Modified fibroblast growth factor chimeric protein and use thereof

Abstract

To provide a modified fibroblast growth factor chimeric protein enabling maintenance of superior cell growth-promoting activity under a temperature-retaining condition for approximately three days.SOLUTION: The modified fibroblast growth factor chimeric protein according to the present disclosure comprises an FGF chimeric polypeptide (FGFC) in which the sequence at positions 41 to 83 in an amino acid sequence having at least 90% identity to the amino acid sequence of an FGF1 polypeptide of the fibroblast growth factor (FGF) family is replaced by an amino acid sequence having at least 90% identity to the sequence at positions 43 to 85 in the amino acid sequence of an FGF2 polypeptide of the FGF family, and further comprises a GAG-added sequence polypeptide (PG polypeptide) to which a glycosaminoglycan (GAG) sugar chain is bound.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a modified chimeric protein obtained by modifying a chimeric protein of fibroblast growth factor (FGF) and its uses, and in particular to a modified fibroblast growth factor chimeric protein that has good stability and can suppress adsorption to containers, etc., and its uses, including its production method, etc. [Background technology]

[0002] The fibroblast growth factor (FGF) family is a group of polypeptide growth factors ranging in size from 18 to 35 kDa, with 22 members known in humans and mice. Each FGF family member (FGF protein) not only promotes the proliferation of various cell types, including fibroblasts, neurons, and epithelial cells, but also contributes to cell migration and differentiation. FGF proteins exert their functions by binding to receptors present on the surface of target cells. However, they have a strong affinity for heparan sulfate, and the coexistence of heparan sulfate is known to be necessary for FGF protein activity.

[0003] FGF proteins are thought to be useful in treating skin ulcers (bedsores, pressure sores), gastric ulcers, repairing surgical wounds or burns, establishing skin grafts, repairing periodontal tissue, repairing spinal injuries, etc. Some FGF proteins are already in practical use for treating skin ulcers, as therapeutic agents for promoting wound healing, or as therapeutic agents for periodontal disease.

[0004] However, several issues are known to exist in the practical application of FGF proteins. First, FGF proteins are easily degraded by proteolytic enzymes present in the body, and their activity is unstable in the temperature range of approximately 20 to 40°C, which corresponds to room temperature to body temperature. Therefore, when administered to the body, FGF proteins quickly lose their activity. Furthermore, from the perspective of formulating FGF proteins, they tend to adsorb to the walls of storage containers and form aggregates, which easily disappear from the formulation solution, making it difficult to maintain their activity.

[0005] Based on these problems, the present inventors have focused on the FGF1 protein in particular among the proteins of the FGF family, and have proposed a chimeric FGF protein with better activity and its use.

[0006] Specifically, Patent Document 1 proposes an FGF1 chimeric protein as a heparin-binding protein to which a sugar chain of a sulfated polysaccharide, a glycosaminoglycan, or a combination of these is covalently bound. Patent Document 2 proposes an FGF1 chimeric protein consisting of a specific amino acid sequence to which a sulfated glycosaminoglycan sugar chain, of which 90% or more of the composition is covalently bound, is heparan sulfate sugar chain. Patent Document 3 proposes a pharmaceutical composition containing as an active ingredient an FGF1 / FGF2 chimeric protein in which a specific partial region of the FGF1 protein is substituted with the corresponding region of the FGF2 protein, a member of the FGF family. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 3318602 [Patent Document 2] Patent No. 4505631 [Patent Document 3] Patent No. 5004250 Summary of the Invention [Problem to be solved by the invention]

[0008] The FGF1 chimeric proteins disclosed in Patent Documents 1 to 3 all have good cell proliferation-promoting activity. In particular, it has been revealed that the FGF1 / FGF2 chimeric protein disclosed in Patent Document 3 can achieve very good cell proliferation-promoting activity.

[0009] However, as a result of intensive research by the present inventors, it became clear that even with the FGF1 / FGF2 chimeric protein disclosed in Patent Document 3, there is room for further investigation regarding the maintenance of cell proliferation-promoting activity in an incubation environment for approximately 3 days.

[0010] The present invention has been made to solve these problems, and aims to provide a modified fibroblast growth factor chimeric protein that can maintain good cell proliferation-promoting activity even in an incubation environment for approximately 3 days. [Means for solving the problem]

[0011] In order to solve the above-mentioned problems, the modified fibroblast growth factor chimeric protein of the present disclosure comprises an FGF chimeric polypeptide (FGFC) in which the sequence of positions 41 to 83 in an amino acid sequence that is at least 90% identical to the amino acid sequence of an FGF1 polypeptide in the fibroblast growth factor (FGF) family is replaced with an amino acid sequence that is at least 90% identical to the sequence of positions 43 to 85 in the amino acid sequence of an FGF2 polypeptide in the FGF family, and further comprises a glycosaminoglycan (GAG) addition sequence polypeptide to which a GAG sugar chain is bound.

[0012] According to the above configuration, the modified fibroblast growth factor chimeric protein not only contains an FGF chimeric polypeptide but also a GAG addition sequence polypeptide (PG polypeptide), and thus undergoes glycosylation by binding of glycosaminoglycan to the PG polypeptide. This allows the modified fibroblast growth factor chimeric protein to exhibit good cell growth-promoting activity without aggregation and to maintain good cell growth-promoting activity even under an incubating environment for approximately three days. Furthermore, the glycosylation of glycosaminoglycan also effectively reduces the adsorption of the modified fibroblast growth factor chimeric protein to the container. This effectively prevents a decrease in the concentration of the modified fibroblast growth factor chimeric protein, achieving even better handling.

[0013] In the modified fibroblast growth factor chimeric protein having the above-described configuration, the GAG ​​addition sequence polypeptide may have the glycosaminoglycan bound to a serine molecule contained in the sequence.

[0014] Furthermore, in the modified fibroblast growth factor chimeric protein having the above configuration, the FGF chimeric polypeptide (FGFC) may be configured to consist of the amino acid sequence shown in SEQ ID NO:7.

[0015] Furthermore, in the modified fibroblast growth factor chimeric protein having the above configuration, the GAG ​​addition sequence polypeptide may be configured to consist of the amino acid sequence shown in SEQ ID NO:5.

[0016] Furthermore, in the modified fibroblast growth factor chimeric protein having the above configuration, the sequence from positions 41 to 83 in the amino acid sequence of the FGF1 polypeptide may be replaced with an amino acid sequence that is at least 95% identical to the sequence from positions 43 to 85 in the amino acid sequence of the FGF2 polypeptide.

[0017] The present disclosure also includes methods for producing modified fibroblast growth factor chimeric proteins. Specifically, a method for producing a modified fibroblast growth factor chimeric protein according to the present disclosure may include the steps of: ligating DNA encoding an FGF chimeric polypeptide (FGFC) in which the sequence at positions 41 to 83 in an amino acid sequence at least 90% identical to the amino acid sequence of an FGF1 polypeptide of the fibroblast growth factor (FGF) family is replaced with an amino acid sequence at positions 43 to 85 in the amino acid sequence of an FGF2 polypeptide of the FGF family; and DNA encoding a GAG addition sequence polypeptide to which a glycosaminoglycan (GAG) sugar chain is attached, thereby constructing a modified FGF chimeric DNA encoding the modified FGF chimeric protein; incorporating the obtained modified FGF chimeric DNA into an expression vector; introducing the expression vector into a host cell having a glycosaminoglycan sugar chain addition pathway; and expressing in the host cell the modified FGF chimeric protein and a protein having the GAG ​​addition sequence polypeptide and in which a glycosaminoglycan sugar chain has been attached to the GAG ​​addition sequence polypeptide.

[0018] In the method for producing a modified fibroblast growth factor chimeric protein having the above configuration, the DNA encoding the modified FGF chimeric protein may have the base sequence shown in SEQ ID NO:8.

[0019] Furthermore, in the method for producing a modified fibroblast growth factor chimeric protein having the above configuration, the DNA encoding the GAG ​​addition sequence polypeptide may have the base sequence shown in SEQ ID NO:6.

[0020] Furthermore, the present disclosure also includes DNA comprising a base sequence encoding a modified fibroblast growth factor chimeric protein of the above configuration, an expression vector comprising DNA of the above configuration, a transformant obtained by introducing DNA of the above configuration or an expression vector of the above configuration into a host cell, a composition comprising a modified fibroblast growth factor chimeric protein of the above configuration, and a method for culturing stem cells comprising a step of adding a composition of the above configuration to a culture medium during stem cell culture. [Effects of the Invention]

[0021] The present invention, with the above-described configuration, has the effect of providing a modified fibroblast growth factor (FGF) chimeric protein that can maintain good cell proliferation-promoting activity even in an incubation environment for approximately 3 days. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 is a schematic diagram showing a representative example of the structure of a modified fibroblast growth factor chimeric protein according to the present disclosure. [Figure 2] FIG. 1 is a schematic diagram showing an example of a variation of a modified fibroblast growth factor chimeric protein according to the present disclosure. [Figure 3] 1 is a graph showing the results of Experimental Result 1, a representative example of the present disclosure, showing the cell proliferation-promoting activity of a modified fibroblast growth factor chimeric protein according to this example when the protein is in a PBS solution. [Figure 4] 1 is a graph showing the results of the cell proliferation-promoting activity of comparative FGF protein 1 (PG-FGF1), which is Comparative Result 1 in an Example of the present disclosure. [Figure 5] 1 is a graph showing the results of Experimental Result 2 in an Example of the present disclosure, showing the results of the cell proliferation-promoting activity when the modified fibroblast growth factor chimeric protein of this Example is in a medium solution. [Figure 6] 1 is a graph showing the results of the cell proliferation-promoting activity of comparative FGF protein 2 (FGFC), which is Comparative Result 2 in an Example of the present disclosure. [Figure 7]This is experimental result 3 in an example of the present disclosure, a graph showing the results of evaluating the adsorption to a container of the modified fibroblast growth factor chimeric protein of this example, comparative FGF protein 1 (PG-FGF1), and comparative FGF protein 2 (FGFC). [Figure 8] 10 is a graph showing the results of evaluating the adsorption of comparative FGF protein 1 (PG-FGF1) to a container, which is Comparative Result 3 in an Example of the present disclosure. [Figure 9] 10 is a graph showing the results of evaluating the adsorption of comparative FGF protein 2 (FGFC) to a container, which is Comparative Result 4 in an Example of the present disclosure. [Figure 10] This is a graph showing the results of Experimental Result 4, a representative example of the present disclosure, which shows the results of evaluating the adsorption to a container of the modified fibroblast growth factor chimeric protein of this example, comparative FGF protein 1 (PG-FGF1), comparative FGF protein 2 (FGFC), and FGF1 protein. DETAILED DESCRIPTION OF THE INVENTION

[0023] [How this disclosure came to be] Fibroblast growth factors (FGFs) have a wide range of applications as pharmaceutical compositions, and are particularly effective in promoting wound healing and preventing and treating radiation damage to the intestinal tract and bone marrow. They are also essential additives to in vitro cell culture media, where they are used to promote the proliferation and regulate the differentiation of stem cells, such as iPS cells and mesenchymal stem cells (MSCs).

[0024] However, FGF family proteins are easily degraded by protease enzymes present in the body, and their activity is unstable in the temperature range of approximately 20-40°C, which corresponds to temperatures from room temperature to body temperature, so they quickly lose their activity when administered to the body.Furthermore, FGF family proteins are prone to adsorption to the walls of storage containers and form aggregates, which easily disappear from solution, making it difficult to maintain their activity.

[0025] Based on these technical challenges, the present inventors invented the FGF chimeric proteins disclosed in the aforementioned Patent Documents 1 to 3. Among them, the FGF1 / FGF2 chimeric protein (FGFC) disclosed in Patent Document 3 has broad receptor specificity, and has been shown to have improved temperature stability and resistance to proteolytic enzymes, as well as improved stability when kept warm for approximately one day (24 hours).

[0026] For example, when culturing stem cells such as iPS cells or MSCs, as described above, FGF proteins are often added to the culture medium for the purpose of differentiation control, etc. However, because the culture medium is kept warm in an incubator maintained at 37°C, it is expected that the FGF proteins added to the culture medium will quickly lose their cell proliferation-promoting activity.

[0027] Furthermore, disposable plastic culture dishes are now used for cell culture. Hydrophobic substances tend to nonspecifically adsorb to the walls of such culture dishes, so when FGF proteins are added to the culture medium, it is necessary to consider the loss due to this adsorption.

[0028] For these reasons, the standard protocol for stem cell culture is to change the culture medium daily, especially when FGF proteins are added. However, because the culture medium must be changed even during weekends, weekend changes are a major bottleneck in stem cell culture protocols. Therefore, to eliminate weekend changes, FGF proteins are required to maintain their cell proliferation-promoting activity for approximately three days.

[0029] As a result of intensive research by the present inventors, it was found that when glycosaminoglycans are added to FGFC, cell proliferation-promoting activity at 37°C can be maintained for approximately 3 days, as will be explained in the Examples below. Furthermore, it was also found that the addition of glycosaminoglycans can effectively suppress adsorption of FGFC to containers without the need for formulation. In this way, the present inventors have completed the modified fibroblast growth factor (FGF) chimeric protein according to the present disclosure.

[0030] Representative embodiments of the present invention will be described below with reference to the drawings. In the following description, except for the title or claims, the modified fibroblast growth factor (FGF) chimeric protein according to the present disclosure will be referred to as the "modified FGF chimeric protein" for convenience of explanation. [Modified fibroblast growth factor chimeric protein] The modified FGF chimeric protein according to the present disclosure is, for example, a chimeric protein, as shown schematically in FIG. 1 as "PG-FGFC," which comprises an FGF chimeric polypeptide (FGFC) in which the sequence from positions 41 to 83 in an amino acid sequence that is at least 90% identical to the amino acid sequence of an FGF1 polypeptide of the fibroblast growth factor (FGF) family is replaced with an amino acid sequence that is at least 90% identical to the sequence from positions 43 to 85 in the amino acid sequence of an FGF2 polypeptide of the FGF family, and further comprises a glycosaminoglycan (GAG) addition sequence polypeptide to which a GAG sugar chain is bound.

[0031] As shown in Figure 1, among the FGF family proteins, FGF1 protein and FGF2 protein can be synthesized using four cassettes, cassettes 1 to 4 (see Patent Document 3). Furthermore, if a peptide consisting of 21 amino acids at the N-terminus is deleted from the FGF1 protein, the expression level is high and the protein is easy to handle (see Patent Document 3).

[0032] Therefore, the FGF chimeric protein (FGFC) disclosed in Patent Document 3 is not the full-length type of FGF1 protein shown in FIG. 1 (referred to as FGF1 (full length) in FIG. 1), but rather uses a truncated isoform of FGF1 protein (referred to as FGF1 truncated in FIG. 1) in which the first cassette is a truncated first cassette in which the N-terminal peptide has been deleted, and has a configuration in which the second cassette of this FGF1 truncated isoform is replaced with a second cassette of FGF2 protein. In the present disclosure, a "polypeptide" is referred to as a "protein" when it constitutes an independent molecule, and as a "polypeptide" or "peptide" when it is contained in a molecule. For example, the FGF chimeric protein (FGFC) shown in FIG. 1 is referred to as an FGF chimeric polypeptide when it is contained in a modified chimeric FGF protein (PG-FGFC) according to the present disclosure.

[0033] In the present disclosure, an amino acid sequence that is at least 90% identical to the amino acid sequence of an FGF1 polypeptide may be a polypeptide that has an amino acid sequence that is 90% identical to the amino acid sequence of a known FGF1 protein and that retains the cell proliferation-promoting activity of the FGF1 protein. Furthermore, an amino acid sequence that is at least 90% identical to the sequence of positions 43 to 85 in the amino acid sequence of an FGF2 polypeptide may be an amino acid sequence in the second cassette of the FGF protein that is 90% identical to the amino acid sequence of the second cassette of the known FGF2 amino acid sequence and that, when constituting a modified chimeric FGF protein, enables the protein to exhibit cell proliferation-promoting activity.

[0034] An amino acid sequence that is at least 90% identical to the amino acid sequence of an FGF1 polypeptide may be 95% or more identical, 96% or more identical, 97% or more identical, 98% or more identical, or 99% identical. Furthermore, an amino acid sequence that is at least 90% identical to the sequence of positions 43 to 85 in the amino acid sequence of an FGF2 polypeptide may be 95% or more identical, 96% or more identical, 97% or more identical, 98% or more identical, or 99% identical.

[0035] The amino acid sequences of the reference FGF1 and FGF2 proteins can be those listed in the sequence listing of Patent Document 3. The contents of Patent Document 3 (Japanese Patent No. 5004250) are incorporated herein by reference. Regarding the identity of amino acid sequences, for example, the expression "90% identical" can be replaced with the expression "90% homology."

[0036] Furthermore, the modified FGF chimeric protein according to the present disclosure has a configuration in which a GAG addition sequence polypeptide is bound to this FGF chimeric protein (FGFC). In the example shown in FIG. 1, the GAG ​​addition sequence polypeptide is bound to the N-terminus of the FGF chimeric protein. As described above, such a chimeric protein according to the present disclosure not only maintains cell proliferation-promoting activity longer than an FGF chimeric protein, but also has the function (action) of effectively suppressing adsorption to a container. This function (action) will be specifically explained in the Examples described below. For convenience of explanation, the GAG-added polypeptide will be referred to as a "PG polypeptide" except in the title or claims. Furthermore, the modified chimeric protein according to the present disclosure can be referred to as a "PG-FGFC" because it contains a PG polypeptide.

[0037] As described above, the modified FGF chimeric protein according to the present disclosure may be configured to contain an FGF chimeric polypeptide and a PG polypeptide, and the specific configuration is not particularly limited. In the present disclosure, the FGF1 protein or FGF2 protein from which the FGF chimeric polypeptide is derived, and the protein from which the PG polypeptide is derived, may be proteins of mammalian origin. Examples of such mammals include, but are not limited to, humans, mice, rats, cows, horses, etc. Generally, from the viewpoint of avoiding undesirable immune system reactions, it is sufficient that the proteins are of the same species as the mammal to which they are to be used.

[0038] More specifically, the FGF chimeric polypeptide (FGFC) included in the modified FGF chimeric protein according to the present disclosure may be a polypeptide prepared using the amino acid sequences of human FGF1 protein and human FGF2 protein. A typical example is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 7 in the Sequence Listing, i.e., the amino acid sequence shown in SEQ ID NO: 5 (abbreviated as "Reference 3 SEQ ID NO: 5") or SEQ ID NO: 7 (abbreviated as "Reference 3 SEQ ID NO: 7") in Patent Document 3.

[0039] The amino acid sequence shown in SEQ ID NO: 7 in the present disclosure is a sequence that does not have the first methionine (M, Met) of the amino acid sequence shown in "Reference 3, SEQ ID NO: 5," and is a sequence in which the first methionine of the amino acid sequence shown in "Reference 3, SEQ ID NO: 7" is replaced with alanine (A, Ala). The modified FGF chimeric protein according to the present disclosure is not limited to the amino acid sequence shown in SEQ ID NO: 7, and may have additions, deletions, substitutions, or modifications in part of the amino acid sequence as long as it can exhibit its function.

[0040] Furthermore, the PG polypeptide contained in the modified FGF chimeric protein of the present disclosure may have a structure in which a glycosaminoglycan (GAG) is bound, and the specific structure is not particularly limited. A representative structure is one in which a glycosaminoglycan is bound to a serine molecule contained in the amino acid sequence of the PG polypeptide, such as a PG polypeptide derived from a mammalian syndecan.

[0041] Furthermore, the PG polypeptide may be any polypeptide as long as it is structured to bind to a glycosaminoglycan, but it is preferable that the glycosaminoglycan be a heparan sulfate. A more specific example of the PG polypeptide is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5. As will be explained in the Examples below, the amino acid sequence shown in SEQ ID NO: 5 is an amino acid sequence derived from human syndecan-4 protein.

[0042] Furthermore, the modified FGF chimeric protein according to the present disclosure desirably has a secretory signal peptide (SS peptide), as shown in FIG. 1. The presence of the SS peptide enables efficient glycosylation. Therefore, the modified FGF chimeric protein according to the present disclosure may have a configuration containing a PG polypeptide and an FGF chimeric polypeptide, and a preferred example is a configuration containing an SS peptide, a PG polypeptide, and an FGF chimeric polypeptide. Note that the example shown in FIG. 1 shows a configuration in which the peptides are bound in the following order from the N-terminus: SS peptide, PG polypeptide, and FGF chimeric polypeptide. However, as will be described later, the order of binding of the PG peptide, FGF chimeric polypeptide (and further, the peptide tag) is not particularly limited.

[0043] 1 also shows a schematic representation of PG-FGF1, which is used as comparative FGF protein 1 in the Examples described below. PG-FGF1 is configured such that an SS peptide and a PG polypeptide are linked to an FGF1 truncated form in order from the N-terminus, similar to the modified FGF chimeric proteins of the present disclosure. Furthermore, in FIG. 1, the numbers below the FGF1 (full-length) protein, FGF2 protein, FGF1 truncated protein, and FGF chimeric protein (FGFC) are ordinal numbers in the amino acid sequence.

[0044] In the Examples described below, for the convenience of purifying the expressed chimeric protein, Hisx6-PA (abbreviated as "HisPA" for convenience of explanation), an amino acid sequence that serves as a peptide tag, is included between the SS peptide and the PG polypeptide. In other words, the modified FGF chimeric protein according to the Examples described below is configured such that, from the N-terminus, SS-HisPA-PG-FGFC is arranged in this order. This is also true for comparative FGF protein 1 (PG-FGF1).

[0045] Therefore, in the modified FGF chimeric protein according to the present disclosure, another peptide such as HisPA may be present between the SS peptide and the PG polypeptide. Similarly, in the modified FGF chimeric protein according to the present disclosure, another peptide may be present between the PG polypeptide and the FGF chimeric polypeptide, as long as the function of the other peptide can be exerted.

[0046] In other words, the modified FGF chimeric protein according to the present disclosure may contain at least an FGF chimeric polypeptide and a PG polypeptide in its amino acid sequence. Therefore, the modified FGF chimeric protein according to the present disclosure may contain known peptides other than the FGF chimeric polypeptide and the PG polypeptide, and the positions of the peptides or other peptides that make up the modified FGF chimeric protein are not particularly limited.

[0047] As long as the modified FGF chimeric protein according to the present disclosure has a HisPA tag, a PG polypeptide, and an FGF chimeric polypeptide (FGFC), the order in which these peptides are linked is not particularly limited, and examples of variations include those shown in FIG. 2. As mentioned above, the "original" shown in the top row of FIG. 2 is a structure in which HisPA-PG-FGFC are linked in this order from the N-terminus, while "construct A" is a structure in which HisPA-FGFC-PG are linked in this order from the N-terminus, "construct B" is a structure in which FGFC-PG-HisPA are linked in this order from the N-terminus, and "construct C" is a structure in which PG-FGFC-HisPA are linked in this order from the N-terminus. Thus, the modified FGF chimeric protein according to the present disclosure includes not only the "original" shown in FIG. 2 but also "constructs A" to "construct C."

[0048] The SS peptide used in the Examples described below is a secretory signal peptide derived from human interleukin 2 (IL2) and has the amino acid sequence shown in SEQ ID NO: 1. The peptide tag used in the Examples described below has the amino acid sequence shown in SEQ ID NO: 3. However, the secretory signal peptides or peptide tags used in the present disclosure are not limited to these, and any peptide tag having a known amino acid sequence can be appropriately selected and used.

[0049] The present disclosure also includes DNA (modified FGF chimeric DNA) having a nucleotide sequence encoding the modified FGF chimeric protein of the above configuration. Specifically, the modified FGF chimeric DNA of the present disclosure may be DNA containing DNA (FGFC-DNA) having a nucleotide sequence encoding the FGF chimeric polypeptide of the above configuration and DNA (PG-DNA) having a nucleotide sequence encoding the PG polypeptide. Examples of FGFC-DNA include DNA having the nucleotide sequence shown in SEQ ID NO: 8 in the Sequence Listing, and examples of PG-DNA include DNA having the nucleotide sequence shown in SEQ ID NO: 6. DNA having the nucleotide sequence shown in SEQ ID NO: 8 encodes the amino acid sequence shown in SEQ ID NO: 7, and DNA having the nucleotide sequence shown in SEQ ID NO: 6 encodes the amino acid sequence shown in SEQ ID NO: 5.

[0050] Here, the modified FGF chimeric DNA according to the present disclosure is not limited to DNA having the nucleotide sequence shown in SEQ ID NO: 8 and DNA having the nucleotide sequence shown in SEQ ID NO: 6. For example, it may be DNA having a nucleotide sequence homologous to the nucleotide sequence shown in SEQ ID NO: 8 and also having a nucleotide sequence homologous to the nucleotide sequence shown in SEQ ID NO: 6. Alternatively, it may be DNA having another nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 7 and another nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 5.

[0051] Furthermore, at least one of the nucleotide sequences shown in SEQ ID NO: 8 and SEQ ID NO: 6 may have a nucleotide sequence in which one or more nucleotides are deleted, substituted, or added, as long as the function of the encoded FGF chimeric polypeptide or PG polypeptide is maintained. Furthermore, the modified FGF chimeric DNA of the present disclosure may be at least 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% identical to the nucleotide sequence shown in SEQ ID NO: 8 or SEQ ID NO: 6, as long as the function of the encoded polypeptide is maintained.

[0052] In the examples described below, as mentioned above, a peptide having the amino acid sequence shown in SEQ ID NO: 1 is used as the SS peptide, and a peptide having the amino acid sequence shown in SEQ ID NO: 3 is used as the peptide tag. DNA having the base sequence shown in SEQ ID NO: 2 in the sequence listing encodes the SS peptide having the amino acid sequence shown in SEQ ID NO: 1, and DNA having the base sequence shown in SEQ ID NO: 4 encodes the peptide tag having the amino acid sequence shown in SEQ ID NO: 3. It goes without saying that the DNA encoding the signal peptide or peptide tag is not limited to the base sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, and may have other known base sequences.

[0053] [Method for producing modified fibroblast growth factor chimeric protein] The method for producing the modified FGF chimeric protein according to the present disclosure is not particularly limited. A representative example is a method in which the modified FGF chimeric DNA (DNA containing at least FGFC-DNA and PG-DNA) described above is introduced into a self-replicating vector to prepare a replicable recombinant DNA, which is then introduced into a host cell to produce the modified FGF chimeric protein.

[0054] As described above, in the present disclosure, the FGF chimeric polypeptides (FGFCs) are derived from mammalian (e.g., human) FGF1 and FGF2 proteins, and the PG polypeptides are derived from mammalian (e.g., human) syndecan-4 proteins. Furthermore, glycosaminoglycans are bound to the PG polypeptides. Therefore, the host cells used in the present disclosure are not particularly limited, as long as they can suitably express these mammalian proteins and have a sugar chain synthesis system.

[0055] Representative host cells include cells having at least a sugar chain biosynthesis system, such as yeasts of the genus Saccharomyces (or Saccharomyces species) such as Saccharomyces cerevisiae, Pichia species such as Schizzosaccharomyces pombe and Pichia pastoris, Kluyveromyces species such as Kluyveromyces lactis, and Candida species such as Candida albicans; animal cells such as COS cells, CHO cells, BHK cells, NIH3T3 cells, BALB / c3T3 cells, and HUVE cells; and insect cells such as Sf9 cells, Sf21 cells, and H5 cells. In the present disclosure, for example, cells derived from mammals can be suitably used, and COS-1 cells are used in the examples described below.

[0056] Representative examples of self-replicating (autonomously replicating) vectors include plasmid vectors that are replicated and maintained within host cells. For example, if the host cell is yeast, various yeast expression vectors may be used; if the host cell is animal cell, various animal cell expression vectors may be used; and if the host cell is insect cell, various insect cell expression vectors may be used.

[0057] In the present disclosure, for example, mammalian-derived host cells can be preferably used, and therefore mammalian expression vectors can also be preferably used as plasmid vectors. Representative examples include the pcDNA3 series and pCMV series of plasmid vectors. In the examples described below, a commercially available mammalian expression vector from the pcDNA3 series is used.

[0058] In the present disclosure, the method for preparing replicable recombinant DNA is not particularly limited, and known methods can be suitably used. For example, the modified FGF chimeric DNA can be prepared by artificial synthesis or by cloning using genetic engineering techniques. In this case, the modified FGF chimeric DNA can be cDNA. Furthermore, the method for incorporating such a modified FGF chimeric DNA as an insert into an autonomously replicating vector to prepare an expression vector is also not particularly limited, and known methods or conditions can be used.

[0059] In the examples described below, DNA having the base sequence shown in SEQ ID NO: 2 (encoding an SS peptide having the amino acid sequence shown in SEQ ID NO: 1), DNA having the base sequence shown in SEQ ID NO: 4 (encoding a peptide tag having the amino acid sequence shown in SEQ ID NO: 3), DNA having the base sequence shown in SEQ ID NO: 6 (encoding a PG polypeptide having the amino acid sequence shown in SEQ ID NO: 5 / PG-DNA), and DNA having the base sequence shown in SEQ ID NO: 8 (encoding an FGF chimeric polypeptide having the amino acid sequence shown in SEQ ID NO: 7 / FGFC-DNA) were artificially synthesized using PCR, and these were linked by the overlap extension method to produce modified FGF chimeric DNA, which was then inserted into a plasmid vector, a mammalian expression vector, using a known method to produce an expression vector.

[0060] In the present disclosure, the method for obtaining a transformant by transformation with an expression vector containing a modified FGF chimeric DNA is not particularly limited. If the host cell is yeast, a method of partially removing the cell wall of the yeast cell to form spheroplasts, or the lithium acetate method, etc., can be used. Furthermore, if the host cell is an animal cell, examples of methods include introducing the expression vector into the animal cell when it is in a growth phase, etc., using the calcium phosphate method, lipofection, or electroporation. In the examples described below, the lipofection method using a commercially available kit is used.

[0061] The method for culturing the obtained transformant is not particularly limited. For example, the medium used for culturing may be a medium commonly used for the specific host cells. If the host cells are animal cells, for example, DMEM (Dulbecco's Modified Eagle Medium) supplemented with animal serum can be used. In the examples described below, a serum-free culture medium is used to facilitate the subsequent purification step, and the medium is replaced with serum-free culture medium several times approximately every 48 hours, thereby increasing the yield and maintaining the cell condition.

[0062] The culture conditions for transformants are not particularly limited and are generally those used for the respective host cells. For animal cells, examples of culture conditions include a temperature range of approximately 32-37°C, 5% carbon dioxide (CO2), and 100% humidity for approximately 24 hours to 2 weeks. These culture conditions can be changed or modified as needed, such as by changing the gas phase conditions or adding agitation, as long as they do not interfere with the culture.

[0063] The culture scale of the transformant is not particularly limited. For example, when a liquid medium (culture solution) is used, the culture may be small-scale culture using a test tube or a flask, large-scale culture using a jar fermenter, or large-scale culture using a tank on an industrial scale.

[0064] The present disclosure also includes the modified FGF chimeric DNA prepared as described above, i.e., DNA (modified FGF chimeric DNA) containing a nucleotide sequence encoding the modified FGF chimeric protein of the present disclosure. Similarly, the present disclosure also includes expression vectors containing the modified FGF chimeric DNA prepared as described above. Furthermore, the present disclosure also includes transformants obtained by introducing the modified FGF chimeric DNA or expression vector into host cells.

[0065] By culturing the transformant described above, the modified FGF chimeric protein according to the present disclosure is expressed intracellularly. The method for recovering the modified FGF chimeric protein from the cultured transformant (cells) is not particularly limited, and known methods can be used. When the expressed modified FGF chimeric protein accumulates intracellularly, the cultured cells are recovered (or collected), disrupted by a known method to obtain a crude protein solution, and this crude protein solution can be purified or concentrated by a known method to obtain the modified FGF chimeric protein according to the present disclosure. When purification or concentration is not necessary, the crude protein solution can be used as the modified FGF chimeric protein according to the present disclosure.

[0066] As described above, in the present disclosure, as a representative example, a chimeric protein is constructed in which a PG polypeptide is linked to an FGF chimeric polypeptide, and an SS peptide (e.g., the amino acid sequence shown in SEQ ID NO: 1) is further linked to the PG polypeptide. The presence of the SS peptide, i.e., secretory signal peptide, allows efficient glycosylation of the modified FGF chimeric protein and promotes highly efficient extracellular secretion. Therefore, the modified FGF chimeric protein can be recovered by recovering the culture medium. In the examples described below, as described above, conditioned medium is obtained by medium exchange with serum-free culture medium in order to increase the recovery yield and maintain the cell state.

[0067] The method for purifying the modified FGF chimeric protein from the conditioned medium is not particularly limited, and known separation and purification methods can be used in combination as needed. Specific separation and purification methods include, for example, salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, SDS-polyacrylamide gel electrophoresis, ion exchange chromatography, affinity chromatography, reversed-phase high-performance liquid chromatography, and isoelectric focusing.

[0068] As described above, in the present disclosure, a peptide tag (e.g., the amino acid sequence shown in SEQ ID NO: 3) has been introduced into the modified FGF chimeric protein, enabling advanced purification using metal chelate affinity chromatography or an anti-PA antibody. Furthermore, affinity chromatography using heparin sepharose as a carrier can be applied to purify the modified FGF chimeric protein according to the present disclosure, as with other FGF chimeric proteins.

[0069] The modified FGF chimeric protein of the present disclosure thus obtained may generally be stored in a refrigerated state at 4°C or below in a buffer solution adjusted to a predetermined pH, but the storage conditions are not particularly limited. For example, the modified FGF chimeric protein of the present disclosure may be frozen at -20°C or below, provided that the cell growth-promoting activity of the modified FGF chimeric protein of the present disclosure is not impaired. Alternatively, the modified FGF chimeric protein of the present disclosure may be stored as a dry powder by lyophilization after dialysis for desalting and buffer exchange. Note that a desalting column or ultrafiltration filter may be used instead of dialysis for desalting and buffer exchange.

[0070] Thus, the method for producing a modified FGF chimeric protein according to the present disclosure may comprise the following steps: (1) constructing a modified FGF chimeric DNA encoding a modified FGF chimeric protein by ligating DNA encoding an FGF chimeric polypeptide (FGFC) in which the amino acid sequence at positions 41 to 83 in the amino acid sequence of an FGF1 polypeptide of the FGF family has been replaced with the amino acid sequence at positions 43 to 85 in the amino acid sequence of an FGF2 polypeptide of the FGF family, with DNA encoding a PG polypeptide having a glycosaminoglycan (GAG) sugar chain attached thereto; (2) incorporating the obtained modified FGF chimeric DNA into an expression vector; (3) introducing the expression vector into a host cell having a glycosaminoglycan sugar chain addition pathway; and (4) expressing in the host cell the modified FGF chimeric protein and a protein having a PG polypeptide and a glycosaminoglycan sugar chain attached to the PG polypeptide.

[0071] In the method for producing a modified FGF chimeric protein, the FGF1 polypeptide used in step (1) may not only be 100% identical to the amino acid sequence of the FGF1 polypeptide (FGF1 protein), but also, as described above, may have an amino acid sequence that is at least 90% identical to that of the FGF1 polypeptide. Similarly, in step (1), the sequence of positions 43 to 85 in the amino acid sequence of the FGF2 polypeptide to be substituted may not only be 100% identical, but may also be at least 90% identical to that of positions 43 to 85, as described above.

[0072] [Use of modified fibroblast growth factor chimeric proteins, etc.] The modified FGF chimeric protein according to the present disclosure can be produced (manufactured) by the method for producing a modified FGF chimeric protein described above, and the modified FGF chimeric protein produced in this manner can be used as a pharmaceutical formulation. That is, the present disclosure also includes pharmaceutical compositions containing the modified FGF chimeric protein described above.

[0073] The pharmaceutical composition according to the present disclosure can be used as a pharmaceutical composition having pharmacological effects equivalent to or greater than those of conventional pharmaceutical compositions having cell proliferation-promoting activity. Specific examples include pharmaceutical compositions used as a promoter of wound healing, a therapeutic agent for periodontal disease, a promoter of fracture healing, and for preventing or treating radiation-induced damage to various organs, including the intestinal epithelium or bone marrow.

[0074] As shown in the Examples below, the modified FGF chimeric protein according to the present disclosure can maintain good cell proliferation-promoting activity even in an incubating environment for about three days. As mentioned above, the task of changing the culture medium on weekends during stem cell culture is a major bottleneck in stem cell culture protocols. The modified FGF chimeric protein according to the present disclosure can maintain cell proliferation-promoting activity even in an incubating environment for about three days, and therefore can be suitably used as a pharmaceutical composition for promoting stem cell proliferation.

[0075] Furthermore, as shown in the results of the Examples described below, the modified FGF chimeric protein of the present disclosure contains a PG polypeptide to which a glycosaminoglycan (GAG) sugar chain is bound, which not only enables the protein to maintain good cell growth-promoting activity even in an incubation environment for approximately three days, as described above, but also, as shown in the Examples described below, effectively suppresses adsorption to containers. Therefore, the modified FGF chimeric protein of the present disclosure can achieve excellent pharmaceutical formulation properties, such as a low concentration in solution even when stored in a container. This allows for the production of a stable, highly active pharmaceutical composition.

[0076] Additionally, the modified FGF chimeric protein according to the present disclosure is thought to have properties such as high stability at room temperature (25°C) and high resistance to proteases at body temperature (37°C). Therefore, pharmaceutical compositions containing the modified FGF chimeric protein according to the present disclosure as an active ingredient are expected to minimize the effects of inactivation by proteases contained in exudates in various biological disorders.

[0077] Furthermore, the modified FGF chimeric protein according to the present disclosure can be more suitably used in the field of stem cell culture because it can suppress loss due to adsorption. Therefore, the present disclosure also includes a method for culturing stem cells, which includes the step of adding a composition containing a modified FGF chimeric protein to a culture medium during stem cell culture.

[0078] The formulation of the pharmaceutical composition according to the present disclosure is not particularly limited. Typically, pharmaceutical compositions may be prepared using pharmaceutically acceptable solvents, excipients, carriers, adjuvants, etc., in accordance with standard pharmaceutical manufacturing methods, such as liquids, injections, powders, granules, tablets, suppositories, enteric-coated formulations, and capsules. The content of the modified FGF chimeric protein, which is the active ingredient, in the pharmaceutical composition according to the present disclosure is also not particularly limited, but typically should be within the range of 0.000001 to 1.0% by mass of the total amount of the pharmaceutical composition.

[0079] The specific method of use of the pharmaceutical composition according to the present disclosure is not particularly limited. Typically, the pharmaceutical composition according to the present disclosure can be safely administered parenterally or orally to mammals such as humans, mice, rats, rabbits, dogs, and cats as a liver cell proliferation agent or a neuronal differentiation and survival promoter. The dosage of the pharmaceutical composition according to the present disclosure can be varied as appropriate depending on the dosage form, administration route, symptoms, and the like. For example, when administered to mammals, including humans, the dosage can be in the range of approximately 0.01 to 10 mg / kg of body weight of the modified FGF chimeric protein according to the present disclosure per day.

[0080] Furthermore, the modified FGF chimeric proteins of the present disclosure can be used not only in the pharmaceutical field but also in other fields where their cell proliferation-promoting activity can be utilized, such as in research reagents, but also in various other fields such as agriculture and the chemical industry.

[0081] In particular, the modified FGF chimeric protein of the present disclosure contains a PG polypeptide, to which glycosaminoglycan is bound, thereby stabilizing the cell growth-promoting activity of the FGF chimeric polypeptide, thereby further improving its handleability and effectively suppressing adsorption to containers. Therefore, the composition of the present disclosure may be suitably used in a wide range of industrial applications outside of the pharmaceutical field. Therefore, the composition of the present disclosure is not limited to a pharmaceutical composition, as long as it contains a modified FGF chimeric protein as an active ingredient.

[0082] As mentioned above, the present disclosure also includes modified FGF chimeric DNA encoding a modified FGF chimeric protein or an expression vector containing the same. Therefore, the present disclosure can be used in a variety of fields, including direct use of the modified FGF chimeric DNA, such as gene therapy, using an expression vector that can be administered to a mammal, such as a human, to be treated. [Example]

[0083] The present disclosure will be described in more detail based on representative examples, but the present disclosure is not limited thereto. Those skilled in the art may make various changes, modifications, and alterations without departing from the scope of the present disclosure.

[0084] Example 1: Sample preparation [Expression of modified FGF chimeric proteins according to this example] The secretory signal used was the secretory signal IL2ss of human interleukin 2 (human IL2). The amino acid sequence of IL2ss is shown in SEQ ID NO: 1, and the DNA sequence encoding the amino acid sequence of IL2ss is shown in SEQ ID NO: 2.

[0085] The tag sequence used was Hisx6-PA, which consists of six consecutive histidine residues, Hisx6, and a PA tag manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. The PA tag is a peptide tag that utilizes the PLAG sequence of human podoplanin. The amino acid sequence of Hisx6-PA is shown in SEQ ID NO: 3, and the DNA sequence encoding the Hisx6-PA amino acid sequence is shown in SEQ ID NO: 4.

[0086] The PG polypeptide used was tr.PG, a polypeptide containing a portion of the amino acid sequence of human syndecan 4. The amino acid sequence of tr.PG is shown in SEQ ID NO:5, and the DNA sequence encoding the amino acid sequence of tr.PG is shown in SEQ ID NO:6.

[0087] The FGF chimeric polypeptide (FGFC) used was one having the amino acid sequence shown in SEQ ID NO: 5 in Patent Document 3, which was prepared by the method described in Patent Document 3. The amino acid sequence of FGFC is shown in SEQ ID NO: 7, and the DNA sequence encoding the amino acid sequence of FGFC is shown in SEQ ID NO: 8.

[0088] DNA encoding these polypeptides was synthesized using PCR. The resulting DNA was ligated by overlap extension to create the modified FGF chimeric DNA insert. This insert was then introduced into the mammalian vector pcDNA3.4 (product name, Thermo Fisher Scientific) to construct the expression vector for the modified FGF chimeric protein of this example (IL2ss-Hisx6PA-tr.PG-FGFC / pcDNA3.4).

[0089] The resulting expression vector was introduced into COS-1 cells as host cells. FuGENE HD (Promega) was used as the transfection reagent, and Opti-MEM (Thermo Fisher Scientific) was used as the transfection medium. 24 hours after transfection, the COS-1 cell culture medium was changed from Opti-MEM to serum-free medium ASF104 (Ajinomoto Co., Inc.), which led to the expression and secretion of the modified FGF chimeric protein into the medium. The serum-free medium ASF104 was changed every 48 hours, and the conditioned medium from three to five times was pooled and used for concentration and purification.

[0090] The sequences of the secretion signal, tag sequence, GAG addition sequence, and FGFC polypeptides, as well as the DNA encoding them, are summarized in Table 1 below.

[0091] [Table 1]

[0092] [Concentration, purification, and quantification of modified FGF chimeric proteins] The conditioned medium obtained as described above was concentrated and purified using various columns. First, the conditioned medium was loaded onto an anion exchange column, HiTrap-DEAE FF (product name, manufactured by GE Life Sciences), and then eluted with a salt gradient. Elution was monitored by absorbance at 280 nm and quantification by ELISA. The eluted fraction was loaded onto an anti-PA antibody column and then eluted by adding 2 M MgCl2. The resulting eluted fraction was concentrated and desalted using a centrifugal ultrafiltration filter, Amicon Ultra (product name, manufactured by Merck), and the buffer was exchanged with PBS.

[0093] The resulting concentrated and purified product (PBS solution) was designated as the modified FGF chimeric protein (PG-FGFC, see Figure 1) of this example. The resulting modified FGF chimeric protein (PG-FGFC) was quantified by ELISA. For this quantification, a Human FGF acidic / FGF1 Quantikine ELISA Kit (product name, manufactured by R&D Systems) was used.

[0094] Example 2: Cell proliferation test [Cell proliferation promoting activity of modified FGF chimeric protein (PBS)] The cell proliferation-promoting activity of the modified FGF chimeric protein (PG-FGFC) according to this example was evaluated based on the method described in Non-Patent Document 1: Motomura K, Hagiwara A, Komi-Kuramochi A, Hanyu Y, Honda E, Suzuki M, Kimura M, Oki J, Asada M, Sakaguchi N, Nakayama F, Akashi M, Imamura T. Biochim Biophys Acta. 2008 Dec;1780(12):1432-1440.

[0095] The modified FGF chimeric protein (PG-FGFC) of this example was used as a test sample at a concentration of 500 ng / ml. A portion of this test sample was kept at 37°C, while the other portion was immersed in ice water, and each was kept for 72 hours (3 days). These test samples (37°C-heated sample and ice water-immersed sample) were quantified by ELISA and then diluted with PBS to concentrations of 10 ng / mL, 30 ng / mL, and 100 ng / mL, respectively, to prepare evaluation samples SB10, SB30, and SB100.

[0096] These evaluation samples were mixed with Ba / F3 cells that stably express the FGF receptor R1c, seeded on a 96-well plate, and cultured for 2 days at 37°C in the presence of 5% CO. When the evaluation samples were mixed with Ba / F3 cells, two groups were prepared: one in which heparin (5 μg / mL) was added (HP+) according to the standard method, and one in which heparin was not added (HP-).

[0097] After culturing, a cell counting kit, Cell Counting Kit-8 (product name, Dojindo Laboratories, Inc.), was added to each evaluation sample, and the water-soluble formazan produced from the reduced color-developing reagent WST-8 (a water-soluble tetrazolium salt) was measured at absorbance at 450 nm to evaluate the cell proliferation-promoting activity of the modified FGF chimeric protein of this example (PG-FGFC). The results are shown in Figure 3 as Experimental Result 1.

[0098] [Cell proliferation promoting activity of modified FGF chimeric proteins (culture medium)] The cell proliferation-promoting activity of the modified FGF chimeric protein of this example was evaluated in the same manner as in [Cell proliferation-promoting activity of modified FGF chimeric protein (PBS)] above, except that the test sample was diluted with RPMI1640 cell culture medium (Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare evaluation samples SM10, SM30, and SM100 at concentrations of 10 ng / mL, 30 ng / mL, and 100 ng / mL. The results are shown in Figure 5 as Experimental Result 2.

[0099] [Comparative cell proliferation promoting activity of FGF protein 1] In a comparative experiment, a comparative FGF protein 1 (PG-FGF1, see FIG. 1 ), which has a structure in which FGFC in the modified FGF chimeric protein (PG-FGFC) is replaced with FGF1 protein, was prepared as an evaluation sample in the same manner as in Example 1, and its cell proliferation-promoting activity was evaluated in the same manner as in the above-mentioned [Cell proliferation-promoting activity of modified FGF chimeric protein (PBS)]. The results are shown in FIG. 4 as comparative result 1.

[0100] [Comparative cell proliferation promoting activity of FGF protein 2] In a comparative experiment, FGFC alone was used as comparative FGF protein 2, and evaluation samples SB30 and SB100 were evaluated using only ice-water-immersed samples. The cell proliferation-promoting activity was evaluated in the same manner as described above for [Cell proliferation-promoting activity of modified FGF chimeric proteins (PBS)], except that evaluation was performed using only ice-water-immersed samples. The results are shown in Figure 6 as comparative result 2.

[0101] Example 3: Adsorption Test [Adsorption of modified FGF chimeric protein (PBS)] As a container for evaluating adsorption, a microtube of ProteoSave SS (product name, manufactured by Sumitomo Bakelite Co., Ltd.), which is a container with low protein adsorption, was used.

[0102] The modified FGF chimeric protein of this example (PG-FGFC), comparative FGF protein 1 (PG-FGF1, see Figure 1), and comparative FGF protein 2 (FGFC) prepared in Example 1 were each prepared as 500 ng / mL PBS solutions, and a 500 ng / mL PBS solution of FGF1 protein was also prepared as a control. For each PBS solution, a set was prepared with and without 1 mg / mL heparin (+HP).

[0103] These PBS solutions were dispensed into the microtubes used for evaluating adsorptive properties and stored at 37°C or 0°C for 3 days. A portion of the PBS solution was then collected from each microtube, and the amount of FGF protein remaining in the PBS solution was measured by ELISA to evaluate the adsorptive properties of these FGF proteins. The results are shown in Figure 7 as Experimental Result 3.

[0104] [Comparison of FGF protein 1 adsorption (PBS)] A comparative FGF protein 1 (PG-FGF1) was prepared as a 500 ng / mL PBS solution, and a 500 ng / mL PBS solution of FGF1 protein was also prepared as a control. These PBS solutions were dispensed into the microtubes used for assessing adsorption and stored at 37°C or 4°C for 3 days. A portion of the PBS solution was then taken from each microtube, and the amount of remaining FGF protein in the PBS solution was measured by ELISA to evaluate the adsorption of these FGF proteins. The results are shown in Figure 8 as Comparative Result 3.

[0105] [Comparison of FGF protein 2 adsorption (PBS)] 500 ng / mL PBS solutions of comparative FGF protein 1 (PG-FGF1) and comparative FGF protein 2 (FGFC) were prepared, and a 500 ng / mL PBS solution of FGF1 protein was also prepared as a control. For each PBS solution, a set with and without 1 mg / mL heparin (HP+) was prepared.

[0106] These PBS solutions were dispensed into the microtubes used for evaluating adsorptive properties and stored at 37°C or 4°C for 3 days. A portion of the PBS solution was then collected from each microtube, and the amount of FGF protein remaining in the PBS solution was measured by ELISA to evaluate the adsorptive properties of these FGF proteins. The results are shown in Figure 9 as Comparative Result 4.

[0107] [Adsorption of modified FGF chimeric proteins and comparative FGF proteins (medium)] The modified FGF chimeric protein of this example (PG-FGFC), comparative FGF protein 1 (PG-FGF1), comparative FGF protein 2 (FGFC), and control FGF1 protein were all diluted with RPMI1640 cell culture medium (Fujifilm Wako Pure Chemical Industries, Ltd.) rather than PBS to prepare a medium solution with a concentration of 500 ng / mL. For each medium solution, a set was prepared with and without 1 mg / mL heparin (+HP).

[0108] These medium solutions were dispensed into the microtubes used for assessing adsorption and stored at 37°C or 0°C for 3 days. A portion of the PBS solution was then collected from each microtube, and the amount of FGF protein remaining in the medium solution was measured by ELISA to assess the adsorption of these FGF proteins. The results are shown in Figure 10 as Experimental Result 4.

[0109] (Example Results) As shown in Figure 3, which shows Experimental Result 1, it was confirmed that the modified FGF chimeric protein (PG-FGFC) of this example retained its cell proliferation-promoting activity in all of the evaluation samples SB10, SB30, and SB100, which were PBS solutions, in the 37°C-incubated samples compared to the control (ice water-immersed sample), although the cell proliferation-promoting activity was only about 40-50%.

[0110] As shown in Figure 4 (Comparative Result 1), the cell proliferation-promoting activity of comparative FGF protein 1 (PG-FGF1) was significantly impaired after incubation for 3 days at 37°C in all of the evaluation samples SB10, SB30, and SB100, which were PBS solutions, regardless of whether heparin was added or not. Here, because comparative FGF protein 1 (PG-FGF1) itself retains GAG sugar chains, it showed sufficient cell proliferation-promoting activity in the high-concentration control (ice water-immersed sample) even without the addition of heparin during the assay.

[0111] Furthermore, as shown in Figure 6 (Comparison Result 2), because comparative FGF protein 2 (FGFC) is a simple protein, its cell proliferation-promoting activity could not be observed without the addition of heparin during the assay. For evaluation sample SB10, the addition of heparin during the assay demonstrated equivalent cell proliferation-promoting activity in both the control (ice-water immersion sample) and the 37°C incubation (37°C-incubated sample). In other words, incubation at 37°C for 3 days is considered to have no effect on the cell proliferation-promoting activity of the FGFC protein itself. Note that, in Comparison Result 2, evaluation samples SB30 and SB100 could not be prepared as 37°C-incubated samples, and therefore their cell proliferation-promoting activity was not evaluated.

[0112] Furthermore, as shown in Figure 5, which shows Experimental Result 2, it was confirmed that the modified FGF chimeric protein (PG-FGFC) of this example maintained cell proliferation-promoting activity at approximately the same level as the control (ice water-immersed sample) in the samples incubated at 37°C in all of the evaluation samples SM10, SM30, and SM100, which are medium solutions.

[0113] Next, as shown in Figure 8 (Comparison Result 3), the remaining amount of FGF1 protein in the control FGF1 significantly decreased after 3 days of storage at 4°C, and the remaining amount of FGF1 protein decreased even more significantly after 3 days of storage at 37°C. In contrast, the comparative FGF protein 1 (PG-FGF1) maintained a sufficient remaining amount after 3 days of storage at 4°C, and even after 3 days of storage at 37°C, a considerable remaining amount was confirmed, although it was reduced compared to 4°C.

[0114] Furthermore, as shown in Figure 9 (Comparative Result 4), the residual amount of control FGF1 was significantly reduced regardless of whether heparin was added (even when stored at 4°C with heparin, the residual amount was reduced to nearly half). The residual amount of comparative FGF protein 2 (FGFC) was significantly reduced when stored at 37°C without heparin. In contrast, the residual amount of comparative FGF protein 1 (PG-FGF1) was not significantly reduced regardless of whether heparin was added, even when stored at 4°C or 37°C.

[0115] Furthermore, as shown in Figure 7 (Experimental Result 3), it was confirmed that the residual amount of FGF1 protein, comparative FGF protein 2 (FGFC), and comparative FGF protein 1 (PG-FGF1) all decreased or remained the same as in Comparative Result 4. Furthermore, in the case of the modified FGF chimeric protein of this example (PG-FGFC), no significant decrease in the residual amount was confirmed, regardless of whether heparin was added or not, even when stored at 4°C or 37°C, as with comparative FGF protein 1 (PG-FGF1).

[0116] Similarly, as shown in Figure 10 (Experimental Result 4), it was confirmed that the residual amounts of FGF1 protein, comparative FGF protein 2 (FGFC), comparative FGF protein 1 (PG-FGF1), and the modified FGF chimeric protein of this example (PG-FGFC) all decreased or remained the same, as in the case of the PBS solution in Experimental Result 3. In particular, when stored at 37°C, the modified FGF chimeric protein of this example (PG-FGFC) maintained a residual amount comparable to or greater than that of comparative FGF protein 1 (PG-FGF1), regardless of the presence or absence of heparin.

[0117] Thus, Comparative Results 3 and 4 and Experimental Results 3 and 4 demonstrate that the addition of glycosaminoglycans to FGF1 protein or FGFC using PG polypeptide significantly reduces adsorption to containers. In particular, the modified FGF chimeric protein (PG-FGFC) of this example demonstrates that adsorption can be reduced to a level equal to or greater than that of comparative FGF protein 1 (PG-FGF1), even when stored at 37°C for 3 days, regardless of the presence or absence of heparin.

[0118] The present invention is not limited to the description of the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments or multiple modified examples are also included in the technical scope of the present invention. [Industrial Applicability]

[0119] The present invention can be widely and publicly used in the fields of medicines, research reagents, medical products, etc. that utilize cell proliferation-promoting activity, and can also be widely and suitably used in fields such as agriculture and the chemical industry.

Claims

1. The present invention provides an FGF chimeric polypeptide (FGFC) in which the sequence of positions 41 to 83 in an amino acid sequence that is at least 90% identical to the amino acid sequence of an FGF1 polypeptide of the fibroblast growth factor (FGF) family is replaced with an amino acid sequence that is at least 90% identical to the sequence of positions 43 to 85 in the amino acid sequence of an FGF2 polypeptide of the FGF family, and Furthermore, the present invention is characterized in that it has a GAG addition sequence polypeptide to which a glycosaminoglycan (GAG) sugar chain is bound. Modified fibroblast growth factor chimeric protein.

2. The GAG ​​addition sequence polypeptide has the glycosaminoglycan bound to a serine molecule contained in the sequence. The modified fibroblast growth factor chimeric protein of claim 1.

3. the sequence of positions 41 to 83 in the amino acid sequence of the FGF1 polypeptide is replaced with an amino acid sequence that is at least 95% identical to the sequence of positions 43 to 85 in the amino acid sequence of the FGF2 polypeptide; The modified fibroblast growth factor chimeric protein of claim 1.

4. The FGF chimeric polypeptide (FGFC) consists of the amino acid sequence shown in SEQ ID NO:

7. The modified fibroblast growth factor chimeric protein of claim 1.

5. The GAG ​​addition sequence polypeptide consists of the amino acid sequence shown in SEQ ID NO:

5. The modified fibroblast growth factor chimeric protein of claim 1.

6. a step of linking DNA encoding an FGF chimeric polypeptide (FGFC) in which the sequence of positions 41 to 83 in an amino acid sequence that is at least 90% identical to the amino acid sequence of an FGF1 polypeptide of the fibroblast growth factor (FGF) family is replaced with an amino acid sequence that is at least 90% identical to the sequence of positions 43 to 85 in the amino acid sequence of an FGF2 polypeptide of the FGF family, to DNA encoding a GAG addition sequence polypeptide to which a glycosaminoglycan (GAG) sugar chain is bound, thereby constructing a modified FGF chimeric DNA that encodes a modified FGF chimeric protein; incorporating the obtained modified FGF chimeric DNA into an expression vector; introducing the expression vector into a host cell having a glycosaminoglycan glycosylation pathway; and expressing in the host cell the modified FGF chimeric protein and the GAG ​​addition sequence polypeptide, wherein the GAG ​​addition sequence polypeptide has a glycosaminoglycan sugar chain attached thereto. Methods for producing modified fibroblast growth factor chimeric proteins.

7. The DNA encoding the modified FGF chimeric protein has the base sequence shown in SEQ ID NO:

8. A method for producing the modified fibroblast growth factor chimeric protein of claim 6.

8. The DNA encoding the GAG ​​addition sequence polypeptide has the base sequence shown in SEQ ID NO:

6. A method for producing the modified fibroblast growth factor chimeric protein of claim 6.

9. A DNA comprising a base sequence encoding the modified fibroblast growth factor chimeric protein according to any one of claims 1 to 5.

10. An expression vector comprising the DNA of claim 9.

11. A transformant obtained by introducing the DNA according to claim 9 or the expression vector according to claim 10 into a host cell.

12. A composition comprising the modified fibroblast growth factor chimeric protein of any one of claims 1 to 5.

13. A method for culturing stem cells, comprising the step of adding the composition according to claim 12 to a culture medium during stem cell culture.

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