to a medium can promote cell culture. The shape of the microcarriers may be spherical, polygonal, conical, pyramidal, fractured, needle-like, or the like. However, spherical particles are preferred from the viewpoint of providing a wide surface for cell adsorption 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.

[0035] 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.

[0036] The method for recovering target cells of the present invention includes the following steps: coating a scaffold containing chitins with the above-mentioned 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 types of chitins with the above-mentioned 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 above-mentioned fusion protein of the present invention, incubating cells on the scaffold, and purifying the target cells captured by the fusion protein, wherein the protein of the fusion protein of the present invention that is capable of adhering to cells specifically binds to the target cells.

[0037] 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, for example, 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 peptide capable of adhering to chitins and the peptide or protein capable of adhering to cells, or at the N-terminus or C-terminus of the fusion protein; more specifically, it may contain a purification tag such as a FLAG (registered trademark) tag or a 6xHis tag.

[0038] 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.

[0039] 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.

[0040] Other steps are as described in the section on the method for coating the scaffold of the present invention.

[0041] The method of the present invention for growing target cells comprises the following steps: coating a scaffold containing chitins with the above-mentioned 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 types of chitins with the above-mentioned 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 above-mentioned fusion protein of the present invention, incubating cells on the scaffold, purifying the target cells captured by the fusion protein, and growing the cells on the scaffold, wherein the cell-adherent peptide of the fusion protein of the present invention specifically binds to the target cells.

[0042] 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.

[0043] In the method of the present invention for growing cells of interest, the step of growing cells on a scaffold 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] Example 1 (Coating of Chitosan Surfaces with the Fusion Protein of the Present Invention) 20 μL of a 10 mg / mL chitosan solution was added to a portion of a 24-well plate and allowed to stand for at least 1 hour. Subsequently, a solution of fluorescein-labeled peptides (Peptide 1 and Peptide 2 below) was added and allowed 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 chitin" is linked to a "peptide capable of adhering to cells" via a peptide linker sequence). A control without peptide was also prepared. After washing with PBS, the area around the boundary between the coated and uncoated wells was observed under a fluorescence microscope (Figure 3). Fluorescence was observed in the presence of peptide 1 and peptide 2, confirming that the peptides were successfully coated onto the 24-well plate via chitosan.

[0048] Example 2 (Human Cell Adhesion After Coating of Chitosan Surface 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, under Condition 1, peptide 2 was added and allowed to stand for at least 1 hour. Under Condition 2, no peptide was added. Under 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 culture, the supernatant was discarded, 4% paraformaldehyde was added, and the plate was 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 number was counted. Under condition 1, in which peptide 2 was added, the cells spread and many cells adhered, whereas under the condition in which peptide 2 was not added, the cells did not spread and sufficient cell adhesion was not observed (FIGS. 4A and 4B).

[0049] Example 3 (Human Cell Adhesion after Coating of Chitosan Surface 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, the following peptide 3 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, 4% paraformaldehyde was added, and the plate was 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 number was counted. Many cells adhered under the condition where peptide 3 was added, but no sufficient cell adhesion was observed under the condition where peptide 3 was not added (FIGS. 5A and 5B).

[0050] Peptide 3: anti-HER2 antibody sequence + DYKDHDGDYKDHDIDYKDDDDKLEHHHHHHGGGGS TTNPGVSAWQVNTAYTAGQLVTYNGKTYK (SEQ ID NO: 26) (sequence of SEQ ID NO: 2 with a FLAG (registered trademark) tag and a 6xHis tag added to the N-terminus)

[0051] Example 4 (Coating of calcium alginate beads with the fusion protein of the present 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 observed only in the bead solution under Condition 1, which contained chitosan, confirming that the calcium alginate was successfully coated with the peptide.

[0052] Example 5 (Human cell proliferation after coating calcium alginate beads with the fusion protein of the present invention) A chitosan solution was added to calcium alginate beads and allowed to stand for at least one hour. Then, 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). The results showed that cell proliferation was superior when peptide 2 was added (Figure 7).

[0053] Example 6 (Human cell proliferation after coating calcium alginate beads with the fusion protein of the present invention) A 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 was added was 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 of adding peptide 3 (Figure 8).

[0054] Example 7 (Human Cell Proliferation After Coating of Chitosan Surface 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, a solution of peptide 2 or peptide 3 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. After washing, the plates were 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).

[0055] Example 8 (Proliferation of bovine cells after coating of chitosan surface 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, 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 cells confirmed that cells had adhered to the coated area (Figure 10A). Culture medium was added and culture was continued for another 6 days, and proliferation of the adhered cells was observed (Figure 10B).

Claims

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 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 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 TTNPGVSAWQVNTAYTAGQLVTYNGKTYK (SEQ ID NO: 2) or TTNPGVSAWQVNTAYTAGQLVIYNGKTYK (SEQ ID NO: 3).

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

6. The peptide or protein capable of adhering to cells is selected from the group consisting of 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), and the like. 4), 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), KGGPQVTRGDVFTMP (SEQ ID NO: 28), polylysine, polyarginine, or polyornithine.

7. The fusion protein according to claim 1, wherein the peptide or 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, 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, a sequence for the purpose of purification or detection, a sequence for improving solubility or hydrophilicity / hydrophobicity, or a sequence for the purpose of serving as a spacer / linker.

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 coating a scaffold containing chitin with the fusion protein of claim 1, 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 1, or coating a scaffold that is a substance that interacts with chitin with a complex of chitin and the fusion protein of claim 1.

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

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

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

14. A method for recovering target cells, 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 types of 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 scaffold coated with the fusion protein; and purifying the target cells captured by the fusion protein, wherein the cell-adherent protein of the fusion protein of claim 8 specifically binds to the target cells.

15. A method for growing target cells, 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 types of 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 scaffold coated with the fusion protein; purifying the target cells captured by the fusion protein; and growing cells on the scaffold coated with the fusion protein, wherein the cell-adherent protein of the fusion protein of claim 8 specifically binds to the target cells.

16. A composition for adhering cells on a scaffold, comprising a fusion protein comprising a region consisting of at least one or more peptides capable of adhering to chitins and a region consisting of at least one or more peptides capable of adhering to cells, wherein the peptide capable of adhering to chitins is a peptide consisting of the amino acid sequence represented by TTNPGVSAWQVNTAYTAGQLVTYNGKTYK (SEQ ID NO: 2) or TTNPGVSAWQVNTAYTAGQLVIYNGKTYK (SEQ ID NO: 3).

17. A composition for adhering cells on a scaffold material, comprising a fusion protein comprising a region consisting of at least one or more peptides capable of adhering to chitins, a region consisting of at least one or more proteins capable of adhering to cells, and a purification / detection tag or a spacer or linker sequence present between the region consisting of the peptides capable of adhering to chitins and the region consisting of the protein capable of adhering to cells, wherein the peptides capable of adhering to chitins are peptides consisting of the amino acid sequence represented by TTNPGVSAWQVNTAYTAGQLVTYNGKTYK (SEQ ID NO: 2) or TTNPGVSAWQVNTAYTAGQLVIYNGKTYK (SEQ ID NO: 3).

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