Carrier for imparting physiological activity to fibrin gel in thrombin-independent manner and use thereof

Abstract

Provided is a protein having a hexamer structure in which two molecules of a heterotrimeric protein associate at the N-terminus side, wherein the heterotrimeric protein is composed of (1) a fibrinogen Aα chain deficient in fibrinopeptide A, (2) a fibrinogen Bβ chain deficient in fibrinopeptide B and losing the ability to bind to B knob, and (3) a fibrinogen γ chain losing the ability to bind to A knob. The protein of the present invention can be used as a carrier for imparting physiological activity to fibrin gel in a thrombin-independent manner.

Description

Carrier for imparting physiological activity to fibrin gel in a thrombin-independent manner and use thereof

[0001] The present invention relates to a carrier for imparting physiological activity to a fibrin gel in a thrombin-independent manner, and to the use thereof.

[0002] Fibrin gel, formed during blood coagulation, is more biocompatible and biodegradable than synthetic polymer gels, and is therefore being used as a scaffold for embedding cells in cell transplantation therapy and a scaffold for sustained release of bioactive molecules. Taking advantage of its cytocompatible properties, it is also expected to be used as a scaffold for three-dimensional cell culture and cell transplantation, particularly for stem cells.

[0003] In recent years, research into the use of mini-organs known as organoids in regenerative medicine and disease modeling for drug discovery has progressed rapidly. Fibrin gel is also expected to be used as a scaffold for organoid production. In organoid production, a basement membrane-like gel called Matrigel has been used almost exclusively as a cell culture scaffold. However, Matrigel is a crude extract of murine tumor tissue called EHS tumor, and is inevitably contaminated with components derived from different species (murine), making its medical application difficult. Therefore, the development of a cell culture scaffold with basement membrane-like scaffolding activity equivalent to or superior to Matrigel and suitable for medical applications is an urgent issue. Since fibrin gel is already used as a fibrin glue in surgery, conferring basement membrane-like scaffolding activity to it could potentially create a medical scaffold for a variety of cells, including organoids.

[0004] Previously, methods have been reported in which bioactive peptides are attached to fibrin gel using cross-linking reagents or transglutaminase to confer new scaffolding activity (Patent Documents 1 and 2). However, these methods involve nonspecifically attaching single-stranded oligopeptides, such as Arg-Gly-Asp (RGD)-containing peptides or Tyr-Ile-Gly-Ser-Arg (YIGSR, SEQ ID NO: 33), to fibrin or its precursor, fibrinogen, using cross-linking reagents or transglutaminase. These methods cannot be used with adhesive proteins such as laminin, which require a three-dimensional structure formed by multiple subunit chains for activity. Furthermore, the cell adhesive active site of laminin contains multiple exposed basic amino acid residues necessary for activity, and the use of cross-linking reagents or transglutaminase that react with these basic amino acid residues is likely to reduce the activity of laminin.

[0005] The present inventors have developed a method for imparting the cell adhesive activity of laminin to a fibrin gel by preparing a chimeric protein by linking an N-terminal fragment of heterotrimeric fibrinogen with a C-terminal fragment (E8 fragment) of heterotrimeric laminin (Patent Document 3). This method requires the addition of thrombin to a mixture of fibrinogen and the chimeric protein to prepare the fibrin gel, and the chimeric protein cannot be incorporated into an already prepared fibrin gel.

[0006] US 2019 / 0192739 A1US 2003 / 0119186 A1WO2021 / 225171

[0007] An objective of the present invention is to provide a protein that can be used as a carrier for imparting physiological activity to fibrin gel in a thrombin-independent manner, and that has a hexameric structure in which two fibrinogen molecules in a heterotrimeric structure associate at their N-terminal ends.

[0008] In order to achieve the above object, the present invention encompasses the following inventions: <1> A protein having a hexameric structure in which two molecules of a heterotrimeric protein composed of (1) a fibrinogen Aα chain lacking fibrinopeptide A, (2) a fibrinogen Bβ chain lacking fibrinopeptide B and having lost its ability to bind to knob B, and (3) a fibrinogen γ chain having lost its ability to bind to knob A are associated at their N-termini. <2> The protein according to <1>, wherein the fibrinogen Aα chain of (1) is a fragment containing at least 116 amino acids from the amino acid sequence shown in SEQ ID NO: 1, the fibrinogen Bβ chain of (2) is a fragment containing at least 150 amino acids from the amino acid sequence shown in SEQ ID NO: 3, and the fibrinogen γ chain of (3) is a fragment containing at least 107 amino acids from the amino acid sequence shown in SEQ ID NO: 5, and having at least 27 amino acids from the amino acid sequence shown in SEQ ID NO: 5. <3> The protein according to <1> or <2>, further comprising a non-fibrin bioactive protein linked to at least one of the fibrinogen chains (1) to (3). <4> The protein according to <3>, wherein the non-fibrin bioactive protein is selected from the group consisting of cell adhesion proteins, growth factors, growth factor-binding proteins, cytokines, chemokines, anti-adhesion proteins, immunostimulatory proteins, immunomodulatory proteins, protein-binding proteins, nucleic acid-binding proteins, carbohydrate-binding proteins, virus-binding proteins, cytotoxic proteins, enzyme proteins, and protease-inhibitory proteins. <5> The protein according to <3>, wherein the non-fibrin bioactive protein is a cell adhesion protein and / or a growth factor-binding protein. <6> A kit for producing the protein according to <1>, comprising a nucleic acid encoding the fibrinogen Aα chain of (1) or an expression vector comprising the nucleic acid, a nucleic acid encoding the fibrinogen Bβ chain of (2) or an expression vector comprising the nucleic acid, and a nucleic acid encoding the fibrinogen γ chain of (3) or an expression vector comprising the nucleic acid.<7> A method for imparting physiological activity to a fibrin gel, comprising the steps of adding the protein described in <3> above to a fibrinogen solution to bind the protein to fibrinogen, and adding thrombin to the resulting solution to prepare a fibrin gel. <8> A method for imparting physiological activity to a fibrin gel, comprising the step of contacting the protein described in <3> above with the fibrin gel to bind the protein to fibrin. <9> A method for imparting physiological activity to fibrinogen, comprising the step of contacting the protein described in <3> above with fibrinogen to bind the protein to fibrinogen.

[0009] The protein provided by the present invention has a hexameric structure in which two heterotrimeric fibrinogen molecules associate at their N-terminal ends, and can be used as a carrier for imparting physiological activity to fibrinogen and fibrin gel in a thrombin-independent manner. Furthermore, the present invention can provide a method for imparting physiological activity to a fibrin gel and a method for imparting physiological activity to fibrinogen.

[0010] The figure shows the results of quantifying the bound Fbg-N and ΔFbg-N after adding eight concentrations of recombinant fibrinogen N-terminal self-association domain protein (Fbg-N) and its fibrinopeptide-deleted form (ΔFbg-N) to a fibrinogen-coated plate. (A) shows the results of quantifying the bound Fbg-N and ΔFbg-N. TM(A) is an image of a 647-labeled fibrin gel observed with a confocal laser scanning microscope; (B) is an image of a fibrin gel containing ΔFbg-N-EGFP observed with a confocal laser scanning microscope; (C) is a superimposed image of (A) and (B). ΔFbg-N-FN, in which the fibronectin cell adhesion domain FNIII7-10 is linked to the C-terminus of the α-chain of ΔFbg-N, or ΔFbg-N without the fibronectin cell adhesion domain FNIII7-10, was captured on a fibrinogen-coated plate. HT-1080 cells were seeded on each plate and incubated at 37°C / 5% CO for 30 minutes, and cell adhesion to the plate was observed. (A) is an image of a plate in which ΔFbg-N-FN was captured on fibrinogen; (B) is an image of a plate in which ΔFbg-N was captured on fibrinogen. ΔFbg-N (ΔFbg-N-LM) containing the cell adhesion domain of human laminin-511, or ΔFbg-N (ΔFbg-N-LM(γ1EQ)) containing a mutant of the cell adhesion domain of human laminin-511 with inactivated integrin-binding activity, was captured on a fibrinogen-coated plate. HT-1080 cells were seeded on each plate and incubated at 37°C / 5% CO2 for 30 minutes, and cell adhesion to the plate was observed. (A) is an image of a plate with ΔFbg-N-LM captured on fibrinogen, and (B) is an image of a plate with ΔFbg-N-LM(γ1EQ) captured on fibrinogen. Fibrin gel coated on a plate was treated with ΔFbg-N (ΔFbg-N-LM), which contained the cell adhesion domain of human laminin-511, or with ΔFbg-N (ΔFbg-N-LM(γ1EQ)), which contained a mutant version of the cell adhesion domain of human laminin-511 with inactivated integrin-binding activity. HT-1080 cells were seeded on each plate and incubated at 37°C / 5% CO for 30 minutes. Cell adhesion and spreading were observed. (A) is an image of cells on a fibrin gel treated with ΔFbg-N-LM, (B) is an image of cells on a fibrin gel treated with ΔFbg-N-LM(γ1EQ), and (C) is an image of cells on an untreated fibrin gel.FIG. 1 is a diagram showing the mechanism by which thrombin acts on hexameric fibrinogen to form a fibrin gel.

[0011] [Protein Having a Hexameric Structure in Which Two Heterotrimeric Fibrinogen Molecules are Associated at Their N-Terminal Sides] The present invention provides a protein having a hexameric structure in which two heterotrimeric fibrinogen molecules are associated at their N-terminus (hereinafter referred to as the "protein of the present invention"). The protein of the present invention is a protein having a hexameric structure in which two molecules of a heterotrimeric protein are associated at their N-terminus, the two molecules being composed of (1) a fibrinogen Aα chain lacking fibrinopeptide A, (2) a fibrinogen Bβ chain lacking fibrinopeptide B and having lost the ability to bind to the B knob, and (3) a fibrinogen γ chain having lost the ability to bind to the A knob. The protein of the present invention can be used as a carrier for imparting physiological activity to fibrin gel in a thrombin-independent manner.

[0012] Fibrinogen is a glycoprotein that is converted to fibrin by the action of thrombin in the final stage of the blood coagulation reaction and is involved in blood coagulation, hemostasis, clot formation, wound healing, inflammation, angiogenesis, and cell-extracellular matrix interactions. Fibrinogen is a heterotrimeric molecule consisting of three subunit chains, the Aα chain, the Bβ chain, and the γ chain, assembled in a coiled-coil configuration. Two of these heterotrimeric molecules associate at their N-termini to form a hexamer (Aα-Bβ-γ)2. The mass numbers of the Aα chain, the Bβ chain, and the γ chain are 67 kDa, 56 kDa, and 47.5 kDa, respectively, and the mass number of the hexamer (Aα-Bβ-γ)2 is 340 kDa. Thrombin cleaves fibrinogen between Arg35 and Gly36 of the Aα chain and between Arg44 and Gly45 of the Bβ chain, generating the A knob and the B knob, respectively, which are then converted to fibrin monomers. Knob A and knob B bind to the γ-hole in the C-terminal region of the γ-chain and the β-hole in the C-terminal region of the Bβ-chain of fibrinogen (fibrin), respectively, and fibrin monomers polymerize to form a fibrin gel (see Figure 6).

[0013] As described above, the protein of the present invention is a "protein having a hexameric structure in which two molecules of a heterotrimeric protein associated at their N-terminus are composed of (1) a fibrinogen Aα chain lacking fibrinopeptide A, (2) a fibrinogen Bβ chain lacking fibrinopeptide B and having lost its ability to bind to knob B, and (3) a fibrinogen γ chain having lost its ability to bind to knob A," and therefore can bind to fibrinogen without the action of thrombin. Furthermore, the protein of the present invention can bind directly to fibrin gel.

[0014] The fibrinogen B β chain that has lost its ability to bind to knob B may be one that has lost its ability to bind to knob B by introducing a mutation into the β-hole, or may be one that has lost the C-terminal region containing the β-hole.The fibrinogen γ chain that has lost its ability to bind to knob A may be one that has lost its ability to bind to knob A by introducing a mutation into the γ-hole, or may be one that has lost the C-terminal region containing the γ-hole.

[0015] The protein of the present invention can be produced as a recombinant protein by appropriately using known gene recombination techniques based on the nucleotide sequence information and amino acid sequence information of the gene encoding wild-type fibrinogen. Specifically, an expression vector containing DNA encoding the Aα chain (1), an expression vector containing DNA encoding the Bβ chain (2), and an expression vector containing DNA encoding the γ chain (3) are co-transfected into appropriate host cells and cultured to express a heterotrimeric protein, and two molecules of the heterotrimeric protein associate at their N-termini to form a hexameric structure. The protein of the present invention can be produced by purifying the hexameric protein by known methods.

[0016] The proteins of the present invention may be obtained by modifying fibrinogen from any organism, or may be obtained by modifying fibrinogen from a mammal. Examples of mammals include humans, mice, rats, cows, and pigs. When the proteins of the present invention are applied to human medical treatment, it is preferable that the proteins of the present invention are produced based on the genetic information of human fibrinogen. Table 1 shows the accession numbers of the nucleotide sequences and amino acid sequences of the genes encoding each chain constituting human fibrinogen. Each fibrinogen chain constituting the proteins of the present invention may contain an affinity tag, such as a His tag, an HA tag, or a FLAG tag. The proteins of the present invention may also be labeled with a known protein-labeling substance.

[0017]

[0018] When the protein of the present invention is a modified human fibrinogen, (1) the fibrinogen Aα chain lacking fibrinopeptide A may be, for example, a fragment containing at least 116 amino acids from the amino acid sequence shown in SEQ ID NO: 1, with the 36th amino acid at the N-terminus, (2) the fibrinogen Bβ chain lacking fibrinopeptide B and having lost the ability to bind to knob B may be, for example, a fragment containing at least 150 amino acids from the amino acid sequence shown in SEQ ID NO: 3, with the 45th amino acid at the N-terminus, and (3) the fibrinogen γ chain may be a fragment containing at least 107 amino acids from the amino acid sequence shown in SEQ ID NO: 5, with the 27th amino acid at the N-terminus. By combining such a fibrinogen Aα chain fragment, a fibrinogen Bβ chain fragment, and a fibrinogen γ chain fragment, a heterotrimeric fibrinogen fragment lacking fibrinopeptides A and B and having lost the ability to bind to knob A and knob B can be constructed.

[0019] The protein of the present invention may comprise a non-fibrin bioactive protein linked to at least one fibrinogen chain selected from the above (1) to (3). The non-fibrin bioactive protein is not particularly limited and may be any protein having the bioactivity to be imparted to the fibrin gel. Specific examples include, but are not limited to, cell adhesion proteins, growth factors, growth factor-binding proteins, cytokines, chemokines, anti-adhesion proteins, immunostimulatory proteins, immunomodulatory proteins, protein-binding proteins, nucleic acid-binding proteins, carbohydrate-binding proteins, virus-binding proteins, cytotoxic proteins, enzyme proteins, and protease-inhibitory proteins. The non-fibrin bioactive protein may be a full-length protein or a fragment containing a domain essential for the expression of bioactivity.

[0020] The cell adhesion protein may be a molecule involved in adhesion between cells and extracellular matrix or between cells, such as laminin, fibronectin, collagen, vitronectin, nephronectin, osteopontin, MAEG, tenascin, SVEP1, TGF-β1, TGF-β3, type I collagen, type IV collagen, E-cadherin, N-cadherin, and P-cadherin.

[0021] Examples of growth factors include epidermal growth factor (EGF), insulin-like growth factor (IGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), transforming growth factor (TGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), erythropoietin (EPO), angiopoietin (Ang), and thrombopoietin (TPO).

[0022] The growth factor binding protein is not particularly limited as long as it is a molecule capable of binding to a growth factor involved in the proliferation of cultured cells. Specific examples include heparan sulfate proteoglycans. Examples of heparan sulfate proteoglycans include perlecan, agrin, type XVIII collagen, syndecans 1 to 4, and glypicans 1 to 6. Examples of growth factor binding proteins other than heparan sulfate proteoglycans include latent TGF-β binding proteins 1 to 4. Examples of fragments containing the growth factor-binding site of a growth factor-binding protein include domains I to III of perlecan (the region from the N-terminus of the perlecan amino acid sequence (NP_005520) from valine at position 22 to proline at position 1676) and a region containing residues 1 to 8 of the follistatin (FS) domain of agrin (Uwe Winzen, Gregory J. Cole, and Willi Halfter, "Agrin is a chimeric proteoglycan with the attachment sites for heparan sulfate / chondroitin sulfate located in two multiple serine-glycine clusters," The Journal of Biological Chemistry, 278, 30106-30114, 2008).

[0023] The cytokines and chemokines are not particularly limited, and known cytokines and chemokines can be used, including, but not limited to, interleukin (IL), tumor necrosis factor (TNF), platelet-activating factor (PAF), interferon (IFN), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), CC chemokines, CXC chemokines, C chemokines, and CX3C chemokines.

[0024] The anti-adhesion protein is not particularly limited as long as it is a protein that resists cell adhesion, i.e., a protein that functions as a negative regulator of cell adhesion. Examples of anti-adhesion proteins include thrombospondin. Anti-adhesion proteins are useful for preventing scar formation and preventing cell migration and cell infiltration.

[0025] The immunostimulatory and immunomodulatory proteins may be any proteins that act as immunostimulatory and immunomodulatory molecules, and are not particularly limited thereto. For example, one of the domains of flagellin is known to bind to members of the toll-like receptor family to activate the maturation of dendritic cells, leading to more effective antigen presentation and the maturation of immune responses. Proteins other than flagellin include, but are not limited to, bacterial coat proteins, mannose receptor ligands, and viral coat proteins.

[0026] The protein-binding protein may be any protein that contains a binding domain for another protein, and is not particularly limited. The nucleic acid-binding protein may be any protein that contains a DNA-binding domain or an RNA-binding domain, and is not particularly limited. The glycan-binding protein may be any protein that binds to a glycan, and is not particularly limited. The virus-binding protein may be any protein that binds to a viral coat protein, and is not particularly limited. The cytotoxic protein may be any protein that acts on a cell surface receptor to induce cytotoxicity, and is not particularly limited. For example, Fas ligand, which acts on a cell surface receptor to induce apoptosis, etc. The enzyme protein may be any protein that has enzymatic activity, and is not particularly limited. The protease inhibitor protein may be any protein that has protease inhibitory activity, and is not particularly limited.

[0027] The non-fibrin physiologically active protein can be produced as a recombinant protein by appropriately using known gene recombination techniques. Information on the nucleotide sequence and amino acid sequence of the non-fibrin physiologically active protein can be obtained from known databases such as NCBI.

[0028] The non-fibrin physiologically active protein contained in the protein of the present invention may be linked to at least one of the fibrinogen chains (1) to (3) above. That is, the non-fibrin physiologically active protein may be linked to the fibrinogen Aα chain (1) above, the fibrinogen Bβ chain (2) above, or the fibrinogen γ chain (3) above. Furthermore, the non-fibrin physiologically active protein may be linked to two chains, the Aα chain and the Bβ chain, two chains, the Aα chain and the γ chain, two chains, the Bβ chain and the γ chain, or three chains, the Aα chain, the Bβ chain, and the γ chain.

[0029] The protein of the present invention may contain one or more types of non-fibrin physiologically active proteins. Therefore, when a non-fibrin physiologically active protein is linked to two chains, one type of non-fibrin physiologically active protein may be linked to two chains, or two types of non-fibrin physiologically active proteins may each be linked to a separate chain. When a non-fibrin physiologically active protein is linked to three chains, one type of non-fibrin physiologically active protein may be linked to three chains, one of two types of non-fibrin physiologically active proteins may be linked to two chains and the other to the remaining chain, or three types of non-fibrin physiologically active proteins may each be linked to a separate chain. Alternatively, two or more types of non-fibrin physiologically active proteins may be produced as a fusion protein or a protein linked via a linker, and multiple types of non-fibrin physiologically active proteins may be linked to a single chain.

[0030] In the protein of the present invention, the form of linkage between the fibrinogen chain and the non-fibrin physiologically active protein is not particularly limited, and any linkage may be used as long as it does not inhibit the functions of both. For example, the non-fibrin physiologically active protein may be linked directly to the C-terminus of the fibrinogen chain as a fusion protein, or may be linked via a spacer peptide or linker to the C-terminus of the fibrinogen chain or any position other than the C-terminus.

[0031] [Method for imparting physiological activity to a fibrin gel] The present invention provides a method for imparting physiological activity to a fibrin gel. A first embodiment of the method for imparting physiological activity to a fibrin gel of the present invention may comprise step 1, in which a protein of the present invention, including a non-fibrin physiologically active protein, is added to a fibrinogen solution to bind the protein of the present invention to fibrinogen, and step 2, in which thrombin is added to the resulting solution to prepare a fibrin gel. Since the protein of the present invention lacks fibrinopeptide A and fibrinopeptide B, the A knob and B knob of the protein of the present invention are exposed by the deletion of fibrinopeptide A and fibrinopeptide B. By adding the protein of the present invention to the fibrinogen solution in step 1, the A knob and B knob of the protein of the present invention can bind to the γ-hole and β-hole of fibrinogen, respectively. Subsequently, in step 2, thrombin is added to the solution obtained in step 1 to prepare a fibrin gel. In step 2, a lysine analogue, such as tranexamic acid or ε-aminocaproic acid, may be added as a fibrin stabilizer.

[0032] In the first embodiment, the protein of the present invention can be bound to fibrinogen in step 1 without the fibrinogen forming a gel, thereby increasing the binding efficiency of the protein of the present invention to fibrinogen compared to a method in which a protein that does not lack fibrinopeptide A and fibrinopeptide B is added to a fibrinogen solution simultaneously with thrombin.

[0033] In step 1 of the first embodiment, the molar ratio of the protein of the present invention to fibrinogen is preferably 1:5 to 1:20,000. The concentration of the protein of the present invention added to the fibrinogen solution is not particularly limited, but may be 0.5 nM to 1,000 nM. The concentration of the protein of the present invention added to the fibrinogen solution may be 1 nM or more, 1.5 nM or more, 2 nM or more, 3 nM or more, 5 nM or more, 10 nM or more, 20 nM or more, 30 nM or more, 50 nM or more, 70 nM or more, 100 nM or more, or 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, or 400 nM or less. The fibrinogen concentration in the fibrinogen solution is not particularly limited, but may be 0.1 mg / mL to 50 mg / mL. The fibrinogen concentration of the fibrinogen solution may be 0.5 mg / mL or more, 1.0 mg / mL or more, 1.5 mg / mL or more, 2 mg / mL or more, 2.5 mg / mL or more, 3 mg / mL or more, 5 mg / mL or more, 7 mg / mL or more, 10 mg / mL or more, 15 mg / mL or more, 20 mg / mL or more, 25 mg / mL or more, or 45 mg / mL or less, 40 mg / mL or less, 35 mg / mL or less, or 30 mg / mL or less.

[0034] In step 2 of the first embodiment, the thrombin concentration is not particularly limited and can be appropriately selected depending on the fibrinogen concentration. For example, the thrombin concentration in the mixture may be 0.01 NIH unit / mL to 250 NIH unit / mL. The thrombin concentration in the mixture may be 0.05 NIH unit / mL or more, 0.1 NIH unit / mL or more, 0.5 NIH unit / mL or more, 1 NIH unit / mL or more, 2 NIH unit / mL or more, 3 NIH unit / mL or more, 5 NIH unit / mL or more, 7 NIH unit / mL or more, or 10 NIH unit / mL or more, or 200 NIH unit / mL or less, 150 NIH unit / mL or less, 125 NIH unit / mL or less, 100 NIH unit / mL or less, 70 NIH unit / mL or less, or 50 NIH unit / mL or less.

[0035] A second embodiment of the method of the present invention for imparting physiological activity to a fibrin gel may include a step of contacting a fibrin gel with a protein of the present invention, including a non-fibrin physiologically active protein, to bind the protein of the present invention to the fibrin gel. The second embodiment can be carried out, for example, when physiological activity is to be imparted afterwards to a fibrin gel that has already been coated on a plate or applied to a target object.

[0036] In the second embodiment of the method of the present invention, the amount of the protein of the present invention to be added to the fibrin gel is not particularly limited, and may be, for example, 1 nM or more, 1.5 nM or more, 2 nM or more, 3 nM or more, 5 nM or more, 10 nM or more, 20 nM or more, 30 nM or more, 50 nM or more, 70 nM or more, 100 nM or more, or 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, or 400 nM or less.

[0037] The fibrin gel imparted with physiological activity obtained by the method of the present invention can be used for the intended medical application, etc., depending on the physiological activity imparted. For example, a fibrin gel imparted with cell adhesion activity and / or growth factor binding activity can be used as a three-dimensional gel to replace Matrigel for three-dimensional culture of cells or tissue fragments.

[0038] [Method for imparting physiological activity to fibrinogen] The present invention provides a method for imparting physiological activity to fibrinogen. The method for imparting physiological activity to fibrinogen of the present invention may include a step of contacting a protein of the present invention, including a non-fibrin physiologically active protein, with fibrinogen to bind the protein of the present invention to fibrinogen. The method for imparting physiological activity to fibrinogen of the present invention can be carried out, for example, when fibrinogen that has already been coated on a plate or applied to a target object is to be subsequently imparted with physiological activity. The fibrinogen that has been imparted with physiological activity by the method of the present invention can be used for the desired medical application, etc., depending on the imparted physiological activity.

[0039] [Kit] The present invention provides a kit for producing the protein of the present invention. The kit of the present invention may comprise the nucleic acid encoding the fibrinogen Aα chain (1) or an expression vector containing said nucleic acid, the nucleic acid encoding the fibrinogen Bβ chain (2) or an expression vector containing said nucleic acid, and the nucleic acid encoding the fibrinogen γ chain (3) or an expression vector containing said nucleic acid, which constitute the protein of the present invention. The protein of the present invention can be produced by co-transfecting the three expression vectors included in the kit of the present invention into suitable host cells and culturing them.

[0040] The protein of the present invention for imparting physiological activity to fibrin gel can be produced by further incorporating a nucleic acid encoding a non-fibrin physiologically active protein into the expression vector included in the kit of the present invention in an expressible manner.

[0041] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0042] Example 1: Preparation, purification, and activity evaluation of a recombinant protein of fibrinogen N-terminal self-association region (Fbg-N) and its fibrinopeptide-deleted form (ΔFbg-N) (1) Construction of an expression vector for human fibrinogen N-terminal self-association region To prepare a recombinant protein of the N-terminal self-association region of human fibrinogen (Fbg-N), a vector encoding the mouse Ig-κ chain V-J2-C signal peptide / human fibrinogen α chain N-terminal sequence (Ala 20 -Glu 191 ) / c-myc tag (hereinafter referred to as "Fbg-N(α)"), mouse Ig-κ chain V-J2-C signal peptide / human fibrinogen β chain N-terminal sequence (Gln 31 -Ser 230 ) / 6×His tag (hereinafter referred to as “Fbg-N(β)”), mouse Ig-κ chain V-J2-C signal peptide / human fibrinogen γ chain N-terminal sequence (Tyr 27 -Thr 168 ) / FLAG tag (hereinafter referred to as "Fbg-N(γ)") was constructed.

[0043] (1-1) Construction of Fbg-N(α) expression vector Human fibrinogen α chain (FBGα(Met 1 -Pro 644 PCR was performed using the following primer set with the mouse Ig-κ chain V-J2-C signal peptide / human fibrinogen α chain N-terminal sequence (Ala) as a template. 20 -Glu 191 The DNA fragments encoding the mouse Ig-κ chain V-J2-C signal peptide / human fibrinogen α chain N-terminal sequence (Ala) were amplified. 20 -Glu 191(ii) Primer set for amplifying the c-myc tag: 5'-GGAGACCCAAGCTGGCTAGCCACCATGGAG-3' (forward, SEQ ID NO: 7) 5'-TTCACGAGCTAAAGCCCTACTGCATGACC-3' (reverse, SEQ ID NO: 8) (ii) Primer set for amplifying the c-myc tag: 5'-GCAGTAGGGCTTTAGCTCGTGAAGAACAAAAACTCATCTCAGAAG-3' (forward, SEQ ID NO: 9) 5'-TCGGGCCCTCCTCGAGCGGCCGCCGATCTACAGATCCTCTTCTGAGATGAGTTTTTGTTC-3' (reverse, SEQ ID NO: 10)

[0044] The two DNA fragments obtained were ligated and amplified by extension PCR using the following primer set: The amplified DNA fragment was inserted into pcDNA3.4+MCS (see Patent Document 1) digested with restriction enzymes NheI and NotI. TM The Fbg-N(α) expression vector was constructed using the Seamless Cloning and Assembly Kit (Thermo Fisher Scientific, #A13288). (iii) Primer set for amplifying Fbg-N(α): 5'-GGAGACCCAAGCTGGCTAGCCACCATGGAG-3' (forward, SEQ ID NO: 7) and 5'-TCGGGCCCTCCTCGAGCGGCCGCCGATCTACAGATCCTCTTCTGAGATGAGTTTTTGTTC-3' (reverse, SEQ ID NO: 10).

[0045] (1-2) Construction of Fbg-N(β) expression vector Human fibrinogen β expression vector chain (FBGβ(Met 1 -Gln 491 PCR was performed using the following primer set and an expression vector (see Patent Document 1) as a template to amplify a DNA fragment encoding Fbg-N(β). The amplified DNA fragment was inserted into pcDNA3.4+MCS digested with restriction enzymes NheI and NotI using GeneArt TMThe Fbg-N(β) expression vector was constructed using the Seamless Cloning and Assembly Kit (Thermo Fisher Scientific). (iv) Primer set for amplifying Fbg-N(β): 5'-GGAGACCCAAGCTGGCTAGCCACCATGG-3' (forward, SEQ ID NO: 11) and 5'-TCGGGCCCTCCTCGAGCGGCCGCGATCTAATGGTGATGGTGATGATGACTGACAGTGCATGGGGTGCGAC-3' (reverse, SEQ ID NO: 12).

[0046] (1-3) Construction of Fbg-N(γ) expression vector Human fibrinogen γ chain (FBGγ (Met 1 -Val 437 PCR was performed using the following primer set and an expression vector (see Patent Document 1) as a template to amplify a DNA fragment encoding Fbg-N(γ). The amplified DNA fragment was inserted into pcDNA3.4+MC digested with restriction enzymes NheI and NotI using GeneArt TM The Fbg-N(γ) expression vector was constructed using the Seamless Cloning and Assembly Kit (Thermo Fisher Scientific). (v) Primer set for amplifying Fbg-N(γ): 5'-GGAGACCCAAGCTGGCTAGCCACCATGGAG-3' (forward, SEQ ID NO: 7) and 5'-TCGGGCCCTCCTCGAGCGGCCGCGATCTACTTGTCATCGTCGTCCTTGTAATCCGTGTCTTTGCAAGGTTCCTGG-3' (reverse, SEQ ID NO: 13).

[0047] (2) Construction of an expression vector for fibrinopeptide-deleted human fibrinogen N-terminal self-association region. To express the recombinant protein (ΔFbg-N) of the N-terminal self-association region of human fibrinogen, we constructed a vector containing the mouse Ig-κ chain V-J2-C signal peptide / fibrinopeptide-deleted human fibrinogen α chain N-terminal sequence (Gly 36 -Glu 191) / c-myc tag (hereinafter referred to as "ΔFbg-N(α)"), human Ig-κ light chain signal peptide / fibrinopeptide-deleted human fibrinogen β chain N-terminal sequence (Gly 45 -Ser 230 An expression vector for ΔFbg-N(β) / 6×His tag (hereinafter referred to as “ΔFbg-N(β)”) was constructed.

[0048] (2-1) Construction of ΔFbg-N(α) Expression Vector Using the same human fibrinogen α expression vector as in (1-1) above as a template, PCR was performed with the following primer set to generate a vector encoding the mouse Ig-κ chain V-J2-C signal peptide (Asp 21 fibrinopeptide-deleted human fibrinogen α-chain N-terminal sequence (Gly 36 -Glu 191 ), and a DNA fragment encoding the c-myc tag was amplified. (vi) Primer set for amplifying mouse Ig-κ chain V-J2-C signal peptide: 5'-GGAGACCCAAGCTGGCTAGCCACCATGGAG-3' (forward, SEQ ID NO: 7) 5'-CACAACCCTTGGGCCACCAGTGGAACCTGGAACCCAG-3' (reverse, SEQ ID NO: 14). (vii) Primer set for amplifying fibrinopeptide-deleted human fibrinogen α chain N-terminal sequence (Gly 36 -Glu 191 (viii) Primer set for amplifying c-myc tag: 5'-GGCCCAAGGGTTGTGGAAAGACATCAATCTGC-3' (forward, SEQ ID NO: 15) 5'-TTCACGAGCTAAAGCCCTACTGCATGACC-3' (reverse, SEQ ID NO: 8) (viii) Primer set for amplifying c-myc tag: 5'-GCAGTAGGGCTTTAGCTCGTGAAGAACAAAAACTCATCTCAGAAG-3' (forward, SEQ ID NO: 9) 5'-TCGGGCCCTCCTCGAGCGGCCGCCGATCTACAGATCCTCTTCTGAGATGAGTTTTTGTTC-3' (reverse, SEQ ID NO: 10)

[0049] The three DNA fragments obtained were ligated and amplified by extension PCR using the following primer set: The amplified DNA fragments were inserted into pcDNA3.4 + MCS digested with restriction enzymes NheI and NotI using GeneArt TM The ΔFbg-N(α) expression vector was constructed using the Seamless Cloning and Assembly Kit (Thermo Fisher Scientific). (ix) Primer set for ΔFbg-N(α) amplification: 5'-GGAGACCCAAGCTGGCTAGCCACCATGGAG-3' (forward, SEQ ID NO: 7) and 5'-TCGGGCCCTCCTCGAGCGGCCGCCGATCTACAGATCCTCTTCTGAGATGAGTTTTTGTTC-3' (reverse, SEQ ID NO: 10).

[0050] (2-2) Construction of ΔFbg-N(β) Expression Vector Using the same human fibrinogen β expression vector as in (1-2) above as a template, PCR was performed with the following primer set to first amplify a DNA fragment encoding the human Ig-κ light chain signal peptide. (x) Primer set for amplifying human Ig-κ light chain signal peptide (first step): 5'-GGAGACCCAAGCTGGCTAGCCACCATGG-3' (forward, SEQ ID NO: 11) 5'-GGTATCTGAAACTGGGAGCCAGAGTAGCAGGAGGAAGAGAAGCTGCGCTGGGGTTTCCATGGTGGCTAGCCAGCTTGG-3' (reverse, SEQ ID NO: 16)

[0051] Next, PCR was performed using a DNA fragment encoding the human Ig-κ light chain signal peptide and the same human fibrinogen β expression vector as in (1-2) above as a template to generate a fragment encoding a part of the human Ig-κ light chain signal peptide / fibrinopeptide-deleted human fibrinogen β chain N-terminal sequence and a fragment encoding a fibrinopeptide-deleted human fibrinogen β chain N-terminal sequence (Gly 45 -Ser 230(xi) Primer set for amplifying a portion of the N-terminal sequence of human Ig-κ light chain signal peptide / fibrinopeptide-deleted human fibrinogen β chain: 5'-GGAGACCCAAGCTGGCTAGCCACCATGG-3' (forward, SEQ ID NO: 11) 5'-AAGGGGTCGATGACCGGTATCTGAAACTGGGAGCCAG-3' (reverse, SEQ ID NO: 17) (xii) DNA fragments encoding the N-terminal sequence of fibrinopeptide-deleted human fibrinogen β chain (Gly 45 -Ser 230 ) / 6xHis tag amplification primer set 5'-GGTCATCGACCCCTTGACAAGAAGAG-3' (forward, SEQ ID NO: 18) 5'-TCGGGCCCTCCTCGAGCGGCCGCGATCTAATGGTGATGGTGATGATGACTGACAGTGCATGGGGTGCGAC-3' (reverse, SEQ ID NO: 12)

[0052] The two DNA fragments obtained were ligated and amplified by extension PCR using the following primer set: The amplified DNA fragment was inserted into pcDNA3.4 + MCS digested with restriction enzymes NheI and NotI using GeneArt TM The ΔFbg-N(β) expression vector was constructed using the Seamless Cloning and Assembly Kit (Thermo Fisher Scientific). (xiii) Primer set for ΔFbg-N(α) amplification: 5'-GGAGACCCAAGCTGGCTAGCCACCATGG-3' (forward, SEQ ID NO: 11) and 5'-TCGGGCCCTCCTCGAGCGGCCGCGATCTAATGGTGATGGTGATGATGACTGACAGTGCATGGGGTGCGAC-3' (reverse, SEQ ID NO: 12).

[0053] (3) Expression and Purification of Fbg-N and ΔFbg-N. Fbg-N and ΔFbg-N were expressed by transfecting the expression vectors for each of the constructed chains into FreeStyle 293-F cells (Thermo Fisher Scientific, #R79007, hereafter referred to as "293-F cells"). For Fbg-N, the expression vectors for Fbg-N(α), Fbg-N(β), and Fbg-N(γ) were transfected into 293-F cells. For ΔFbg-N, the expression vectors for ΔFbg-N(α), ΔFbg-N(β), and Fbg-N(γ) were transfected into 293-F cells. 3.0 × 10 8 293-F cells (1.0 x 10 6 cells / mL) with transfection reagent 293fectin TM 120 μg of each chain expression vector was co-transfected using Transfection Reagent (Thermo Fisher Scientific, #12347019) and Opti-MEM (Thermo Fisher Scientific, #31985070). After culturing for 3 days, the culture medium was harvested.

[0054] The harvested culture medium was centrifuged at 1,000 × g for 5 minutes, and the resulting supernatant was further centrifuged at 12,000 × g for 30 minutes to remove cells and insoluble material. 2 M Tris-HCl solution (pH 8.0; final concentration: 20 mM) and 2 M imidazole solution (final concentration: 5 mM) were added to the resulting culture supernatant. Six mL of a 50% slurry of Ni-NTA agarose solution (QIAGEN, #30230) was added to the culture supernatant and incubated overnight to adsorb the target protein. The Ni-NTA agarose was recovered and washed with Tris-buffered saline (pH 7.4) containing 20 mM Tris-HCl (pH 7.4) and 137 mM NaCl, hereafter referred to as "TBS(-)"), and then eluted with TBS(-) (pH 7.4) containing 200 mM imidazole. The eluted fractions were confirmed by measuring the absorbance at A280.

[0055] The eluted fractions containing the target protein were concentrated using Amicon Ultra-15 Centrifugal Filter Units (Merck Millipore, #UFC901024) and then subjected to gel filtration chromatography using Superose 6 Increase 10 / 300GL (Cytiva, #29091596). The elution was performed with D-PBS(-), pH 7.4, at a flow rate of 0.5 mL / min. The amount of target protein in the eluted fractions was confirmed by absorbance at A280 and SDS-PAGE. The purified product after gel filtration chromatography was sterilized with a 0.22 μm disk syringe filter (Merck Millipore, #SLGV033RS) and stored at -80°C. Protein concentration was determined using Pierce TM Quantification was performed using a BCA Protein Assay Kit (Thermo Fisher Scientific, #23223) based on a standard curve prepared with bovine serum albumin.

[0056] (4) Fibrinogen Binding Assay (4-1) Plate Coating Human plasma-derived fibrinogen solution (Enzyme Research Laboratories, #FIB3) was diluted with HEPES-buffered saline (buffered saline containing 20 mM HEPES-NaOH (pH 7.4) and 137 mM NaCl, hereafter referred to as "HBS(-) (pH 7.4)") adjusted to pH 7.4 to a final concentration of 100 nM. 50 μL of this solution was added to each well of a 96-well plate (Thermo Fisher Scientific, #442404) and coated overnight at room temperature with gentle shaking.

[0057] (4-2) Fibrinogen Binding Assay: Fibrinogen-coated 96-well plates were washed with 200 μL / well of a solution containing 1% (weight / volume) bovine serum albumin (BSA; Sigma-Aldrich, #A7906) in 20 mM Tris buffer, pH 7.4 (hereafter referred to as "TBS(-)-T") containing 0.1% Tween-20 (Sigma, #P1379) and 137 mM NaCl (hereafter referred to as "1% BSA / TBS(-)-T"). Next, 200 μL / well of 1% BSA / TBS(-)-T was added and the plates were blocked at room temperature for 1 hour. After draining the 1% BSA / TBS(-)-T from the plate, 25 μL of HBS(-) was added to each well. Then, 25 μL of a solution of Fbg-N and ΔFbg-N diluted with HBS (7.4) to final concentrations of 0.4 nM, 0.8 nM, 1.6 nM, 3.1 nM, 6.3 nM, 12.5 nM, 25 nM, and 50 nM was added to each well. The plates were incubated at room temperature for 1 hour while being shaken on a shaker.

[0058] (4-3) Measurement of Bound Fbg-N and ΔFbg-N. Fibrinogen-bound Fbg-N and ΔFbg-N were quantified using anti-c-Myc antibody (Abcam, #ab62928). Anti-c-Myc antibody 9E10, diluted 2,000-fold in 1% BSA / TBS(-)-T, was added at 50 μL / well and incubated for 1 hour on a shaker at room temperature. After washing three times with 200 μL / well of TBS-T, 50 μL / well of ο-phenylenediamine (Fujifilm Wako Pure Chemical Industries, #158-01671) dissolved in 25 mM citric acid / 50 mM NaHPO buffer containing 0.04% HO to a final concentration of 0.4 mg / mL was added and incubated for 6 minutes. The color reaction was stopped by adding 100 μL / well of 2.5 M H2SO4, and the absorbance of the colorimetric substrate was measured at 490 nm using a microplate reader (Molecular Devices, Emax).

[0059] The results are shown in Figure 1. ΔFbg-N bound to fibrinogen in a dose-dependent manner in the absence of thrombin, whereas no binding was observed with Fbg-N. This result indicates that ΔFbg-N, designed to expose knobs A and B, binds to the γ- and β-holes of fibrinogen coated on the plate.

[0060] Example 2: Preparation of Enhanced Green Fluorescence Protein (EGFP)-introduced ΔFbg-N and evaluation of its incorporation into fibrin gel (1) Construction of an expression vector for C-terminal EGFP-fused Fbg-N(α) (Fbg-N(α)-EGFP) To prepare a recombinant protein (Fbg-N-EGFP) in which EGFP was introduced into the N-terminal self-association region of human fibrinogen, a vector containing the mouse Ig-κ chain V-J2-C signal peptide / human fibrinogen α chain N-terminal sequence (Ala 20 -Glu 191 ) / EGFP / c-myc tag (hereinafter referred to as "Fbg-N(α)-EGFP") expression vector was constructed.

[0061] PCR was performed using the Fbg-N(α) expression vector and pEGFP-N1 (TaKaRa Bio, #6085-1) as templates with the following primer set: 20 -Glu 191 (xiv) DNA fragments encoding mouse Ig-κ chain V-J2-C signal peptide / human fibrinogen α chain N-terminal sequence (Ala 20 -Glu 191(xv) Primer set for amplifying the N-terminal side of EGFP: 5'-CTTTAGCTCGTGAAATGGTGAGCAAGGGCGAGGAG-3' (forward, SEQ ID NO: 20) 5'-GCTCAGTTTGGACTGGGTGCTCAGGTAGTG-3' (reverse, SEQ ID NO: 21) (xvi) Primer set for amplifying the C-terminal side of EGFP and c-myc tag: 5'-CCAGTCCAAACTGAGCAAAGACCCCAACGAG-3' (forward, SEQ ID NO: 22) 5'-TCTGAGATGAGTTTGTTCCTTGTACAGCTCGTCCATGC-3' (reverse, SEQ ID NO: 23)

[0062] The three DNA fragments obtained were ligated and amplified by extension PCR using the following primer set: The amplified DNA fragments were inserted into pcDNA3.4 + MCS digested with restriction enzymes NheI and NotI using GeneArt TM The Fbg-N(α) / C(EGFP) expression vector was constructed using the Seamless Cloning and Assembly Kit (Thermo Fisher Scientific). (xvii) Primer set for amplifying Fbg-N(α) / C(EGFP): 5'-GGAGACCCAAGCTGGCTAGCCACCATGG-3' (forward, SEQ ID NO: 11) and 5'-TCGGGCCCTCCTCGAGCGGCCGCCGATCTACAGATCCTCTTCTGAGATGAGTTTTTGTTC-3' (reverse, SEQ ID NO: 10).

[0063] (2) Construction of an expression vector for C-terminal EGFP-fused ΔFbg-N(α) (ΔFbg-N(α)-EGFP). To prepare a recombinant protein (ΔFbg-N-EGFP) in which EGFP was introduced into the N-terminal self-association region of fibrinopeptide-deleted human fibrinogen, we cloned the mouse Ig-κ chain V-J2-C signal peptide / fibrinopeptide-deleted human fibrinogen α chain N-terminal sequence (Gly 36 -Glu 191 ) / EGFP / c-myc tag (hereinafter referred to as "ΔFbg-N(α)-EGFP") was constructed.

[0064] PCR was performed using the Fbg-N(α)-EGFP expression vector as a template and the following primer set to amplify the mouse Ig-κ chain V-J2-C signal peptide and the fibrinopeptide-deleted human fibrinogen α chain N-terminal sequence (Gly 36 -Glu 191 (xviii) Primer set for amplifying mouse Ig-κ chain V-J2-C signal peptide: 5'-GGAGACCCAAGCTGGCTAGCCACCATGG-3' (forward, SEQ ID NO: 11) 5'-CACAACCCTTGGGCCACCAGTGGAACCTGGAACCCAG-3' (reverse, SEQ ID NO: 14) (xix) Fibrinopeptide-deleted human fibrinogen α chain N-terminal sequence (Gly 36 -Glu 191 Primer set for amplifying the c-myc tag: 5'-GGCCCAAGGGTTGTGGAAAGACATCAATCTGC-3' (forward, SEQ ID NO: 15) 5'-TCGGGCCCTCCTCGAGCGGCCGCCGATCTACAGATCCTCTTCTGAGATGAGTTTTTGTTC-3' (reverse, SEQ ID NO: 10)

[0065] The two DNA fragments obtained were ligated and amplified by extension PCR using the following primer set: The amplified DNA fragment was inserted into pcDNA3.4 + MCS digested with restriction enzymes NheI and NotI using GeneArt TMThe ΔFbg-N(α)-EGFP expression vector was constructed using the Seamless Cloning and Assembly Kit (Thermo Fisher Scientific). (xx) Primer set for amplifying Fbg-N(α)-EGFP: 5'-GGAGACCCAAGCTGGCTAGCCACCATGG-3' (forward, SEQ ID NO: 11) and 5'-TCGGGCCCTCCTCGAGCGGCCGCCGATCTACAGATCCTCTTCTGAGATGAGTTTTTGTTC-3' (reverse, SEQ ID NO: 10).

[0066] (3) Expression and purification of ΔFbg-N-EGFP ΔFbg-N-EGFP was expressed by introducing the expression vectors for each of the constructed chains into 293-F cells. First, the expression vectors for ΔFbg-N(α), ΔFbg-N(β), and Fbg-N(γ) were introduced into 293-F cells. 5.0 × 10 8 293-F cells (1.0 x 10 6 cells / mL) with transfection reagent 293fectin TM Using Transfection Reagent (Thermo Fisher Scientific, #12347019) and Opti-MEM (Thermo Fisher Scientific, #31985070), 200 μg of each chain expression vector was co-transfected and cultured for 3 days, after which the culture medium was harvested.

[0067] The collected culture medium was centrifuged at 1,000 × g for 5 minutes, and the resulting supernatant was further centrifuged at 12,000 × g for 30 minutes to remove cells and insoluble material. 2 M Tris-HCl (pH 8.0) and 2 M imidazole were added to the resulting culture supernatant to final concentrations of 20 mM and 5 mM, respectively. Six mL of a 50% slurry of Ni-NTA agarose solution (QIAGEN, #30230) was added to the culture supernatant and incubated overnight to adsorb the target protein. The Ni-NTA agarose was collected, washed with TBS(-), and then eluted with TBS(-) containing 200 mM imidazole. The eluted fraction was confirmed by measuring the absorbance at A280.

[0068] The eluted fraction containing the target protein was concentrated using Amicon Ultra-15 Centrifugal Filter Units (Merck Millipore, #UFC901024) and then subjected to gel filtration chromatography using Superose 6 Increase 10 / 300GL (Cytiva, #29091596). The eluate was eluted with D-PBS(-) at a flow rate of 0.5 mL / min. The amount of target protein in the eluted fraction was confirmed by absorbance at A280 and SDS-PAGE. The purified product after gel filtration chromatography was sterilized with a 0.22 μm disc syringe filter (Merck Millipore, #SLGV033RS) and stored at -80°C. Protein concentration was determined using Pierce TM Quantification was performed using a BCA Protein Assay Kit (Thermo Fisher Scientific, #23223) based on a standard curve prepared with bovine serum albumin.

[0069] (4) Preparation and observation of fibrin gel containing ΔFbg-N / EGFP. Purified ΔFbg-N-EGFP and human fibrinogen solution (Enzyme Research Laboratories, #FIB3) were mixed in a 1.7 mL plastic tube with Alexa Fluor Fluor Fluorescence Imaging (Fluorescence Indicator). TMA 100 μL 2x mixture of ΔFbg-N / EGFP and human fibrinogen was prepared by diluting 647-labeled human fibrinogen solution (Thermo Fisher Scientific, #F-35200) with HBS(-) to 500 nM, 4.75 mg / mL, and 0.25 mg / mL, respectively. Next, in a separate 1.7 mL plastic tube, human thrombin (Sigma-Aldrich, #T4393) was diluted with HBS(-) to 1 NIH unit / mL, preparing 100 μL of 2x thrombin solution. 100 μL of 2x thrombin solution was added to 100 μL of the 2x mixture, mixed by inversion, and 100 μL of the mixture was added to one well of a 24-well glass-bottom plate, SensoPlate (Greiner, #662892). The mixture was incubated at 37°C / 5% CO2 for 10 minutes and then washed twice with HBS(-). The prepared gel was observed using a confocal laser scanning microscope, FLUOVIEW FV1200 system and UPLSAPO60XS (NA: 1.30).

[0070] The results are shown in Figure 2. (A) shows Alexa Fluor TM (B) is a fluorescent image of 647-labeled fibrin gel, (C) is a fluorescent image of ΔFbg-N-EGFP-containing fibrin gel, and (C) is a superimposed image of (A) and (B). Co-localization of the two was observed in (C). This result indicates that ΔFbg-N-EGFP is incorporated into polymerized fibrinogen polymers.

[0071] Example 3: Preparation of ΔFbg-N incorporating the cell adhesion domain of human fibronectin (FNIII7-10) and evaluation of cell adhesion activity (1) Construction of an expression vector for ΔFbg-N(α) (ΔFbg-N(α)-FN) in which the cell adhesion domain of fibronectin, FNIII7-10, is linked to the C-terminus. To prepare a recombinant protein (ΔFbg-N-FN) in which the cell adhesion domain of fibronectin, FNIII7-10, is linked to the N-terminal self-association region of human fibrinogen, the mouse Ig-κ chain V-J2-C signal peptide (Asp 21 ) and fibrinopeptide-deleted human fibrinogen α chain N-terminal sequence (Gly36 -Glu 191 ) / FNIII7-10 (Pro 1173 -Thr 1631 ) / c-myc tag (hereinafter referred to as "ΔFbg-N(α)-FN") expression vector was constructed.

[0072] PCR was performed using the ΔFbg-N(α) expression vector and the FNIII7-10 fused human laminin γ1E8 fragment expression vector (see WO2012 / 137970) as templates with the following primer set to generate a gene encoding the mouse Ig-κ chain V-J2-C signal peptide / fibrinopeptide-deleted human fibrinogen α chain N-terminal sequence (Gly 36 -Glu 191 ) and FNIII7-10 (Pro 1173 -Thr 1631 (xxi) DNA fragments encoding mouse Ig-κ chain V-J2-C signal peptide / fibrinopeptide-deleted human fibrinogen α chain N-terminal sequence (Gly 36 -Glu 191 ) Amplification primer set: 5'-GGAGACCCAAGCTGGCTAGCCACCATGG-3' (forward, SEQ ID NO: 11) 5'-TGGTGGAGACAATGGTTCACGAGCTAAAGCCCTACTGCATGAC-3' (reverse, SEQ ID NO: 24) (xxii) FNIII7-10 (Pro 1173 -Thr 1631 ) / c-myc tag amplification primer set 5'-GCTTTAGCTCGTGAACCATTGTCTCCACCAACAAACTTGC-3' (forward, SEQ ID NO: 25) 5'-TCTGAGATGAGTTTTTGTTCTGTTCGGTAATTAATGGAAATTGG-3' (reverse, SEQ ID NO: 26)

[0073] The two DNA fragments obtained were ligated and amplified by extension PCR using the following primer set: The amplified DNA fragment was inserted into pcDNA3.4+MC digested with restriction enzymes NheI and NotI using GeneArt TMThe ΔFbg-N(α)-FN expression vector was constructed using the Seamless Cloning and Assembly Kit (Thermo Fisher Scientific). (xxiii) Primer set for amplifying Fbg-N(α)-FN: 5'-GGAGACCCAAGCTGGCTAGCCACCATGG-3' (forward, SEQ ID NO: 11) and 5'-TCGGGCCCTCCTCGAGCGGCCGCCGATCTACAGATCCTCTTCTGAGATGAGTTTTTGTTC-3' (reverse, SEQ ID NO: 10).

[0074] (2) Expression and purification of ΔFbg-N-FN ΔFbg-N / FN was expressed by introducing the expression vectors for each of the constructed chains into 293-F cells. First, the expression vectors for ΔFbg-N(α)-FN, ΔFbg-N(β), and Fbg-N(γ) were introduced into 293-F cells. 5.0 × 10 293-F cells (1.0 × 10 6 cells / mL) with transfection reagent 293fectin TM Using Transfection Reagent (Thermo Fisher Scientific, #12347019) and Opti-MEM (Thermo Fisher Scientific, #31985070), 200 μg of each chain expression vector was co-transfected and cultured for 3 days, after which the culture medium was harvested.

[0075] The collected culture medium was centrifuged at 1,000 × g for 5 minutes, and the resulting supernatant was further centrifuged at 12,000 × g for 30 minutes to remove cells and insoluble material. 2 M Tris-HCl (pH 8.0) and 2 M imidazole were added to the resulting culture supernatant to final concentrations of 20 mM and 5 mM, respectively. Six mL of a 50% slurry of Ni-NTA agarose solution (QIAGEN, #30230) was added to the culture supernatant and incubated overnight to adsorb the target protein. The Ni-NTA agarose was collected, washed with TBS(-), and then eluted with TBS(-) containing 200 mM imidazole. The eluted fraction was confirmed by measuring the absorbance at A280.

[0076] The eluted fraction containing the target protein was concentrated using Amicon Ultra-15 Centrifugal Filter Units (Merck Millipore, #UFC901024) and then subjected to gel filtration chromatography using Superose 6 Increase 10 / 300GL (Cytiva, #29091596). The eluate was eluted with D-PBS(-) at a flow rate of 0.5 mL / min. The amount of target protein in the eluted fraction was confirmed by absorbance at A280 and SDS-PAGE. The purified product after gel filtration chromatography was sterilized with a 0.22 μm disc syringe filter (Merck Millipore, #SLGV033RS) and stored at -80°C. Protein concentration was determined using Pierce TM Quantification was performed using a BCA Protein Assay Kit (Thermo Fisher Scientific, #23223) based on a standard curve prepared with bovine serum albumin.

[0077] (3) Cell Adhesion Activity of ΔFbg-N-FN Entrapped in Fibrinogen (3-1) Subculture of Human Fibrosarcoma-Derived HT-1080 Cells HT-1080 cells (JCRB Cell Bank, #JCRB9113) were maintained and cultured in Dulbecco's Modified Eagle Medium (DMEM; Nacalai, #08456) containing 10% (vol / vol) heat-inactivated fetal bovine serum (Sigma-Aldrich, #172012-500ML) (hereafter referred to as "10% FBS / DMEM"). 10 cm diameter cell culture-treated dishes (Corning, #353003) were used as culture vessels.

[0078] (3-2) HT-1080 Cell Adhesion Assay (3-2-1) Plate Coating Human plasma-derived fibrinogen solution (Enzyme Research Laboratories, #FIB3) was diluted with D-PBS(-) (pH 7.4) to a final concentration of 100 nM, and then 350 μL was added to each well of a 24-well cell culture plate (Corning, #353047). The plate was then coated overnight at room temperature with gentle shaking.

[0079] (3-2-2) Capture of ΔFbg-N-FN and ΔFbg-N by fibrinogen. The fibrinogen-coated 24-well plate was washed with 1% (wt / vol) BSA (Sigma-Aldrich, #A7906) in D-PBS(-) (Nacalai, #14249) (hereafter referred to as "1% BSA / D-PBS(-)") at 1 mL / well. Blocking was then performed at 37°C / 5% CO2 for 1 hour. After the 1% BSA / D-PBS(-) solution was removed, the plate was washed with 1 mL of D-PBS(-) per well. ΔFbg-N-FN and ΔFbg-N solutions, each diluted to 5 nM in D-PBS(-), were added at 350 μL per well and incubated at 37°C for 2 hours under 5% CO2. After washing the plate with 1 mL of DMEM containing 20 mM HEPES-NaOH (pH 7.4) (Nacalai, #17557-94) and 1% (wt / vol) BSA (hereafter referred to as "assay medium"), the plate was then added at 1 mL per well and incubated at 37°C for 2 hours under 5% CO2.

[0080] (3-2-3) Seeding, fixation, and observation of cells After removing the medium from the HT-1080 cells that had been maintained and subcultured, 10 mL of D-PBS(-) containing 1 mM EDTA (hereinafter referred to as "EDTA / D-PBS(-)") was added to the dish to wash the cells. TMCells were detached by adding 1 mL of Select Enzyme (1X) (Thermo Fisher Scientific, #12563011) per dish and allowing the dish to stand at 37°C / 5% CO2. After confirming cell detachment under a microscope, 4 mL of 10% FBS / DMEM was added per dish to stop the enzyme reaction. The detached cells were suspended and physically monodispersed using a pipette. The resulting cell suspension was then allowed to stand in a floating state at 37°C / 5% CO2 for 30 minutes to promote cell exocytosis. Cells were collected by centrifugation and then washed with assay medium. After collecting the cells again by centrifugation, they were suspended in assay medium and the cell density was quantified using a hemocytometer. The cell density was adjusted to 2 x 10 in assay medium. 5 After adjusting the concentration to cells / mL, ΔFbg-N-FN and ΔFbg-N were seeded at 1 mL / well onto plates containing the respective antibodies. After incubation at 37°C / 5% CO2 for 30 minutes, the plates were removed and washed twice with 1 mL / well of assay medium. Cells were fixed by adding 500 μL / well of 4% (wt / vol) paraformaldehyde in D-PBS(-) (hereafter referred to as "fixative") and incubating at room temperature for 10 minutes. After removing the fixative, the cells were washed twice with 1 mL / well of D-PBS(-). Cell nuclei were stained by adding 500 μL / well of 200 mM MES-NaOH (pH 6.0) containing 20 μg / mL Hoechst 33342 (Thermo Fisher Scientific, #H3570) in 200 mM MES-NaOH (pH 6.0) and incubating at room temperature for 30 minutes. Fluorescence and phase-contrast images of cells after nuclear staining were taken using a BZ-X710 fluorescence microscope (Keyence).

[0081] The results are shown in Figure 3. (A) is an image of a plate in which ΔFbg-N-FN was captured by fibrinogen, and (B) is an image of a plate in which ΔFbg-N was captured by fibrinogen. HT-1080 cell adhesion was observed in (A), but not in (B). This result indicates that ΔFbg-N, which has the cell adhesive domain of human fibronectin attached, can be incorporated into fibrinogen in the absence of thrombin and exerts cell adhesive activity.

[0082] Example 4: Preparation of ΔFbg-N linked to the cell adhesion domain of human laminin-511 and evaluation of cell adhesion activity (1-1) Construction of ΔFbg-N(β)-LM(α) expression vector Using the Chimera-α5 expression vector (see Patent Document 1) as a template, PCR was performed with the following primer set to amplify a DNA fragment encoding the human Ig-κ light chain signal peptide. (xxiv) Primer set for amplifying human Ig-κ light chain signal peptide: 5'-GGAGACCCAAGCTGGCTAGCCACCATGG-3' (forward, SEQ ID NO: 11) 5'-GGTATCTGAAACTGGGAGCCAGAGTAGCAGGAGGAAGAGAAGCTGCGCTGGGGTTTCCATGGTGGCTAGCCAGCTTGG-3' (reverse, SEQ ID NO: 16)

[0083] Next, PCR was performed using a DNA fragment encoding the human Ig-κ light chain signal peptide and the Chimera-α5 expression vector (see Patent Document 1) as templates to generate a fragment encoding a human Ig-κ light chain signal peptide / fibrinopeptide-deleted portion of the N-terminal sequence of human fibrinogen β chain (Gly 45 -Leu 49 ) and fibrinopeptide-deleted human fibrinogen β-chain N-terminal sequence (Gly 45 -Asn 194) / DNA fragment encoding human laminin α5 (coiled-coil region and part of globular domain 1) was amplified. (xxv) Primer set for amplifying human Ig-κ light chain signal peptide: 5'-GGAGACCCAAGCTGGCTAGCCACCATGG-3' (forward, SEQ ID NO: 11) 5'-AAGGGGTCGATGACCGGTATCTGAAACTGGGAGCCAG-3' (reverse, SEQ ID NO: 17) (xxvi) Fibrinopeptide-deleted human fibrinogen β chain N-terminal sequence (Gly 45 -Asn 194 ) / Primer set for amplifying human laminin α5 (coiled-coil region and part of globular domain 1): 5'-GGTCATCGACCCCTTGACAAGAAGAG-3' (forward, SEQ ID NO: 18) 5'-CTCCCCAATGTCCTCATCGATGCTTAGGACTGC-3' (forward, SEQ ID NO: 27)

[0084] The two DNA fragments obtained were ligated and amplified by extension PCR using the following primer set: The amplified DNA fragment was inserted into a Chimera-α5 expression vector (see Patent Document 1) digested with restriction enzymes NheI and ClaI. TM The ΔFbg-N(β)-LM(α) expression vector was constructed using the Seamless Cloning and Assembly Kit (Thermo Fisher Scientific). (xxvii) Primer set for amplifying Fbg-N(β)-LM(α): 5'-GGAGACCCAAGCTGGCTAGCCACCATGG-3' (forward, SEQ ID NO: 11) and 5'-CTCCCCAATGTCCTCATCGATGCTTAGGACTGC-3' (forward, SEQ ID NO: 27).

[0085] (1-2) Construction of ΔFbg-N(α)-LM(β) Expression Vector Using the Chimera-β1 expression vector (see Patent Document 1) as a template, PCR was performed with the following primer set to amplify the sequence encoding the mouse Ig-κ chain V-J2-C signal peptide and the fibrinopeptide-deleted human fibrinogen α chain N-terminal sequence (Gly 36 -His151 ) / Laminin β1 (Leu 1761 -Leu 1786 (xxviii) Primer set for amplifying mouse Ig-κ chain V-J2-C signal peptide: 5'-GGAGACCCAAGCTGGCTAGCCACCATGGAG-3' (forward, SEQ ID NO: 7) 5'-CACAACCCTTGGGCCACCAGTGGAACCTGGAACCCAG-3' (reverse, SEQ ID NO: 14) (xxix) Fibrinopeptide-deleted human fibrinogen α chain N-terminal sequence (Gly 36 -His 151 ) / Laminin β1 (Leu 1761 -Leu 1786 ) Amplification primer set: 5'-GGCCCAAGGGTTGTGGAAAGACATCAATCTGC-3' (forward, SEQ ID NO: 15) 5'-GGGCCCTCCTCGAGCGGCCGCCACTGTGC-3' (forward, SEQ ID NO: 28)

[0086] The two DNA fragments obtained were ligated and amplified by extension PCR using the following primer set: The amplified DNA fragment was inserted into pcDNA3.4 + MCS digested with restriction enzymes NheI and NotI using GeneArt TM The ΔFbg-N(α)-LM(β) expression vector was constructed using the Seamless Cloning and Assembly Kit (Thermo Fisher Scientific). (xxx) Primer set for amplifying Fbg-N(α)-LM(β): 5'-GGAGACCCAAGCTGGCTAGCCACCATGGAG-3' (forward, SEQ ID NO: 7) and 5'-GGGCCCTCCTCGAGCGGCCGCCACTGTGC-3' (forward, SEQ ID NO: 28).

[0087] (1-3) Construction of Fbg-N(γ)-LM(γ1EQ) Expression Vector. The glutamic acid residue at position 1607 of the human laminin γ1 chain is an acidic residue essential for laminin integrin binding. Substitution of this glutamic acid residue with glutamine significantly reduces integrin binding activity without affecting laminin structure (Ido et al., “The requirement of the glutamic acid residue at the third position from the carboxyl termini of the laminin gamma chains in integrin binding by laminins.” JBC(2007) 282(15):111144-54; Takizawa et al., “Mechanistic basis for the recognition of laminin-511 by α6β1 integrin.” Sci. Adv.(2017) 3(9):e1701497). PCR was performed using the Chimera-γ1 expression vector (see Patent Document 1) as a template and the following primer set to amplify the sequence encoding the mouse Ig-κ chain V-J2-C signal peptide / human fibrinogen γ chain N-terminal sequence (Tyr 27 -Ser 132 ) / Laminin γ1 (Ile 1579 -Pro 1609 / Glu1607Gln) and laminin γ1 (Ile 1579 -Pro 1609 (xxxi) A DNA fragment encoding a portion of the C-terminus of mouse Ig-κ chain V-J2-C signal peptide / human fibrinogen γ chain N-terminal sequence (Tyr 27 -Ser 132 ) / Laminin γ1 (Ile 1579 -Pro 1609Primer set for amplifying laminin γ1 (Ile / Glu1607Gln): 5'-CTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGA-3' (forward, SEQ ID NO: 29) 5'-TTCGATCTAGGGCTTCTGAATGGACGGGGTGTTG-3' (reverse, SEQ ID NO: 30) 1579 -Pro 1609 Primer set for amplifying the C-terminal region of Glu1607Gln: 5'-CAACACCCCGTCCATTCAGAAGCCCTAGATCGAA-3' (forward, SEQ ID NO: 31) 5'-CGGTAGGGATCGAACCCTTGATGGCTGGC-3' (reverse, SEQ ID NO: 32)

[0088] The two DNA fragments obtained were ligated and amplified by extension PCR using the following primer set. The resulting DNA fragment was digested with restriction enzymes NheI and ClaI and inserted into the corresponding restriction enzyme sites of pcDNA3.4+MC to construct the Fbg-N(γ)-LM(γ1EQ) expression vector. (xxxiii) Primer set for amplifying Fbg-N(γ)-LM(γ1EQ): 5'-CTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGA-3' (forward, SEQ ID NO: 29) 5'-CGGTAGGGATCGAACCCTTGATGGCTGGC-3' (reverse, SEQ ID NO: 32)

[0089] (2) Expression and purification of ΔFbg-N-LM and ΔFbg-N-LM(γ1EQ). ΔFbg-N-LM and ΔFbg-N-LM(γ1EQ) were expressed by transfecting the expression vectors for each chain into 293-F cells. First, the expression vectors for ΔFbg-N(β)-LM(α), ΔFbg-N(α)-LM(β), Fbg-N(γ)-LM(γ) or ΔFbg-N(β)-LM(α), ΔFbg-N(α)-LM(β), and Fbg-N(γ)-LM(γ1EQ) were transfected into 293-F cells. 5.0 × 10 8 293-F cells (1.0 x 10 6cells / mL) with transfection reagent 293fectin TM Using Transfection Reagent (Thermo Fisher Scientific, #12347019) and Opti-MEM (Thermo Fisher Scientific, #31985070), 200 μg of each chain expression vector was co-transfected and cultured for 3 days, after which the culture medium was harvested.

[0090] The collected culture medium was centrifuged at 1,000 × g for 5 minutes, and the supernatant was further centrifuged at 10,000 × g for 30 minutes to remove cells and insoluble matter. TM His-Tag Purification Resin (Roche, #5893801001) was added and incubated overnight to adsorb the target protein. TM The His-Tag Purification Resin was recovered and washed with HEPES-buffered saline (HBS(-) (pH 8.0)) adjusted to pH 8.0 (20 mM HEPES and 137 mM NaCl), followed by elution with HBS(-) (pH 8.0) containing 250 mM imidazole. Recovery of the target protein was confirmed by absorbance at A280 and SDS-PAGE.

[0091] The eluted fraction containing the target protein was concentrated using Amicon Ultra-15 Centrifugal Filter Units (Merck Millipore, #UFC901024) and then subjected to gel filtration chromatography using Superose 6 Increase 10 / 300GL (Cytiva, #29091596). The eluate was eluted with HBS(-), pH 7.4, at a flow rate of 0.5 mL / min. The amount of target protein in the eluted fraction was confirmed by SDS-PAGE. The purified product after gel filtration chromatography was sterilized with a 0.22 μm disk syringe filter (Merck Millipore, #SLGV033RS) and stored at -80°C. Protein concentration was determined using PierceTM Quantification was performed using a BCA Protein Assay Kit (Thermo Fisher Scientific, #23223) based on a standard curve prepared with bovine serum albumin.

[0092] (3) Cell Adhesion Activity of ΔFbg-N-LM and ΔFbg-N-LM(γ1EQ) Supplemented with Fibrinogen (3-1) Subculture of Human Fibrosarcoma-Derived HT-1080 Cells. HT-1080 cells (JCRB Cell Bank, #JCRB9113) were maintained in 10% FBS / DMEM. 10 cm diameter cell culture-treated dishes (Corning, #353003) were used as culture vessels. (3-2) HT-1080 Cell Adhesion Assay (3-2-1) Plate Coating. Human plasma-derived fibrinogen solution (Enzyme Research Laboratories, #FIB3) was diluted with D-PBS(-) (pH 7.4) to a final concentration of 100 nM. 350 μL was added to each well of a 24-well cell culture plate (Corning, #353047) and incubated overnight at room temperature with gentle shaking.

[0093] (3-2-2) Capture of ΔFbg-N-LM and ΔFbg-N-LM(γ1EQ) by Fibrinogen. 1% BSA / D-PBS(-) was added to a fibrinogen-coated 24-well plate at 1 mL / well and washed. Next, 1% BSA / D-PBS(-) was added at 1 mL / well and blocked at 37°C / 5% CO2 for 1 hour. After draining the 1% BSA / D-PBS(-), the plate was washed with 1 mL / well of D-PBS(-). After that, ΔFbg-N-LM and ΔFbg-N-LM(γ1EQ) solutions, diluted to 5 nM in D-PBS(-), were added at 350 μL / well to separate wells and incubated at 37°C / 5% CO2 for 2 hours. After washing the plate with 1 mL / well of assay medium, 1 mL / well of assay medium was added and the plate was left to stand under 37°C / 5% CO2 conditions.

[0094] (3-2-3) Cell seeding, fixation, and observation After removing the medium from the HT-1080 cells that had been maintained and subcultured, 10 mL of EDTA / D-PBS(-) was added to the dish to wash the cells. TM Cells were detached by adding 1 mL of Select Enzyme (1X) (Thermo Fisher Scientific, #12563011) per dish and allowing the dish to stand at 37°C / 5% CO2. After confirming cell detachment under a microscope, 4 mL of 10% FBS / DMEM was added per dish to stop the enzyme reaction. The detached cells were suspended and physically monodispersed using a pipette. The resulting cell suspension was then allowed to stand in a floating state at 37°C / 5% CO2 for 30 minutes to promote cell exocytosis. Cells were collected by centrifugation and then washed with assay medium. After collecting the cells again by centrifugation, they were suspended in assay medium and the cell density was quantified using a hemocytometer. The cell density was adjusted to 2 x 10 in assay medium. 5 After adjusting the concentration to cells / mL, ΔFbg-N-LM and ΔFbg-N-LM(γ1EQ) were seeded at 1 mL / well onto plates containing the respective ΔFbg-N-LM and ΔFbg-N-LM(γ1EQ). After 30 minutes at 37°C / 5% CO2, the plates were removed and washed twice with 1 mL / well of assay medium. 500 μL / well of fixative solution was added and incubated at room temperature for 10 minutes to fix the cells. After removing the fixative, the cells were washed twice with 1 mL / well of D-PBS(-). 500 μL / well of 200 mM MES-NaOH (pH 6.0) containing 20 μg / mL Hoechst 33342 (Thermo Fisher Scientific, #H3570) was added and incubated at room temperature for 30 minutes to stain the cell nuclei. Fluorescence and phase-contrast images of the cells after nuclear staining were captured using a BZ-X710 fluorescence microscope (Keyence).

[0095] The results are shown in Figure 4. (A) is an image of a plate in which ΔFbg-N-LM was captured by fibrinogen, and (B) is an image of a plate in which ΔFbg-N-LM(γ1EQ) was captured by fibrinogen. HT-1080 cell adhesion was observed in (A), but not in (B). This result indicates that ΔFbg-N-LM, which has the cell adhesive domain of laminin-511 attached, can be incorporated into fibrinogen in the absence of thrombin and exerts integrin-dependent cell adhesion activity.

[0096] Example 5: Cultivation of HT-1080 cells on fibrin gels carrying ΔFbg-N-LM (1) Subculture of human fibrosarcoma-derived HT-1080 cells HT-1080 cells (JCRB Cell Bank, #JCRB9113) were maintained and cultured in 10% FBS / DMEM. A 10 cm diameter cell culture-treated dish (Corning, #353003) was used as the culture vessel.

[0097] (2) Preparation of fibrin gel and loading of ΔFbg-N-LM. In a 1.7 mL plastic tube, 77 μL of human plasma-derived fibrinogen solution (Enzyme Research Laboratories, #FIB3) was mixed with 51 μL of assay medium and 72 μL of 20 mM phosphate buffer (pH 7.4) containing 1 M NaCl to prepare 200 μL of 2× fibrinogen solution (fibrinogen: 5 mg / mL, NaCl concentration: 388 mM). Next, in a separate 1.7 mL plastic tube, human thrombin (Sigma-Aldrich, #T4393) was diluted with assay medium to a concentration of 1 NIH units / mL to prepare 200 μL of 2× thrombin solution. 200 μL of 2x fibrinogen solution was added to 200 μL of 2x thrombin solution and mixed by inversion. 250 μL of the mixture was added to one well of a 24-well cell culture plate (Corning, #353047). After incubation at 37°C / 5% CO2 for 10 minutes, 1 mL of assay medium was added per well and the plate was incubated at 37°C / 5% CO2 for 60 minutes. The medium was then aspirated, and 1 mL of assay medium was added per well. The plate was then incubated at 37°C / 5% CO2 overnight.

[0098] The medium was removed by aspiration from the wells containing the fibrin gels, and 500 μL of ΔFbg-N-LM solution diluted to 5 nM in assay medium was added to each well. The mixture was incubated at 37°C with 5% CO2 for 2 hours. The medium was then removed by aspiration, and 1 mL of assay medium was added to each well to wash the gels. This washing procedure was repeated twice. After washing, 500 μL of assay medium was added to each well, and the gels were incubated at 37°C with 5% CO2 until cell seeding. As a control, fibrin gels treated with ΔFbg-N-LM(γ1EQ) solution were prepared in the same manner.

[0099] (3) Cell seeding and observation After removing the medium from the HT-1080 cells that had been maintained and subcultured, 10 mL of EDTA / D-PBS(-) was added to the dish to wash the cells. TMCells were detached by adding 1 mL of Select Enzyme (1X) (Thermo Fisher Scientific, #12563011) per dish and allowing the dish to stand at 37°C / 5% CO2. After confirming cell detachment under a microscope, 4 mL of 10% FBS / DMEM was added per dish to stop the enzyme reaction. The detached cells were suspended and physically monodispersed using a pipette. The resulting cell suspension was then allowed to stand in a floating state at 37°C / 5% CO2 for 30 minutes to promote cell exocytosis. Cells were collected by centrifugation and then washed with assay medium. After collecting the cells again by centrifugation, they were suspended in assay medium and the cell density was quantified using a hemocytometer. The cell density was adjusted to 2 x 10 in assay medium. 5 After adjusting the concentration to cells / mL, 1 mL / well of the medium was seeded onto plates containing ΔFbg-N-LM and ΔFbg-N-LM(γ1EQ). After incubation at 37°C / 5% CO2 for 30 minutes, the plates were removed and washed twice with 1 mL / well of assay medium. Another 1 mL / well of assay medium was added, and phase-contrast images were captured using an AxioCam ERc5s digital camera mounted on a Primo Vert (Zeiss).

[0100] The results are shown in Figure 5. (A) Images of cells on fibrin gels treated with ΔFbg-N-LM, (B) Images of cells on fibrin gels treated with ΔFbg-N-LM(γ1EQ), and (C) Images of cells on untreated fibrin gels. HT-1080 cells adhered and spread on fibrin gels loaded with ΔFbg-N-LM (A). On the other hand, no cell adhesion or spreading was observed on fibrin gels loaded with ΔFbg-N-LM(γ1EQ) whose integrin binding activity had been inactivated (B), as with untreated fibrin gels (C). These results indicate that ΔFbg-N-LM can be incorporated into fibrin gels in the absence of thrombin and can confer the cell adhesive activity of laminin-511 to the fibrin gel surface.

[0101] The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in different embodiments. Furthermore, all academic literature and patent documents described in this specification are incorporated herein by reference.

Claims

1. A protein with a hexameric structure in which two molecules of a heterotrimeric protein composed of (1) a fibrinogen Aα chain lacking fibrinopeptide A, (2) a fibrinogen Bβ chain lacking fibrinopeptide B and losing its ability to bind to the B knob, and (3) a fibrinogen γ chain losing its ability to bind to the A knob are associated at the N-terminus.

2. The protein of claim 1, wherein the fibrinogen Aα chain of (1) is a fragment containing at least 116 amino acids from the amino acid sequence shown in SEQ ID NO: 1, with the 36th amino acid at its N-terminus; the fibrinogen Bβ chain of (2) is a fragment containing at least 150 amino acids from the amino acid sequence shown in SEQ ID NO: 3, with the 45th amino acid at its N-terminus; and the fibrinogen γ chain of (3) is a fragment containing at least 107 amino acids from the amino acid sequence shown in SEQ ID NO: 5, with the 27th amino acid at its N-terminus.

3. The protein of claim 1, further comprising a non-fibrin bioactive protein linked to at least one of the fibrinogen chains (1) to (3).

4. The protein of claim 3, wherein the non-fibrin bioactive protein is selected from the group consisting of cell adhesion proteins, growth factors, growth factor binding proteins, cytokines, chemokines, anti-adhesion proteins, immunostimulatory proteins, immunomodulatory proteins, protein-binding proteins, nucleic acid-binding proteins, carbohydrate-binding proteins, virus-binding proteins, cytotoxic proteins, enzyme proteins, and protease-inhibitory proteins.

5. The protein of claim 3, wherein the non-fibrin bioactive protein is a cell adhesion protein and / or a growth factor binding protein.

6. A kit for producing the protein described in claim 1, comprising (1) a nucleic acid encoding the fibrinogen Aα chain or an expression vector containing said nucleic acid, (2) a nucleic acid encoding the fibrinogen Bβ chain or an expression vector containing said nucleic acid, and (3) a nucleic acid encoding the fibrinogen γ chain or an expression vector containing said nucleic acid.

7. A method for imparting physiological activity to a fibrin gel, comprising the steps of adding the protein described in claim 3 to a fibrinogen solution to bind the protein to fibrinogen, and adding thrombin to the resulting solution to prepare a fibrin gel.

8. A method for imparting physiological activity to a fibrin gel, comprising the step of contacting the fibrin gel with the protein of claim 3 to bind the protein to fibrin.

9. A method for imparting physiological activity to fibrinogen, comprising the step of contacting fibrinogen with the protein of claim 3 to bind said protein to fibrinogen.

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