Method for producing peptide having physiological activity, and peptide comprising short linker

Abstract

To provide: a method for producing a complicated peptide molecule, such as a dimeric peptide, in a simpler manner at lower cost compared with the conventional chemical synthesis methods; a dimeric peptide which comprises a specific short linker and has a satisfactory activity; and others.SOLUTION: A peptide molecule comprises: (I) a first physiologically active peptide portion; (II) a linker portion composed of 49 or less amino acid residues; and (III) a second physiologically active peptide portion located on the opposite side of the linker portion from the first physiologically active peptide portion, wherein the first physiologically active peptide portion and the second physiologically active peptide portion may be the same as or different from each other, and at least 90% of an amino acid sequence of the linker portion is composed of amino acid residues selected from alanine (A), proline (P), and serine (S).SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing a peptide having physiological activity, a peptide containing a short-chain linker, and the like.

Background Art

[0002] In recent years, as pharmaceutical modalities, biopharmaceuticals such as antibody drugs, regenerative medicine, nucleic acid drugs, etc. have been studied and are now actually being used. Also, as a new pharmaceutical modality, medium molecules, particularly peptides, have been attracting attention. In particular, a plurality of peptides having a special shape such as cyclic peptides (special peptides) have been developed and are expected to be applied to pharmaceuticals. Under such circumstances, it is considered that the development of dimer peptides and peptide complexes will accelerate in the future. For example, regarding dimer peptides, APL-2 of Apellis Pharmaceuticals is being clinically studied as a therapeutic agent for age-related macular degeneration and the like, and practical application is expected.

[0003] A dimer peptide generally has a structure in which two peptide chains are linked by a short-chain linker, and a peptide complex generally has a structure in which a peptide chain and a drug are linked by a short-chain linker. As such a short-chain linker, a PEG linker is mainly used. However, in the production of dimer peptides and peptide complexes, conventional chemical synthesis methods require chemical synthesis of peptide chains, addition of PEG linkers, and purification, and need to go through many manufacturing steps. Further, when the peptide is a cyclic peptide, cyclization of the peptide chain must be further performed. Therefore, in order to produce dimer peptides and peptide complexes by conventional chemical synthesis methods, it is necessary to go through complicated steps and is very laborious. Moreover, in the synthesis of a dimer peptide formed by binding a first peptide and a second peptide via a PEG linker by conventional chemical synthesis methods, a product in which the C-terminus of the first peptide binds to the linker and the C-terminus of the second peptide also binds to the linker is obtained, and efficient synthesis of a dimer peptide in which the C-terminus of the first peptide binds to the linker and the N-terminus of the second peptide binds to the linker cannot be performed. In addition, the mainly used PEG linker is expensive, and it cannot be said to be economically preferable in conventional chemical synthesis methods.

[0004] Special Table 2013-531480 describes a drug conjugate comprising a biologically active protein and a random coil polypeptide comprising an amino acid sequence consisting of at least 50 proline and alanine amino acid residues. In Patent Document 1, it is described that the random coil polypeptide provides increased blood stability of the drug conjugate. However, Special Table 2013-531480 does not specifically disclose using the random coil polypeptide in place of a short-chain linker such as a PEG linker in dimer peptides and peptide complexes. In addition, when using peptide molecules such as dimer peptides as pharmaceuticals, not only improvement in the stability of peptide molecules such as dimer peptides but also maintenance or improvement in activity are desired. As described above, a PEG linker is mainly used as a short-chain linker that connects two peptide chains, and there are problems in terms of production when using a PEG linker. However, from the viewpoint of the activity of peptide molecules such as dimer peptides, a more useful short-chain linker is eagerly desired.

Summary of the Invention

Problems to be Solved by the Invention

[0005] As described above, in the production of peptide molecules such as dimer peptides, conventional chemical synthesis methods were not simple in operation and required high costs. Therefore, an object of one aspect of the present invention is to provide a method for producing peptide molecules such as dimer peptides simply and inexpensively as compared with conventional chemical synthesis methods.

[0006] As described above, as a short-chain linker that connects two peptide chains, a more useful short-chain linker is desired from the viewpoint of the activity of peptide molecules such as dimer peptides. Therefore, an object of one aspect of the present invention is to provide a peptide molecule such as a dimer peptide having good activity and containing a specific short-chain linker.

Means for Solving the Problems

[0007] The present invention may include, for example, the following aspects. [1] (I) A first bioactive peptide site, (II) A linker site consisting of 49 or fewer amino acid residues, (III) A second bioactive peptide site located on the side opposite to the first bioactive peptide site via the linker site and a peptide molecule comprising The first bioactive peptide site and the second bioactive peptide site may be the same as or different from each other. The peptide molecule, wherein at least 90% of the amino acid sequence of the linker site consists of amino acid residues selected from alanine (A), proline (P), and serine (S). 〔2〕The peptide molecule according to 〔1〕, wherein the first bioactive peptide site forms a cyclic peptide and / or the second bioactive peptide site forms a cyclic peptide. 〔3〕The peptide molecule according to 〔1〕 or 〔2〕, wherein the first bioactive peptide site forms a cyclic peptide having a cyclic structure formed by a disulfide bond and / or the second bioactive peptide site forms a cyclic peptide having a cyclic structure formed by a disulfide bond. 〔4〕The peptide molecule according to any one of 〔1〕 to 〔3〕, wherein the first bioactive peptide site and the second bioactive peptide site each form a cyclic peptide that binds to the c-Met protein, and may be the same as or different from each other. 〔5〕The peptide molecule according to 〔4〕, wherein the cyclic peptide is a cyclic peptide having a cyclic structure formed by a disulfide bond and containing an amino acid sequence selected from the following (a) to (i). (a) CYRQFNRRTHEVWNLDC (SEQ ID NO: 1); (b) CRQFNRRTHEVWNLDC (SEQ ID NO: 2); (c) CYWYYAWDQTYKAFPC (SEQ ID NO: 3); (d) CWYYAWDQTYKAFPC (SEQ ID NO: 4); (e) CYISWNEFNSPNWRFITC (SEQ ID NO: 5); (f) CISWNEFNSPNWRFITC (SEQ ID NO: 6); (g) A peptide in which one or two amino acid residues other than cysteine residues are substituted, deleted, or added in any one of the amino acid sequences of (a) to (f) and that binds to the c-Met protein; (h) A peptide consisting of an amino acid sequence having 90% or more sequence identity with any one of the amino acid sequences of (a) to (f), provided that it has cysteine residues at both ends and binds to the c-Met protein; and (i) A peptide in which at least one amino acid other than the cysteine residues at both ends is modified in any one of the amino acid sequences of (a) to (h) (the modification of the amino acid is phosphorylation, methylation, acetylation, adenylation, ADP ribosylation or glycosylation). [6] The first bioactive peptide site and the second bioactive peptide site each constitute a cyclic peptide that binds to the erythropoietin receptor, and may be the same or different from each other. The peptide molecule according to any one of [1] to [3] above. [7] The cyclic peptide is a cyclic peptide that forms a cyclic structure by a disulfide bond and contains an amino acid sequence selected from the following (j) to (n). The peptide molecule according to [6] above. (j) GGLYACHMGPMTWVCQPLRG (SEQ ID NO: 65); (k) CISWNEFNSPNWRFITC (SEQ ID NO: 66); (l) A peptide in which one or two amino acid residues other than cysteine residues are substituted, deleted or added in the amino acid sequence of (j) or (k) and binds to the erythropoietin receptor; (m) A peptide consisting of an amino acid sequence having 90% or more sequence identity with any one of the amino acid sequences of (j) or (k), provided that it has cysteine residues at the same positions as in (j) or (k) and binds to the erythropoietin receptor; and (n) A peptide in which at least one amino acid other than cysteine residues is modified in any one of the amino acid sequences of (j) to (m) (the modification of the amino acid is phosphorylation, methylation, acetylation, adenylation, ADP ribosylation or glycosylation). 〔8〕The peptide molecule according to any one of 〔1〕to 〔3〕above, wherein the first bioactive peptide site and the second bioactive peptide site each consist of a bioactive peptide that binds to a thrombopoietin receptor, and they may be the same or different from each other. 〔9〕The peptide molecule according to 〔8〕above, wherein the bioactive peptide contains an amino acid sequence selected from the following (o) to (u). (o)IEGPTLRQWLAARA (SEQ ID NO: 67); (p)GGCADGPTLREWISFCGG (SEQ ID NO: 68); (q)GGCTLREWLHGGFCGG (SEQ ID NO: 69); (r)LAIEGPTLRQWLHGNGRDT (SEQ ID NO: 70); (s) A peptide in which one or two amino acid residues other than cysteine residues are substituted, deleted or added in any one of the amino acid sequences of (o) to (r) and which binds to a thrombopoietin receptor; (t) A peptide consisting of an amino acid sequence having 90% or more sequence identity with any one of the amino acid sequences of (o) to (r), provided that when (p) or (q) is used as a reference, it has cysteine residues at the same positions as in (p) or (q), and which binds to a thrombopoietin receptor; and (u) A peptide in which at least one amino acid other than cysteine residues is modified in any one of the amino acid sequences of (o) to (t) (the modification of the amino acid is phosphorylation, methylation, acetylation, adenylation, ADP-ribosylation or glycosylation). 〔10〕The peptide molecule according to any one of 〔1〕to 〔9〕above, further comprising a functional modification site at a position not adjacent to the linker site (II). 〔11〕The linker site (II) is the first linker site, The second bioactive peptide site further comprises a second linker site on the side opposite to the first linker site, The second bioactive peptide site further comprises a third bioactive peptide site located on the side opposite to the second bioactive peptide site via the second linker site, The third bioactive peptide site may be the same as or different from the first bioactive peptide site and / or the second bioactive peptide site. The second linker site consists of 49 or fewer amino acid residues, and at least 90% of the amino acid sequence consists of amino acid residues selected from alanine (A), proline (P), and serine (S). It may be the same as or different from the first linker site. The peptide molecule according to any one of [1] to

[10] above. 〔12〕The linker site (II) contains two or more repetitions of an amino acid sequence selected from the group consisting of AP, AAP, AS, ASP, and ASS. When the second linker site is present, the second linker site contains two or more repetitions of an amino acid sequence selected from the group consisting of AP, AAP, AS, ASP, and ASS. The peptide molecule according to any one of [1] to

[11] above. 〔13〕A method for producing a peptide molecule, Expressing the polynucleotide in a host cell containing the polynucleotide encoding the peptide molecule, or expressing the polynucleotide encoding the peptide molecule in a cell-free expression system to generate the peptide molecule. comprising The peptide molecule includes (I) a first bioactive peptide site, (II) a linker site consisting of amino acid residues, and (III) a second bioactive peptide site and / or an additional amino acid sequence consisting of at least one amino acid residue located on the opposite side of the first bioactive peptide site via the linker site. The first bioactive peptide site and the second bioactive peptide site each have a molecular weight of 10,000 or less and consist of 50 or fewer amino acid residues, and they may be the same as or different from each other. The additional amino acid sequence has a molecular weight of 10,000 or less and consists of 50 or fewer amino acid residues. The manufacturing method as described above. The production method according to

[13] , wherein the first bioactive peptide site forms a cyclic peptide and / or the second bioactive peptide site forms a cyclic peptide. 〔15〕A method for producing a peptide molecule, comprising the step of generating the peptide molecule by expressing the polynucleotide encoding the peptide molecule in a host cell containing the polynucleotide or by expressing the polynucleotide encoding the peptide molecule in a cell-free expression system. comprising the peptide molecule comprising: (I) a first bioactive peptide site forming a cyclic peptide; (II) a linker site composed of amino acid residues; and (III) a second bioactive peptide site and / or an additional amino acid sequence composed of at least one amino acid residue located on the opposite side of the first bioactive peptide site via the linker site. the first bioactive peptide site and the second bioactive peptide site may be the same as or different from each other. the production method according to any one of

[13] to

[16] , wherein the additional amino acid sequence has a molecular weight of 10,000 or less and is composed of 50 or fewer amino acid residues. 〔16〕The production method according to

[15] , wherein the peptide molecule contains the second bioactive peptide site and the second bioactive peptide site forms a cyclic peptide. 〔17〕The production method according to any one of

[13] to

[16] , wherein the linker site has a molecular weight of 10,000 or less and is composed of 50 or fewer amino acid residues. 〔18〕The production method according to any one of

[13] to

[16] , wherein the linker site has a molecular weight of 10,000 or less and is a linker site composed of 49 or fewer amino acid residues, and at least 90% of the amino acid sequence thereof is composed of amino acid residues selected from alanine (A), proline (P), and serine (S). 〔19〕The production method according to any one of 〔13〕to 〔18〕above, wherein the linker portion contains two or more repetitions of an amino acid sequence selected from the group consisting of AP, AAP, AS, ASP, and ASS.

[0008] According to one embodiment of the present invention, a peptide molecule such as a dimer peptide can be produced simply and inexpensively as compared with conventional chemical synthesis methods. According to one embodiment of the present invention, an efficient synthesis of a dimer peptide can be performed in which the C-terminus of the first peptide is bound to the linker and the N-terminus of the second peptide is bound to the linker. According to one embodiment of the present invention, a dimer peptide having good activity can be provided.

Brief Description of the Drawings

[0009]

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Mode for Carrying Out the Invention

[0010] One embodiment of the present invention relates to a peptide molecule. Such a peptide molecule (I) a first bioactive peptide site, (II) a linker site consisting of 49 or fewer amino acid residues, (III) a second bioactive peptide site located on the side opposite to the first bioactive peptide site via the linker site and is a peptide molecule comprising the first bioactive peptide site and the second bioactive peptide site may be the same or different from each other, and at least 90% of the amino acid sequence of the linker site consists of amino acid residues selected from alanine (A), proline (P), and serine (S). In one embodiment of the peptide molecule of the present invention, it may include a first bioactive peptide site on the N-terminal side of the linker site and a second bioactive peptide site on the C-terminal side of the linker site, or it may include a first bioactive peptide site on the C-terminal side of the linker site and a second bioactive peptide site on the N-terminal side of the linker site. Also, in one embodiment of the peptide molecule of the present invention, between the N-terminal side and / or the C-terminal side of the first bioactive peptide site and / or the second bioactive peptide site, or between the linker site, it may include any amino acid sequence within the range that does not inhibit the functions of the first bioactive peptide and the second bioactive peptide. Such arbitrary amino acid sequences may include, for example, sequences useful for enhancing the expression and secretion of the peptide molecule in host cells, or those for simply adjusting the length (such as one, two, three, or one to five Gly, etc.). In the peptide molecule of the present invention, each amino acid may be an L-amino acid or a D-amino acid, and may be a natural type or a non-natural type. Also, in the peptide molecule of the present invention, each amino acid is preferably an α-, β- or γ-amino acid, more preferably an α-amino acid.

[0011] In one embodiment of the peptide molecule of the present invention, the peptide molecule may be configured to have two or more linker sites, such as "first bioactive peptide site" - "linker site" - "second bioactive peptide site" - "linker site" - "arbitrary sequence" from the N-terminal side. Here, the arbitrary sequence may be a third bioactive peptide site (which may be the same as or different from the first bioactive peptide site and / or the second bioactive peptide site), or may be an additional amino acid sequence consisting of at least one amino acid residue. Examples of the additional amino acid sequence include sequences having a molecular weight of 10,000 or less and consisting of 50 or fewer amino acid residues. Such additional amino acid sequences include enzyme recognition sequences such as SortaseA recognition signal and Butelase recognition signal, tag sequences such as FLAG tag, PA tag, and histidine tag, reactive amino acids such as lysine, cysteine, glutamic acid, and aspartic acid, or sequences containing such reactive amino acids, and the like. In one embodiment of the peptide molecule of the present invention, the linker site (II) is the first linker site, the second bioactive peptide site further includes a second linker site on the side opposite to the first linker site, and further includes a third bioactive peptide site located on the side opposite to the second bioactive peptide site via the second linker site, the third bioactive peptide site may be the same as or different from the first bioactive peptide site and / or the second bioactive peptide site, the second linker site consists of 49 or fewer amino acid residues, and at least 90% of the amino acid sequence consists of amino acid residues selected from alanine (A), proline (P), and serine (S), and may be the same as or different from the first linker site.

[0012] As used herein, the term "bioactive peptide" means a peptide capable of exhibiting bioactivity in a living body such as a human or an animal. Here, "bioactivity" refers to the property of acting on the physiological functions of a living body, and may include both the property of enhancing physiological functions and the property of suppressing physiological functions. In this specification, the "bioactive peptide" includes not only the peptide itself capable of exhibiting bioactivity in a living body such as a human or an animal, but also a peptide (i.e., a precursor) that becomes capable of exhibiting bioactivity through modification in the living body. The "bioactive peptide" may consist of a naturally occurring amino acid sequence or a non-naturally occurring amino acid sequence. Examples of the "bioactive peptide" include, but are not limited to, a target-binding peptide, a target-inhibiting peptide, a target-activating peptide, an enzyme-recognizing peptide, and the like.

[0013] As used herein, the term "target-binding peptide" means a bioactive peptide (including those that become capable of exhibiting binding affinity or bioactivity to a target through modification in the living body) that binds to a target (mainly a protein) such as a receptor, an enzyme, an ion channel, or a transporter. Examples of the target-binding peptide include, but are not limited to, an albumin-binding cyclic peptide, an organ-delivery cyclic peptide, and the like. As used herein, the term "target-inhibiting peptide" means a bioactive peptide (including those that become capable of exhibiting an inhibitory action through modification in the living body) that has an action of inhibiting a biological reaction caused by the activation of a target (mainly a protein) such as a receptor, an enzyme, an ion channel, or a transporter, or a physiological function in which the target is involved. This "target-inhibiting peptide" includes not only a bioactive peptide that binds to the target itself and inhibits a biological reaction caused by the activation of the target or a physiological function in which the target is involved, but also a bioactive peptide that binds to a factor that binds to the target, changes the property or shape of the factor, and thereby inhibits a biological reaction caused by the activation of the target or a physiological function in which the target is involved. As used herein, the term "target activating peptide" refers to a bioactive peptide that has the effect of activating a target (mainly a protein) such as a receptor, enzyme, ion channel, transporter, etc., to cause a biological reaction, or enhancing a physiological function in which the target is involved (including those that exhibit an activating effect or enhancing effect by modification in vivo). This "target activating peptide" includes not only a bioactive peptide that binds to the target itself to activate the target and cause a biological reaction or enhance the physiological function in which the target is involved, but also a bioactive peptide that binds to a certain factor, changes the nature or shape of the factor, and based on the fact that the factor binds to the target, activates the target to cause a biological reaction or enhances the physiological function in which the target is involved. Preferably, examples of the target in "target binding peptide", "target inhibitory peptide" and "target activating peptide" include receptors that are activated by dimerization, such as growth factor receptors, growth factor receptors, cytokine receptors, etc. As used herein, the term "enzyme recognition peptide" refers to a bioactive peptide that an enzyme recognizes as a substrate (including those that become recognized as a substrate by an enzyme or exhibit bioactivity by modification in vivo).

[0014] In one embodiment of the peptide molecule of the present invention, the molecular weights of the first bioactive peptide site and the second bioactive peptide site are not particularly limited, but can be, for example, 250 or more, 500 or more, 800 or more, or 1,000 or more, and can be 10,000 or less, 9,000 or less, 8,000 or less, 7,000 or less, 6,000 or less, 5,000 or less, 4,000 or less. In one embodiment of the peptide molecule of the present invention, the lengths of the first bioactive peptide site and the second bioactive peptide site are not particularly limited, but can be, for example, each independently composed of 3 or more, 6 or more, 10 or more, or 12 or more amino acid residues, and can be composed of 50 or less, 45 or less, 40 or less, 35 or less, 30 or less, 25 or less, or 20 or less amino acid residues. In one embodiment of the peptide molecule of the present invention, the first bioactive peptide site and / or the second bioactive peptide site may be linear or branched, and may form a cyclic peptide. The cyclic peptide in the first bioactive peptide site and / or the second bioactive peptide site is not particularly limited as to the binding mode for forming the cyclic structure. For example, it can form a cyclic structure by a disulfide bond between two cysteine residues, by a thioether bond (a bond between a thiol group and a haloalkyl group), or by a 1,2,3-triazole bond (a bond between an azide group and an alkyne group). The disulfide bond may be formed spontaneously in vivo or in vitro by culturing the bacterial cells, or by an enzymatic reaction of the bacteria. In one embodiment of the peptide molecule of the present invention, the first bioactive peptide site may contain two cysteine residues, or at least two cysteine residues. In one embodiment of the peptide molecule of the present invention, the second bioactive peptide site may contain two cysteine residues, or at least two cysteine residues.

[0015] In one embodiment of the peptide molecule of the present invention, preferably, the first bioactive peptide site forms a cyclic peptide. In one embodiment of the peptide molecule of the present invention, preferably, the second bioactive peptide site forms a cyclic peptide. When the peptide molecule of the present invention has a cyclic peptide at the first bioactive peptide site and / or the second bioactive peptide site, the cyclic peptide is not particularly limited. For example, FGFR-binding peptide, c-Met (HGFR)-binding peptide, erythropoietin receptor-binding peptide, thrombopoietin receptor-binding peptide, albumin-binding peptide, EGFR-binding peptide, VEGFR-binding peptide, PDGFR-binding peptide, Axl-binding peptide, PDGFR-binding peptide, SCFR-binding peptide, Flt-3-binding peptide, c-Ret-binding peptide, ROR-binding peptide, Tie-binding peptide, NGFR-binding peptide, Insulin receptor-binding peptide, EphR-binding peptide, Alk-binding peptide, DDR-binding peptide, TGFBR-binding peptide, Activin receptor-binding peptide, BMP receptor-binding peptide, interleukin receptor-binding peptide, T cell receptor-binding peptide, transferrin receptor-binding peptide, lipoprotein receptor-binding peptide, ubiquitin ligase-binding peptide, antibody-binding peptide, complement-binding peptide, GPCR-binding peptide, ion channel-binding peptide, virus-binding peptide, etc. can be mentioned.

[0016] In one embodiment of the peptide molecule of the present invention, as a specific example, the first bioactive peptide site and the second bioactive peptide site each constitute a cyclic peptide that binds to the c-Met (also simply referred to as Met) protein, and they may be the same or different from each other. Examples of the cyclic peptide that binds to the c-Met protein include, but are not limited to, cyclic peptides containing or consisting of an amino acid sequence selected from the following (a) to (i) and forming a cyclic structure by a disulfide bond. (a) CYRQFNRRTHEVWNLDC (SEQ ID NO: 1) (b) CRQFNRRTHEVWNLDC (SEQ ID NO: 2) (c) CYWYYAWDQTYKAFPC (SEQ ID NO: 3) (d) CWYYAWDQTYKAFPC (SEQ ID NO: 4) (e) CYISWNEFNSPNWRFITC (SEQ ID NO: 5) (f) CISWNEFNSPNWRFITC (SEQ ID NO: 6) (g) A peptide in which one or two amino acid residues other than cysteine residues are substituted, deleted or added in any of the amino acid sequences of (a) to (f) and which binds to the c-Met protein. (h) A peptide consisting of an amino acid sequence having 90% or more sequence identity with any of the amino acid sequences of (a) to (f), provided that it has cysteine residues at both ends and binds to the c-Met protein. For example, the sequence identity may be 92% or more, 95% or more, or 98% or more. In the present specification, the amino acid sequence homology can be calculated using known analysis tools. For example, it can be calculated by using the homology algorithm BLAST (Basic local alignment search tool) of the National Center for Biotechnology Information (NCBI) in the United States. Also, for calculating the amino acid sequence homology, the default (initial setting) parameters in the analysis tool may be used. (i) A peptide in which at least one amino acid other than the cysteine residues at both ends is modified in any of the amino acid sequences of (a) to (h). Here, the modification of the amino acid is phosphorylation, methylation, acetylation, adenylation, ADP-ribosylation or glycosylation. Note that Met is the HGF receptor, a tyrosine kinase type receptor, and it is known that such a cyclic peptide that binds to Met (HGFR) functions as a Met (HGFR) agonist by dimerization.

[0017] In this specification, in the expression "substitution, deletion or addition", substitution means substitution with a known amino acid, preferably a conservative amino acid substitution. "Conservative amino acid substitution" is a substitution in which an amino acid residue is substituted by another amino acid residue having a side chain R group with similar chemical properties (for example, charge or hydrophobicity). Examples of amino acid groups having side chains with similar chemical properties include glycine, alanine, valine, leucine, and isoleucine having aliphatic side chains, serine and threonine having aliphatic hydroxyl side chains, asparagine and glutamine having amide-containing side chains, phenylalanine, tyrosine, and tryptophan having aromatic side chains, lysine, arginine, and histidine having basic side chains, and aspartic acid and glutamic acid having acidic side chains. Each amino acid introduced by substitution may be an L-amino acid or a D-amino acid, and may be a natural amino acid or a non-natural amino acid, but preferably an amino acid constituting a protein in humans or animals. In this specification, in the expression "substitution, deletion or addition", addition means that a known amino acid is added to the end of an amino acid sequence, or a known amino acid is inserted between amino acid residues of an amino acid sequence. Each amino acid introduced by addition may be an L-amino acid or a D-amino acid, and may be a natural amino acid or a non-natural amino acid, but preferably an amino acid constituting a protein in humans or animals.

[0018] In one embodiment of the peptide molecule of the present invention, as a specific example, the first bioactive peptide site and the second bioactive peptide site each constitute a cyclic peptide that binds to the erythropoietin receptor, and may be the same or different from each other. Examples of the cyclic peptide that binds to the erythropoietin receptor include, but are not limited to, cyclic peptides that contain or consist of an amino acid sequence selected from the following (j) to (n) and form a cyclic structure by a disulfide bond. (j)GGLYACHMGPMTWVCQPLRG (SEQ ID NO: 65) (k)CISWNEFNSPNWRFITC (SEQ ID NO: 66) (l) A peptide in which one or two amino acid residues other than cysteine residues are substituted, deleted or added in the amino acid sequence of (j) or (k), and which binds to the erythropoietin receptor. (m) A peptide having at least 90% sequence identity with the amino acid sequence of either (j) or (k), provided that it has cysteine residues at the same positions as in (j) or (k), and which binds to the erythropoietin receptor. For example, the sequence identity may be 92% or more, 95% or more, or 98% or more. (n) A peptide in which at least one amino acid other than cysteine residues is modified in the amino acid sequence of any one of (j) to (m). Here, the modification of the amino acid is phosphorylation, methylation, acetylation, adenylation, ADP-ribosylation or glycosylation.

[0019] In one embodiment of the peptide molecule of the present invention, as a specific example, the first bioactive peptide site and the second bioactive peptide site each consist of a bioactive peptide that binds to the thrombopoietin receptor, and they may be the same or different from each other. Examples of the bioactive peptide that binds to the thrombopoietin receptor include, but are not limited to, peptides containing or consisting of the amino acid sequences selected from the following (o) to (u). (o)IEGPTLRQWLAARA (SEQ ID NO: 67) (p)GGCADGPTLREWISFCGG (SEQ ID NO: 68) (q)GGCTLREWLHGGFCGG (SEQ ID NO: 69) (r)LAIEGPTLRQWLHGNGRDT (SEQ ID NO: 70) (s) A peptide in which one or two amino acid residues other than cysteine residues are substituted, deleted or added in the amino acid sequence of any one of (o) to (r), and which binds to the thrombopoietin receptor. A peptide consisting of an amino acid sequence having at least 90% sequence identity with any one of the amino acid sequences of (t)(o)~(r), provided that when based on (p) or (q), it has cysteine residues at the same positions as those in (p) or (q), and binds to the thrombopoietin receptor. For example, the sequence identity may be 92% or more, 95% or more, or 98% or more. A peptide in which at least one amino acid other than cysteine residues is modified in any one of the amino acid sequences of (u)(o)~(t). Here, the modification of the amino acid is phosphorylation, methylation, acetylation, adenylation, ADP ribosylation, or glycosylation. The peptides represented by SEQ ID NO: 68 and SEQ ID NO: 69 are cyclic peptides that form a cyclic structure by disulfide bonds, respectively.

[0020] In one embodiment of the peptide molecule of the present invention, as a specific example, the first bioactive peptide site and the second bioactive peptide site each constitute a cyclic peptide that binds to a vascular endothelial growth factor receptor (VEGFR), and they may be the same or different from each other. Examples of the cyclic peptide that binds to VEGFR include, but are not limited to, cyclic peptides that contain or consist of an amino acid sequence selected from the following (aa)~(ff) and form a cyclic structure by disulfide bonds. (aa)AGPTWCEDDWYYCWLFGT (SEQ ID NO: 71) (bb)VCWEDSWGGEVCWLFGT (SEQ ID NO: 72) (cc)VCWEDSWGGEVCFRYDP (SEQ ID NO: 73) A peptide in which one or two amino acid residues other than cysteine residues are substituted, deleted, or added in any one of the amino acid sequences of (aa)~(cc) and binds to VEGFR. A peptide consisting of an amino acid sequence having 90% or more sequence identity with any one of (ee)(aa) to (cc), provided that it has cysteine residues at their original positions in (aa) to (cc), and binds to VEGFR. For example, the sequence identity may be 92% or more, 95% or more, or 98% or more. A peptide in which at least one amino acid other than a cysteine residue is modified in any one of the amino acid sequences of (ff)(aa) to (ee). Here, the modification of the amino acid is phosphorylation, methylation, acetylation, adenylation, ADP ribosylation, or glycosylation.

[0021] In one embodiment of the peptide molecule of the present invention, the linker site may have a molecular weight of 10,000 or less. In one embodiment of the peptide molecule of the present invention, the linker site may consist of amino acid residues in which at least 90%, at least 95%, or 100% of its amino acid sequence is selected from alanine (A), proline (P), and serine (S). The proline residue may constitute more than 4% and less than 40% in the amino acid sequence of the linker site. For example, the proline residue may constitute more than about 4%, more than about 5%, more than about 6%, more than about 8%, more than about 10%, more than about 15%, or more than about 20% in the amino acid sequence of the linker site, and may also constitute less than about 40%, or less than about 35%. Also, the alanine residue and the serine residue may each independently constitute more than about 4%, more than about 10%, or more than about 20%, and may also constitute less than about 50%. As used herein, the term "about X%" is not limited to the number X, but also includes values of residues that are 10% - 20% more or 10% - 20% less. For example, the term "about 10%" also relates to 11% or 12%, and 9% or 8%, respectively. In the amino acid sequence of the linker portion, amino acid residues different from alanine, serine, and proline include, for example, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val, as well as those obtained by modifying these amino acids or adding a functional group or a protecting group (for example, chloroacetylated amino acids, homocysteine, mercaptonorvaline, mercaptonorleucine, 2-amino-7-mercaptoheptanoic acid, and 2-amino-8-mercaptooctanoic acid, and amino acids in which the SH group of these amino acids is once protected and then the protecting group is deprotected, propargylglycine, homopropargylglycine, 2-amino-6-heptynoic acid, 2-amino-7-octynoic acid, 2-amino-8-nonynoic acid, 4-pentynoylated or 5-hexynoylated amino acids, azidoalanine, 2-amino-4-azidobutanoic acid, azidoptonorvaline, azidonorleucine, 2-amino-7-azidoheptanoic acid, 2-amino-8-azidooctanoic acid, azidoacetylated or 3-azidopentanoylated amino acids, N-(4-aminomethyl-benzoyl)-phenylalanine (AMBF), 4-3-aminomethyltyrosine, 5-hydroxytryptophan (WOH), N-3-chloromethylbenzoyl-L-phenylalanine, N-3-chloromethylbenzoyl-L-tyrosine, N-3-chloromethylbenzoyl-L-tryptophane), and can be selected from the group consisting of. Each amino acid may be an L-amino acid or a D-amino acid.As amino acid residues different from such alanine, serine and proline, those having no hydrophobic side chains such as Val, Ile, Leu, Met, Phe, Tyr or Trp, and / or those having no charged side chains such as Lys, Arg, Asp or Glu are preferred.

[0022] In one embodiment of the peptide molecule of the present invention, the linker site may consist of amino acid residues in which at least 90%, at least 95%, or 100% of its amino acid sequence is selected from alanine (A) and proline (P). In this case, the proline residues may constitute more than 10% and less than 75% in the amino acid sequence of the linker site. For example, the proline residues may constitute more than about 10%, more than about 12%, more than about 14%, more than about 18%, more than about 20%, more than about 22%, more than about 23%, more than about 24%, or more than about 25% in the amino acid sequence of the linker site, and less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 48%, less than about 46%, less than about 44%, less than about 42%, less than about 41%, less than about 40%, less than about 39%, less than about 38%, less than about 37%, less than about 36%, or less than about 35%. Also, the alanine residues may constitute more than about 25%, more than about 30%, more than about 35%, more than about 40%, more than about 45%, more than about 50%, more than about 52%, more than about 54%, more than about 56%, more than about 58%, more than about 59%, more than about 60%, more than about 61%, more than about 62%, more than about 63%, more than about 64%, or more than about 65% in the amino acid sequence of the linker site, and less than about 90%, less than about 88%, less than about 86%, less than about 84%, less than about 82%, less than about 80%, less than about 79%, less than about 78%, less than about 77%, less than about 76%, or less than about 75%. In the amino acid sequence of the linker region, the amino acid residues different from alanine, serine, and proline can be selected from the group consisting of Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val. Such amino acid residues different from alanine, serine, and proline are preferably those having no hydrophobic side chain such as Val, Ile, Leu, Met, Phe, Tyr, or Trp, and / or those having no charged side chain such as Lys, Arg, Asp, or Glu.

[0023] In one embodiment of the peptide molecule of the present invention, the linker region contains a plurality of amino acid repeats, the amino acid repeats consist of alanine (A), proline (P), and serine (S), and the number of consecutive identical amino acid residues in the amino acid repeats can be 6 residues or less, 5 residues or less, 4 residues or less, or 3 residues or less. In one embodiment of the peptide molecule of the present invention, the linker region contains a plurality of amino acid repeats, the amino acid repeats consist of alanine (A) and proline (P), and the number of consecutive identical amino acid residues in the amino acid repeats can be 6 residues or less, 5 residues or less, 4 residues or less, or 3 residues or less. In one embodiment of the peptide molecule of the present invention, the linker region can be one that contains two or more repetitions of an amino acid sequence selected from the group consisting of AP, AAP, AS, ASP, and ASS. In one embodiment of the peptide molecule of the present invention, the linker region can consist of, for example, 5 or more, 6 or more, 7 or more, or 8 or more amino acid residues.

[0024] In one embodiment of the peptide molecule of the present invention, specific examples of the linker site include, but are not limited to, those containing the following amino acid sequences or consisting of the following amino acid sequences: AAPAAPAP (SEQ ID NO: 74), AAPAAPAPAAPAAPAP (SEQ ID NO: 75), AAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPA (SEQ ID NO: 76), SAPSPSSAPASASAPASPASASASASPASSPASASSASPAASSASPASS (SEQ ID NO: 77), AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 7), AAPAAPAPAAPAAPAPAAPAAP (SEQ ID NO: 8), AAPAAPAPAAPAAP (SEQ ID NO: 9), AAPAAAPAPAAPAAPAPAAP (SEQ ID NO: 10), AAAPAAAPAAAPAAAPAAAP (SEQ ID NO: 11), AAPAAPAAPAAPAAPAAPAAPAAP (SEQ ID NO: 12), APAAAPAPAAAPAPAAAPAPAAAP (SEQ ID NO: 13), AAAPAAPAAPPAAAAPAAPAAPPA (SEQ ID NO: 14), and APAPAPAPAPAPAPAPAPAP (SEQ ID NO: 15), ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 16), AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 17), APSSPSPSAPSSPSPASPSS (SEQ ID NO: 18), SAPSSPSPSAPSSPSPASPS (SEQ ID NO: 19), SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 20), AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 21), and ASAAAPAAASAAASAPSAAA (SEQ ID NO: 22), and amino acid sequences selected from the circular permutation variants of these. The circular permutation variant array can be easily prepared, for example, by removing the first alanine of the above array and adding another alanine to the end of the above array, taking AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 7) as an example. Such a circular permutation variant array of SEQ ID NO: 7 becomes APAAPAPAAPAAPAPAAPAA (SEQ ID NO: 23). Further, non-limiting examples of the circular permutation variant array in which the order of amino acid residues is changed circularly in SEQ ID NO: 7 include the following: PAAPAPAAPAAPAPAAPAAA (SEQ ID NO: 24), AAPAPAAPAAPAPAAPAAAP (SEQ ID NO: 25), APAPAAPAAPAPAAPAAAPA (SEQ ID NO: 26), PAPAAPAAPAPAAPAAAPAA (SEQ ID NO: 27), APAAPAAPAPAAPAAAPAAP (SEQ ID NO: 28), PAAPAAPAPAAPAAAPAAPA (SEQ ID NO: 29), AAPAAPAPAAPAAAPAAPAP (SEQ ID NO: 30), APAAPAPAAPAAAPAAPAPA (SEQ ID NO: 31), and PAAPAPAAPAAAPAAPAPAA (SEQ ID NO: 32).

[0025] In one embodiment of the peptide molecule of the present invention, the peptide molecule may further include a functional modification site at a position not adjacent to the linker site. As used herein, the term "functional modification site" means a site that imparts some function to the peptide molecule separately from the function of the bioactive peptide in the peptide molecule, and preferably consists of an amino acid sequence. Examples of the functional modification site include, but are not limited to, a stabilization sequence, a water solubility improvement sequence, an enzyme recognition sequence, a tag sequence, a target binding sequence, and the like. Specific examples of the stabilizing sequence or the water-solubility improving sequence include, but are not limited to, a random coil polypeptide containing an amino acid sequence consisting of at least 50 amino acid residues of proline and alanine as described in JP-T-2013-531480 (the content of which is incorporated herein by reference as part of this specification), a random coil polypeptide containing an amino acid sequence consisting of amino acid residues of proline, alanine and serine as described in JP-T-2010-531139 (the content of which is incorporated herein by reference as part of this specification), and the like. Specific examples of the enzyme recognition sequence include, but are not limited to, Sortase A recognition signal, Butelase recognition signal, Transglutaminase recognition signal and the like. Specific examples of the tag sequence include, but are not limited to, FLAG tag, PA tag, histidine tag and the like. Specific examples of the target binding sequence include, but are not limited to, small molecule antibodies such as VHH and scFv. In one embodiment of the peptide molecule of the present invention, the functional modification site can consist of, for example, 3 or more, 6 or more, 10 or more, or 12 or more amino acid residues, and can consist of 1000 or less, 500 or less, 300 or less, or 150 or less amino acid residues.

[0026] One embodiment of the present invention relates to a method for producing a peptide molecule. The peptide molecule produced by the production method of the present invention (I) a first bioactive peptide site, (II) a linker site consisting of amino acid residues, and (III) a second bioactive peptide site or an additional amino acid sequence consisting of at least one amino acid residue located on the side opposite to the first bioactive peptide site via the linker site and contains the same. In one embodiment of the manufacturing method of the present invention, the peptide molecule may have, for example, a structure of "first bioactive peptide site" - "linker site" - "second bioactive peptide site" from the N-terminal side, or a structure of "first bioactive peptide site" - "linker site" - "added amino acid sequence" from the N-terminal side, but is not limited thereto. Further, the peptide molecule may have two or more linker sites, such as "first bioactive peptide site" - "linker site" - "second bioactive peptide site" - "linker site" - "arbitrary sequence" from the N-terminal side (the first bioactive peptide site, and the second bioactive peptide site and / or an added amino acid sequence consisting of at least one amino acid residue are linked in tandem through two or more linker sites). Here, the arbitrary sequence may be a bioactive peptide site (which may be the same as the first bioactive peptide site and / or the second bioactive peptide site), or an added amino acid sequence consisting of at least one amino acid residue. Specific examples of such a produced peptide molecule include the aforementioned (I) a first bioactive peptide site, (II) a linker site consisting of 49 or fewer amino acid residues, (III) a second bioactive peptide site located on the opposite side of the first bioactive peptide site via the linker site and a peptide molecule containing wherein the first bioactive peptide site and the second bioactive peptide site may be the same as or different from each other, and at least 90% of the amino acid sequence of the linker site consists of amino acid residues selected from alanine (A), proline (P), and serine (S).

[0027] In the peptide molecule produced by the production method of the present invention, the first bioactive peptide site and the second bioactive peptide site can each have a molecular weight of 10,000 or less and may consist of 50 or fewer amino acid residues, and they may be the same or different from each other. Examples of the first bioactive peptide site and / or the second bioactive peptide site include, but are not limited to, FGFR-binding peptide, c-Met (HGFR)-binding peptide, erythropoietin receptor-binding peptide, thrombopoietin receptor-binding peptide, albumin-binding peptide, EGFR-binding peptide, VEGFR-binding peptide, PDGFR-binding peptide, Axl-binding peptide, PDGFR-binding peptide, SCFR-binding peptide, Flt-3-binding peptide, c-Ret-binding peptide, ROR-binding peptide, Tie-binding peptide, NGFR-binding peptide, Insulin receptor-binding peptide, EphR-binding peptide, Alk-binding peptide, DDR-binding peptide, TGFBR-binding peptide, Activin receptor-binding peptide, BMP receptor-binding peptide, interleukin receptor-binding peptide, T cell receptor-binding peptide, transferrin receptor-binding peptide, lipoprotein receptor-binding peptide, ubiquitin ligase-binding peptide, antibody-binding peptide, complement-binding peptide, GPCR-binding peptide, ion channel-binding peptide, virus-binding peptide, etc. In one embodiment of the production method of the present invention, the molecular weights of the first bioactive peptide site and the second bioactive peptide site can be, for example, 9,000 or less, 8,000 or less, 7,000 or less, 6,000 or less, 5,000 or less, 4,000 or less, and can also be 250 or more, 500 or more, 800 or more, or 1,000 or more. In one embodiment of the production method of the present invention, the lengths of the first bioactive peptide site and the second bioactive peptide site can each independently consist of 45 or fewer, 40 or fewer, 35 or fewer, 30 or fewer, 25 or fewer, or 20 or fewer amino acid residues, and can also consist of 3 or more, 6 or more, 10 or more, or 12 or more amino acid residues.

[0028] In one embodiment of the production method of the present invention, the first bioactive peptide site and / or the second bioactive peptide site may be linear or branched, and may form a cyclic peptide. The cyclic peptide in the first bioactive peptide site and / or the second bioactive peptide site is not particularly limited as to the bonding mode of forming the cyclic structure. For example, it can form a cyclic structure by a disulfide bond between two cysteine residues, by a thioether bond (bond between a thiol group and a haloalkyl group), or by a 1,2,3-triazole bond (bond between an azide group and an alkyne group). The disulfide bond may be formed spontaneously inside or outside the bacterial cell by culturing the bacterial cell, or by an enzymatic reaction of the bacterium. In one embodiment of the production method of the present invention, the first bioactive peptide site may contain two cysteine residues, or at least two cysteine residues. In one embodiment of the production method of the present invention, the second bioactive peptide site may contain two cysteine residues, or at least two cysteine residues. Although various cyclic peptides are considered to exist, in the first bioactive peptide site and / or the second bioactive peptide site, the cyclic peptide constituting them is not particularly limited. In one embodiment of the production method of the present invention, when the peptide molecule is a dimer peptide, there may be one having higher bioactivity than the monomer peptide. For example, when the peptide molecule produced by the production method of the present invention is one in which two FGFR-binding cyclic peptides are bound via a linker site, it may have higher FGFR inhibitory activity than one FGFR-binding cyclic peptide.

[0029] Specific examples of the cyclic peptide include, but are not limited to, cyclic peptides that bind to c-Met (also simply referred to as Met) in the peptide molecule obtained by the production method of the present invention. A cyclic peptide that forms a cyclic structure by a disulfide bond and contains or consists of an amino acid sequence selected from the following (a) to (i). (a) CYRQFNRRTHEVWNLDC (SEQ ID NO: 1) (b) CRQFNRRTHEVWNLDC (SEQ ID NO: 2) (c) CYWYYAWDQTYKAFPC (SEQ ID NO: 3) (d) CWYYAWDQTYKAFPC (SEQ ID NO: 4) (e) CYISWNEFNSPNWRFITC (SEQ ID NO: 5) (f) CISWNEFNSPNWRFITC (SEQ ID NO: 6) (g) A peptide in which one or two amino acid residues other than cysteine residues are substituted, deleted or added in any of the amino acid sequences of (a) to (f), and which binds to the c-Met protein. (h) A peptide having at least 90% sequence identity with any of the amino acid sequences of (a) to (f), consisting of an amino acid sequence having cysteine residues at both ends, and which binds to the c-Met protein. For example, the sequence identity may be 92% or more, 95% or more, or 98% or more. In the present specification, amino acid sequence homology can be calculated using known analysis tools. For example, it can be calculated by using the homology algorithm BLAST (Basic local alignment search tool) of the National Center for Biotechnology Information (NCBI). In addition, for calculating amino acid sequence homology, the default (initial setting) parameters in the analysis tool may be used. (i) A peptide in which at least one amino acid other than the cysteine residues at both ends is modified in any of the amino acid sequences of (a) to (h). Here, the modification of the amino acid is phosphorylation, methylation, acetylation, adenylation, ADP ribosylation or glycosylation. Note that Met is the HGF receptor, a tyrosine kinase-type receptor, and it is known that such a cyclic peptide that binds to Met (HGFR) functions as a Met (HGFR) agonist by dimerizing.

[0030] In one embodiment of the production method of the present invention, the first bioactive peptide site and / or the second bioactive peptide site of the produced peptide molecule may constitute a cyclic peptide that binds to the erythropoietin receptor. When both the first bioactive peptide site and the second bioactive peptide site constitute a cyclic peptide that binds to the erythropoietin receptor, these cyclic peptides may be the same or different from each other. Examples of the cyclic peptide that binds to the erythropoietin receptor include, but are not limited to, cyclic peptides that contain or consist of an amino acid sequence selected from the following (j) to (n) and form a cyclic structure by a disulfide bond. (j)GGLYACHMGPMTWVCQPLRG (SEQ ID NO: 65) (k)CISWNEFNSPNWRFITC (SEQ ID NO: 66) (l) A peptide in which one or two amino acid residues other than cysteine residues are substituted, deleted or added in the amino acid sequence of (j) or (k) and that binds to the erythropoietin receptor. (m) A peptide consisting of an amino acid sequence having 90% or more sequence identity with either of the amino acid sequences of (j) or (k), provided that it has cysteine residues at the same positions as in (j) or (k) and that binds to the erythropoietin receptor. For example, the sequence identity may be 92% or more, 95% or more, or 98% or more. (n) A peptide in which at least one amino acid other than cysteine residues is modified in the amino acid sequence of any one of (j) to (m). Here, the modification of the amino acid is phosphorylation, methylation, acetylation, adenylation, ADP ribosylation or glycosylation.

[0031] In one embodiment of the production method of the present invention, the first bioactive peptide site and / or the second bioactive peptide site of the produced peptide molecule may consist of a bioactive peptide that binds to the thrombopoietin receptor. When both the first bioactive peptide site and the second bioactive peptide site consist of a bioactive peptide that binds to the thrombopoietin receptor, these bioactive peptides may be the same as or different from each other. Examples of the bioactive peptide that binds to the thrombopoietin receptor include, but are not limited to, peptides containing or consisting of the amino acid sequences selected from the following (o) to (u). (o)IEGPTLRQWLAARA (SEQ ID NO: 67) (p)GGCADGPTLREWISFCGG (SEQ ID NO: 68) (q)GGCTLREWLHGGFCGG (SEQ ID NO: 69) (r)LAIEGPTLRQWLHGNGRDT (SEQ ID NO: 70) (s) A peptide in which one or two amino acid residues other than cysteine residues are substituted, deleted or added in any of the amino acid sequences of (o) to (r) and which binds to the thrombopoietin receptor. (t) A peptide consisting of an amino acid sequence having 90% or more sequence identity with any of the amino acid sequences of (o) to (r), provided that when (p) or (q) is used as a reference, it has a cysteine residue at the same position as in (p) or (q), and which binds to the thrombopoietin receptor. For example, the sequence identity may be 92% or more, 95% or more, or 98% or more. (u) A peptide in which at least one amino acid other than cysteine residues is modified in any of the amino acid sequences of (o) to (t). Here, the modification of the amino acid is phosphorylation, methylation, acetylation, adenylation, ADP-ribosylation or glycosylation. The peptides represented by SEQ ID NO: 68 and SEQ ID NO: 69 are cyclic peptides that form a cyclic structure by a disulfide bond, respectively.

[0032] In one embodiment of the production method of the present invention, the first bioactive peptide site and / or the second bioactive peptide site of the produced peptide molecule may constitute a cyclic peptide that binds to vascular endothelial growth factor receptor (VEGFR). When both the first bioactive peptide site and the second bioactive peptide site constitute a cyclic peptide that binds to VEGFR, these cyclic peptides may be the same or different from each other. Examples of the cyclic peptide that binds to VEGFR include, but are not limited to, cyclic peptides that contain or consist of an amino acid sequence selected from the following (aa) to (ff) and form a cyclic structure through disulfide bonds. (aa)AGPTWCEDDWYYCWLFGT (SEQ ID NO: 71) (bb)VCWEDSWGGEVCWLFGT (SEQ ID NO: 72) (cc)VCWEDSWGGEVCFRYDP (SEQ ID NO: 73) (dd) A peptide in which one or two amino acid residues other than cysteine residues are substituted, deleted, or added in any of the amino acid sequences of (aa) to (cc) and that binds to VEGFR. (ee) A peptide that has a sequence identity of 90% or more with any of the amino acid sequences of (aa) to (cc), provided that it consists of an amino acid sequence having cysteine residues at the same positions as in (aa) to (cc) and that binds to VEGFR. For example, the sequence identity may be 92% or more, 95% or more, or 98% or more. (ff) A peptide in which at least one amino acid other than cysteine residues is modified in any of the amino acid sequences of (aa) to (ee). Here, the modification of the amino acid is phosphorylation, methylation, acetylation, adenylation, ADP ribosylation, or glycosylation.

[0033] In one embodiment of the production method of the present invention, the linker site may have a molecular weight of 10,000 or less and consist of 50 or fewer amino acid residues. Also, in one embodiment of the production method of the present invention, the linker site can be a linker site having a molecular weight of 10,000 or less and consisting of 50 or less or 49 or less amino acid residues, and at least 90%, at least 95%, or 100% of the amino acid sequence thereof can consist of amino acid residues selected from alanine (A), proline (P), and serine (S). In this case, the proline residue can constitute more than 4% and less than 40% in the amino acid sequence of the linker site. For example, the proline residue may constitute more than about 4%, more than about 5%, more than about 6%, more than about 8%, more than about 10%, more than about 15%, or more than about 20% in the amino acid sequence of the linker site, and may constitute less than about 40% or less than about 35%. Also, the alanine residue and the serine residue may each independently constitute more than about 4%, more than about 10%, or more than about 20%, and may constitute less than about 50%. As used herein, the term "about X%" is not limited to a concise number of percentages, but also includes values of residues that are 10% to 20% more or 10% to 20% less. For example, the term "about 10%" also relates to 11% or 12%, and 9% or 8%, respectively. In the amino acid sequence of the linker site, amino acid residues different from alanine, serine, and proline can be selected from the group consisting of Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val. Such amino acid residues different from alanine, serine, and proline are preferably those having no hydrophobic side chains such as Val, Ile, Leu, Met, Phe, Tyr, or Trp, and / or those having no charged side chains such as Lys, Arg, Asp, or Glu.

[0034] In one embodiment of the production method of the present invention, the linker site may be a linker site having a molecular weight of 10,000 or less and consisting of 50 or less or 49 or less amino acid residues, and at least 90%, at least 95%, or 100% of the amino acid sequence thereof may consist of amino acid residues selected from alanine (A) and proline (P). In this case, the proline residue may constitute more than 10% and less than 75% in the amino acid sequence of the linker site. For example, the proline residue may constitute more than about 10%, more than about 12%, more than about 14%, more than about 18%, more than about 20%, more than about 22%, more than about 23%, more than about 24%, or more than about 25% in the amino acid sequence of the linker site, and less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 48%, less than about 46%, less than about 44%, less than about 42%, less than about 41%, less than about 40%, less than about 39%, less than about 38%, less than about 37%, less than about 36%, or less than about 35%. Also, the alanine residue may constitute more than about 25%, more than about 30%, more than about 35%, more than about 40%, more than about 45%, more than about 50%, more than about 52%, more than about 54%, more than about 56%, more than about 58%, more than about 59%, more than about 60%, more than about 61%, more than about 62%, more than about 63%, more than about 64%, or more than about 65%, and less than about 90%, less than about 88%, less than about 86%, less than about 84%, less than about 82%, less than about 80%, less than about 79%, less than about 78%, less than about 77%, less than about 76%, or less than about 75%. In the amino acid sequence of the linker site, amino acid residues different from alanine, serine, and proline can be selected from the group consisting of Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val. Such amino acid residues different from alanine, serine, and proline are preferably those having no hydrophobic side chain such as Val, Ile, Leu, Met, Phe, Tyr, or Trp, and / or those having no charged side chain such as Lys, Arg, Asp, or Glu.

[0035] In one embodiment of the production method of the present invention, the linker site contains a plurality of amino acid repeats, the amino acid repeats consist of alanine (A), proline (P), and serine (S), and the number of consecutive identical amino acid residues in the amino acid repeats can be 6 residues or less, 5 residues or less, 4 residues or less, or 3 residues or less. In one embodiment of the production method of the present invention, the linker site contains a plurality of amino acid repeats, the amino acid repeats consist of alanine (A) and proline (P), and the number of consecutive identical amino acid residues in the amino acid repeats can be 6 residues or less, 5 residues or less, 4 residues or less, or 3 residues or less. In one embodiment of the production method of the present invention, the linker site may include two or more repetitions of an amino acid sequence selected from the group consisting of AP, AAP, AS, ASP, and ASS. In one embodiment of the production method of the present invention, the linker site may include a sequence composed of glycine (G) and serine (S). In this case, the linker site may repeatedly include an amino acid sequence selected from the group consisting of GGGGS, GGGS, GGS, and GS. In one embodiment of the production method of the present invention, the linker site may continuously contain glycine and may consist only of glycine. In one embodiment of the production method of the present invention, the linker site may repeatedly include an amino acid sequence represented by EAAAK. In one embodiment of the production method of the present invention, the amino acid sequence of the linker site is preferably, but not limited to, one consisting of A, P, and S, one consisting of A and P, or a repetition of the sequence represented by GGGGS. In one embodiment of the production method of the present invention, the length of the linker site can consist of 50 or fewer amino acid residues. For example, the linker site can consist of 49 or fewer, 40 or fewer, 35 or fewer, 30 or fewer, or 25 or fewer amino acid residues. In one embodiment of the manufacturing method of the present invention, the linker site can consist of one or more amino acid residues. For example, the linker site can consist of 4 or more, 8 or more, 12 or more, or 16 or more amino acid residues.

[0036] In one embodiment of the production method of the present invention, specific examples of the linker site include, but are not limited to, those containing the following amino acid sequences or consisting of the following amino acid sequences. AAPAAPAP (SEQ ID NO: 74), AAPAAPAPAAPAAPAP (SEQ ID NO: 75), AAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPA (SEQ ID NO: 76), SAPSPSSAPASASAPASPASASASASPASSPASASSASPAASSASPASS (SEQ ID NO: 77), AAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAP (SEQ ID NO: 80), AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 7), AAPAAPAPAAPAAPAPAAPAAP (SEQ ID NO: 8), AAPAAPAPAAPAAP (SEQ ID NO: 9), AAPAAAPAPAAPAAPAPAAP (SEQ ID NO: 10), AAAPAAAPAAAPAAAPAAAP (SEQ ID NO: 11), AAPAAPAAPAAPAAPAAPAAPAAP (SEQ ID NO: 12), APAAAPAPAAAPAPAAAPAPAAAP (SEQ ID NO: 13), AAAPAAPAAPPAAAAPAAPAAPPA (SEQ ID NO: 14), and APAPAPAPAPAPAPAPAPAP (SEQ ID NO: 15), ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 16),AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 17), APSSPSPSAPSSPSPASPSS (SEQ ID NO: 18), SAPSSPSPSAPSSPSPASPS (SEQ ID NO: 19), SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 20), AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 21), and ASAAAPAAASAAASAPSAAA (SEQ ID NO: 22), and amino acid sequences selected from these circular permutation variant sequences., Taking the circular permutation variant sequence AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 7) as an example, it can be easily prepared, for example, by removing the first alanine of the above sequence and adding another alanine to the end of the above sequence. Such a circular permutation variant sequence of SEQ ID NO: 7 becomes APAAPAPAAPAAPAPAAPAA (SEQ ID NO: 23). Furthermore, non-limiting examples of circular permutation variant sequences in which the order of amino acid residues is changed circularly for SEQ ID NO: 7 include the following: PAAPAPAAPAAPAPAAPAAA (SEQ ID NO: 24), AAPAPAAPAAPAPAAPAAAP (SEQ ID NO: 25), APAPAAPAAPAPAAPAAAPA (SEQ ID NO: 26), PAPAAPAAPAPAAPAAAPAA (SEQ ID NO: 27), APAAPAAPAPAAPAAAPAAP (SEQ ID NO: 28), PAAPAAPAPAAPAAAPAAPA (SEQ ID NO: 29), AAPAAPAPAAPAAAPAAPAP (SEQ ID NO: 30), APAAPAPAAPAAAPAAPAPA (SEQ ID NO: 31), and PAAPAPAAPAAAPAAPAPAA (SEQ ID NO: 32).

[0037] In one embodiment of the production method of the present invention, the peptide molecule produced by the production method of the present invention may include an additional amino acid sequence consisting of at least one amino acid residue on the opposite side of the first bioactive peptide site via a linker site. This additional amino acid sequence has a molecular weight of 10,000 or less and consists of 50 or fewer amino acid residues. Examples of such additional amino acid sequences include enzyme recognition sequences such as Sortase A recognition signal and Butelase recognition signal, tag sequences such as FLAG tag, PA tag, and histidine tag, reactive amino acids such as lysine, cysteine, glutamic acid, and aspartic acid, or sequences containing such reactive amino acids. When the peptide molecule produced by the production method of the present invention includes an additional amino acid sequence consisting of at least one amino acid residue on the opposite side of the first bioactive peptide site via a linker site, and the additional amino acid sequence is a sequence consisting of or containing a reactive amino acid, the peptide molecule can be used as a conjugate reagent for preparing a bioactive peptide-drug conjugate or the like. The peptide molecule produced by the production method of the present invention may include a second bioactive peptide site and an additional amino acid sequence consisting of at least one amino acid residue on the opposite side of the first bioactive peptide site via a linker site. The peptide molecule produced by the production method of the present invention includes a first bioactive peptide site on the N-terminal side of the linker site, and may include a second bioactive peptide site and / or an additional amino acid sequence on the C-terminal side of the linker site. Further, the peptide molecule produced by the production method of the present invention may include a first bioactive peptide site on the C-terminal side of the linker site, and a second bioactive peptide site and / or an additional amino acid sequence on the N-terminal side of the linker site. Furthermore, the peptide molecule produced by the production method of the present invention may include an arbitrary amino acid sequence on the N-terminal side and / or C-terminal side of the first bioactive peptide site and / or the second bioactive peptide site, or between the linker site, within a range that does not inhibit the functions of the first bioactive peptide and the second bioactive peptide. As such an arbitrary amino acid sequence, for example, a sequence useful for enhancing the expression and secretion of the peptide molecule in host cells can be used. In one embodiment of the production method of the present invention, the produced peptide molecule may further include a functional modification site at a position not adjacent to the linker site. The functional modification site is as described above.

[0038] The production method of the present invention includes a step of producing the peptide molecule by expressing the polynucleotide in a host cell containing the polynucleotide encoding the peptide molecule, or by expressing the polynucleotide encoding the peptide molecule in a cell-free expression system. A host cell containing a polynucleotide encoding a peptide molecule can be prepared by well-known techniques. For example, a host cell containing a polynucleotide encoding a peptide molecule can be prepared by introducing a recombinant vector containing the polynucleotide into the host cell. In one embodiment of the production method of the present invention, the host cell is not particularly limited, and examples include prokaryotic cells (bacteria) such as Escherichia genus (e.g., Escherichia coli), Corynebacterium genus (e.g., Corynebacterium glutamicum), fungal cells (fungi) such as yeast, plant cells, insect cells, and animal cells. For the bacteria of the genus Corynebacterium as a host cell, those described in WO2016 / 171224 can be used. In one embodiment of the production method of the present invention, the expression of the polynucleotide in the host cell may be transient or stable. The culture conditions of the host cell can be appropriately selected by those skilled in the art based on common general knowledge according to the type of host cell used. For example, conditions such as the temperature, humidity, and carbon dioxide concentration of the environment for culturing may be any conditions that enable polynucleotide expression in the host cell, and the culture medium used is not particularly limited as long as it is suitable for each host cell and can be selected from conventionally known ones.

[0039] In one embodiment of the production method of the present invention, the synthesis of peptide molecules in a cell-free expression system may use a cell extract or commercially available reagents for cell-free expression and can be carried out based on techniques well-known to those skilled in the art. Components included in the cell-free expression system include nucleic acids (DNA or mRNA) encoding the desired polypeptide as a template, proteins required for transcription, translation, energy regeneration, and post-translational modification (RNA polymerase, ribosome, aminoacyl-tRNA synthetase, translation initiation factor, translation elongation factor, translation termination factor, ribosome recycling factor, nucleoside diphosphate kinase, adenosine kinase, creatine kinase, prolyl isomerase, disulfide bond isomerase, chaperone, etc.), tRNA, aminoacyl-tRNA, natural amino acids, unnatural amino acids, NTP, buffer, etc. Examples of commercially available reagents for cell-free expression include PUREfrex2.0 (Gene Frontier), PURExpress In Vitro Protein Synthesis Kit (New England BioLabs), etc.

[0040] In the production method of the present invention, when generating a peptide molecule, it may include the steps of introducing a polynucleotide encoding the peptide molecule or a recombinant vector containing the polynucleotide into a host cell, and expressing the polynucleotide in the host cell to generate a peptide molecule. The introduction means in the introduction step can be appropriately selected by those skilled in the art based on common general knowledge according to the type of host cell used. As the vector that can be used, any vector can be used as long as it can functionally insert a polynucleotide encoding a peptide molecule and can transport the fragment into the host cell to bring about replication and / or expression of a heterologous nucleic acid fragment in the host cell. Examples include plasmids, phages, cosmids, and viruses. The vector can be appropriately selected by those skilled in the art according to the type of host cell used.

[0041] In one embodiment of the production method of the present invention, the polynucleotide encoding the peptide molecule can be appropriately synthesized by those skilled in the art using conventional polynucleotide synthesis techniques based on the amino acid sequence of the peptide molecule. Examples of the polynucleotide encoding the peptide molecule include those encoding a peptide molecule having a structure of "first bioactive peptide site"-"linker site"-"second bioactive peptide site" or "first bioactive peptide site"-"linker site"-"added amino acid sequence". A peptide molecule can be generated simply and inexpensively from such a polynucleotide. In one embodiment of the production method of the present invention, the polynucleotide encoding the peptide molecule may appropriately contain a promoter sequence or a signal sequence on its N-terminal side. Further, the polynucleotide may contain a restriction enzyme recognition sequence on the N-terminal side and / or the C-terminal side. Such promoter sequences, signal sequences, and restriction enzyme recognition sequences can be appropriately selected by those skilled in the art according to the type of host cell and vector used. In one embodiment of the production method of the present invention, the produced peptide molecule can be appropriately isolated from the host cell, and can also be purified and / or recovered based on the common general knowledge of those skilled in the art. In one embodiment of the production method of the present invention, after the produced peptide molecule is secreted from the host cell, it can be appropriately isolated based on the common general knowledge of those skilled in the art, and can also be purified and / or recovered.

[0042] In one embodiment of the production method of the present invention, a desired peptide molecule such as a dimer peptide can be expressed from a single polynucleotide chain, and compared with conventional chemical synthesis methods, a desired peptide molecule such as a dimer peptide can be produced simply and inexpensively. In one embodiment of the production method of the present invention, when the first bioactive peptide site and / or the second bioactive peptide site constitute a cyclic peptide, a linear peptide molecule is generated from a single polynucleotide chain, and within the host cell or after secretion from the host cell, it is possible to form a cyclic peptide from the linear peptide molecule. Therefore, regarding the synthesis of a peptide molecule containing a cyclic peptide, many steps were required and it was complicated with conventional chemical synthesis methods, but according to the production method of the present invention, a peptide molecule containing a cyclic peptide can be produced simply and inexpensively. Regarding the formation of the cyclic peptide, a cyclic structure can be formed by a disulfide bond between two cysteine residues, but it is not limited thereto.

[0043] One embodiment of the present invention relates to a polynucleotide for encoding a peptide molecule. The peptide molecule generated by the expression of this polynucleotide may include (I) a first bioactive peptide site, (II) a linker site consisting of amino acid residues, and (III) a second bioactive peptide site and / or an additional amino acid sequence consisting of at least one amino acid residue located on the opposite side of the first bioactive peptide site via the linker site. Here, the first bioactive peptide site and the second bioactive peptide site may be the same as or different from each other. Such a polynucleotide can be appropriately synthesized by those skilled in the art using conventional polynucleotide synthesis techniques based on the amino acid sequence of the generated peptide molecule. One embodiment of the polynucleotide of the present invention may be the same as that described in the manufacturing method of the present invention described above. In one embodiment of the polynucleotide of the present invention, the polynucleotide may be for use in the manufacturing method of the present invention described above. In one embodiment of the polynucleotide of the present invention, the first bioactive peptide site, the linker site consisting of amino acid residues, the second bioactive peptide site, and the additional amino acid sequence consisting of at least one amino acid residue in the peptide molecule generated by the expression of the polynucleotide may be the same as those described in the manufacturing method of the present invention described above.

[0044] One embodiment of the present invention relates not only to the above-mentioned peptide molecule but also to the following peptide molecules. This peptide molecule may include (I) a first bioactive peptide site, (II) a linker site consisting of amino acid residues, and (III) a second bioactive peptide site located on the opposite side of the first bioactive peptide site via the linker site. In one embodiment of the peptide molecule of the present invention, the first bioactive peptide site may be included on the N-terminal side of the linker site, and the second bioactive peptide site may be included on the C-terminal side of the linker site, or the first bioactive peptide site may be included on the C-terminal side of the linker site, and the second bioactive peptide site may be included on the N-terminal side of the linker site. Also, in one embodiment of the peptide molecule of the present invention, within a range that does not inhibit the functions of the first bioactive peptide and the second bioactive peptide, an arbitrary amino acid sequence may be included between the N-terminal side and / or the C-terminal side of the first bioactive peptide site and / or the second bioactive peptide site, or between the linker site. As such an arbitrary amino acid sequence, for example, a sequence useful for enhancing the expression and secretion of the peptide molecule in a host cell may be used. In one embodiment of the peptide molecule of the present invention, the first bioactive peptide site and the second bioactive peptide site each constitute a cyclic peptide that binds to the c-Met protein, and they may be the same or different from each other. Examples of such cyclic peptides that bind to the c-Met (also simply referred to as Met) protein include cyclic peptides that form a cyclic structure by disulfide bonds and contain an amino acid sequence selected from the following (a) to (i).

[0045] (a) CYRQFNRRTHEVWNLDC (SEQ ID NO: 1) (b) CRQFNRRTHEVWNLDC (SEQ ID NO: 2) (c) CYWYYAWDQTYKAFPC (SEQ ID NO: 3) (d) CWYYAWDQTYKAFPC (SEQ ID NO: 4) (e) CYISWNEFNSPNWRFITC (SEQ ID NO: 5) (f) CISWNEFNSPNWRFITC (SEQ ID NO: 6) (g) A peptide in which one or two amino acid residues other than cysteine residues are substituted, deleted, or added in any of the amino acid sequences of (a) to (f) and that binds to the c-Met protein. (h) A peptide that has a sequence identity of 90% or more with any of the amino acid sequences of (a) to (f), consists of an amino acid sequence having cysteine residues at both ends, and binds to the c-Met protein. For example, the sequence identity may be 92% or more, 95% or more, or 98% or more. In the present specification, the amino acid sequence homology can be calculated using a known analysis tool. For example, it can be calculated by using the homology algorithm BLAST (Basic local alignment search tool) of the National Center for Biotechnology Information (NCBI) in the United States. Also, for calculating the amino acid sequence homology, the default (initial setting) parameters in the analysis tool may be used. (i) A peptide in which at least one amino acid other than the cysteine residues at both ends is modified in any of the amino acid sequences of (a) to (h). Here, the modification of the amino acid is phosphorylation, methylation, acetylation, adenylation, ADP ribosylation, or glycosylation. In addition, such a cyclic peptide that binds to Met(HGFR) is known to function as a Met(HGFR) agonist by dimerizing.

[0046] In one embodiment of the peptide molecule of the present invention, the linker site can have a molecular weight of 10,000 or less and consist of 50 or fewer amino acid residues. Also, in one embodiment of the peptide molecule of the present invention, the linker site can be a linker site having a molecular weight of 10,000 or less and consisting of 50 or fewer or 49 or fewer amino acid residues, and at least 90%, at least 95%, or 100% of its amino acid sequence can consist of amino acid residues selected from alanine (A), proline (P), and serine (S). In this case, the proline residue can constitute more than 4% and less than 40% in the amino acid sequence of the linker site. For example, the proline residue may constitute more than about 4%, more than about 5%, more than about 6%, more than about 8%, more than about 10%, more than about 15%, or more than about 20% in the amino acid sequence of the linker site, and less than about 40%, or less than about 3%. Also, the alanine residue and the serine residue can each independently constitute more than about 4%, more than about 10%, or more than about 20%, and less than about 50%. In the amino acid sequence of the linker site, the amino acid residues different from alanine, serine, and proline can be selected from the group consisting of Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val. Such amino acid residues different from alanine, serine, and proline are preferably those having no hydrophobic side chains such as Val, Ile, Leu, Met, Phe, Tyr, or Trp, and / or those having no charged side chains such as Lys, Arg, Asp, or Glu.

[0047] In one embodiment of the peptide molecule of the present invention, the linker site may be a linker site having a molecular weight of 10,000 or less and consisting of 50 or fewer or 49 or fewer amino acid residues, and at least 90%, at least 95%, or 100% of the amino acid sequence thereof may consist of amino acid residues selected from alanine (A) and proline (P). In this case, the proline residues may constitute more than 10% and less than 75% in the amino acid sequence of the linker site. For example, the proline residues may constitute more than about 10%, more than about 12%, more than about 14%, more than about 18%, more than about 20%, more than about 22%, more than about 23%, more than about 24%, or more than about 25% in the amino acid sequence of the linker site, and less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 48%, less than about 46%, less than about 44%, less than about 42%, less than about 41%, less than about 40%, less than about 39%, less than about 38%, less than about 37%, less than about 36%, or less than about 35%. Also, the alanine residues may constitute more than about 25%, more than about 30%, more than about 35%, more than about 40%, more than about 45%, more than about 50%, more than about 52%, more than about 54%, more than about 56%, more than about 58%, more than about 59%, more than about 60%, more than about 61%, more than about 62%, more than about 63%, more than about 64%, or more than about 65% in the amino acid sequence of the linker site, and less than about 90%, less than about 88%, less than about 86%, less than about 84%, less than about 82%, less than about 80%, less than about 79%, less than about 78%, less than about 77%, less than about 76%, or less than about 75%. In the amino acid sequence of the linker site, the amino acid residues different from alanine, serine and proline can be selected from the group consisting of Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr and Val. Such amino acid residues different from alanine, serine and proline are preferably those having no hydrophobic side chains such as Val, Ile, Leu, Met, Phe, Tyr or Trp, and / or those having no charged side chains such as Lys, Arg, Asp or Glu.

[0048] In one embodiment of the peptide molecule of the present invention, the linker site contains a plurality of amino acid repeats, the amino acid repeats consist of alanine (A), proline (P) and serine (S), and the number of consecutive identical amino acid residues in the amino acid repeats can be 6 residues or less, 5 residues or less, 4 residues or less, or 3 residues or less. In one embodiment of the peptide molecule of the present invention, the linker site contains a plurality of amino acid repeats, the amino acid repeats consist of alanine (A) and proline (P), and the number of consecutive identical amino acid residues in the amino acid repeats can be 6 residues or less, 5 residues or less, 4 residues or less, or 3 residues or less. In one embodiment of the peptide molecule of the present invention, the linker site may contain two or more repetitions of an amino acid sequence selected from the group consisting of AP, AAP, AS, ASP, and ASS. In one embodiment of the peptide molecule of the present invention, the linker site may contain a sequence composed of glycine (G) and serine (S). In this case, the linker site may repeatedly contain an amino acid sequence selected from the group consisting of GGGGS, GGGS, GGS, and GS. In one embodiment of the production method of the present invention, the linker site may continuously contain glycine and may consist only of glycine. In one embodiment of the production method of the present invention, the linker site may repeatedly contain an amino acid sequence represented by EAAAK. In one embodiment of the production method of the present invention, the amino acid sequence of the linker site is preferably, but not limited to, one consisting of A, P, and S, one consisting of A and P, or one consisting of a repetition of the sequence represented by GGGGS. In one embodiment of the peptide molecule of the present invention, the length of the linker site can consist of 50 or fewer amino acid residues. For example, the linker site can consist of 49 or fewer, 40 or fewer, 35 or fewer, 30 or fewer, or 25 or fewer amino acid residues. In one embodiment of the manufacturing method of the present invention, the linker site may consist of one or more amino acid residues. For example, the linker site can consist of 4 or more, 8 or more, 12 or more, or 16 or more amino acid residues.

[0049] In one embodiment of the peptide molecule of the present invention, specific examples of the linker site include, but are not limited to, those containing the following amino acid sequences or consisting of the following amino acid sequences. AAPAAPAP (SEQ ID NO: 74), AAPAAPAPAAPAAPAP (SEQ ID NO: 75), AAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPA (SEQ ID NO: 76), SAPSPSSAPASASAPASPASASASASPASSPASASSASPAASSASPASS (SEQ ID NO: 77), AAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAP (SEQ ID NO: 80), AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 7), AAPAAPAPAAPAAPAPAAPAAP (SEQ ID NO: 8), AAPAAPAPAAPAAP (SEQ ID NO: 9), AAPAAAPAPAAPAAPAPAAP (SEQ ID NO: 10), AAAPAAAPAAAPAAAPAAAP (SEQ ID NO: 11), AAPAAPAAPAAPAAPAAPAAPAAP (SEQ ID NO: 12), APAAAPAPAAAPAPAAAPAPAAAP (SEQ ID NO: 13), AAAPAAPAAPPAAAAPAAPAAPPA (SEQ ID NO: 14), and APAPAPAPAPAPAPAPAPAP (SEQ ID NO: 15), ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 16),AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 17), APSSPSPSAPSSPSPASPSS (SEQ ID NO: 18), SAPSSPSPSAPSSPSPASPS (SEQ ID NO: 19), SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 20), AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 21), and ASAAAPAAASAAASAPSAAA (SEQ ID NO: 22), and amino acid sequences selected from the circular permutation variant sequences thereof., The circular permutation variant sequence can be easily prepared, for example, by removing the first alanine of the above sequence and adding another alanine to the end of the above sequence, taking AAPAAPAPAAPAAPAPAAPA (SEQ ID NO: 7) as an example. Such a circular permutation variant sequence of SEQ ID NO: 7 becomes APAAPAPAAPAAPAPAAPAA (SEQ ID NO: 23). Furthermore, non-limiting examples of the circular permutation variant sequences in which the order of amino acid residues is changed circularly in SEQ ID NO: 7 include the following. PAAPAPAAPAAPAPAAPAAA (SEQ ID NO: 24), AAPAPAAPAAPAPAAPAAAP (SEQ ID NO: 25), APAPAAPAAPAPAAPAAAPA (SEQ ID NO: 26), PAPAAPAAPAPAAPAAAPAA (SEQ ID NO: 27), APAAPAAPAPAAPAAAPAAP (SEQ ID NO: 28), PAAPAAPAPAAPAAAPAAPA (SEQ ID NO: 29), AAPAAPAPAAPAAAPAAPAP (SEQ ID NO: 30), APAAPAPAAPAAAPAAPAPA (SEQ ID NO: 31), and PAAPAPAAPAAAPAAPAPAA (SEQ ID NO: 32).

Examples

[0050] Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto. Note that all of the HGFs used in the examples are recombinant human HGF (rhHGF, R&D Systems), and in this specification and the drawings, recombinant human HGF is simply denoted as HGF.

[0051] Reference Example 1: Construction of a truncated vector (pPK10) of the vector pPK6 for amplifying the tatABC gene The pPK6 vector described in WO2016 / 171224 is a vector for amplifying the tatABC gene encoding the Tat secretion apparatus. By removing unnecessary sequences from pPK6, a truncated vector was constructed as follows. First, using pPK6 as a template, the 5' side of the removal region was amplified with the primers described in SEQ ID NO: 33 and SEQ ID NO: 34, and the 3' side was amplified with the primers described in SEQ ID NO: 35 and SEQ ID NO: 36, respectively. The two amplified DNA fragments were introduced into the SalI-SalI site of pPK6 by an infusion reaction to obtain a plasmid from which 548 bases had been removed from pPK6. This plasmid was named pPK6b. Next, using pPK6b as a template, the 5' side of the removal region was amplified with the primers described in SEQ ID NO: 37 and SEQ ID NO: 38, and the 3' side was amplified with the primers described in SEQ ID NO: 39 and SEQ ID NO: 40, respectively. The two amplified DNA fragments were introduced into the ClaI-SpeI site of pPK6b by an infusion reaction to construct a plasmid from which an additional 310 bases had been removed from pPK6b. For the infusion reaction, In-Fusion (registered trademark) HD Cloning Kit (Takara Bio) was used, and the reaction conditions followed the protocol recommended by the manufacturer. In this way, a plasmid was constructed by removing 858 unnecessary bases from the pPK6 vector and truncating it. This vector was named pPK10.

[0052] [Table 1]

[0053] Example 1: Design of the FBP-PAS dimer peptide and its secretory expression in C. glutamicum (1-1) Outline of the design of the FBP-PAS dimer peptide A peptide molecule in which two FGFR1-binding cyclic peptides (FBPs) are linked by a PAS linker is expressed in C. glutamicum as a single polypeptide. Therefore, the peptide sequence was designed such that it would be "the first FBP" - "PAS linker" - "the second FBP (the same sequence as the first FBP)". That is, the C-terminus of the first FBP and the N-terminus of the PAS linker, and the N-terminus of the second FBP and the C-terminus of the PAS linker are linked by amide bonds and can be expressed in C. glutamicum as a single polypeptide chain.

[0054] (1-2) Preparation of FBP-PAS dimer peptide The prepared FBP-PAS dimer peptide is as follows. (a) FBP-PAS RPGCGPRKPRTPKKCGSHGAAPAAPAPAAPAAPAPAAPAGRPGCGPRKPRTPKKCGSH (SEQ ID NO: 41)

[0055] (1-3) Expression of FBP-PAS dimer peptide The expression of the FBP-PAS dimer peptide was investigated using Corynex (registered trademark). Hereinafter, examples of the expression investigation using Corynex (registered trademark) will be described.

[0056] (1-4) Construction of a secretion expression plasmid for FBP-PAS dimer peptide using TorA signal sequence As the FBP-PAS dimer peptide, the amino acid sequence of the above (a) FBP-PAS (hereinafter sometimes referred to as "FBP-PAS") was designed, and the nucleotide sequence encoding this protein was designed in consideration of the codon usage frequency of C. glutamicum. Furthermore, the following expression cassette was designed to enable secretion expression by C. glutamicum. FBP-PAS was secreted and expressed as a fusion protein of the 39-amino acid residue signal peptide of TorA derived from E. coli and FBP-PAS (hereinafter referred to as "TorAss-FBP-PAS"). The nucleotide sequence and amino acid sequence encoding the designed TorAss-FBP-PAS are shown in SEQ ID NO: 42 and SEQ ID NO: 43, respectively.

[0057] Nucleotide sequence encoding TorAss-FBP-PAS atgaacaataacgatctctttcaggcatcacgtcggcgttttctggcacaactcggcggcttaaccgtcgccgggatgctggggccgtcattgttaacgccgcgacgtgcgactgcgcgtcctggttgtggtcctcgcaagccccgcacccctaagaaatgtggttctcacggcgccgctcctgccgcacccgcacccgctgctccagcagccccagccccagctgcaccagcaggtcgcccaggctgtggtccccgcaaaccccgcacccctaagaaatgtggctctcactaa(SEQ ID NO: 42) Amino acid sequence of TorAss-FBP-PAS MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATARPGCGPRKPRTPKKCGSHGAAPAAPAPAAPAAPAPAAPAGRPGCGPRKPRTPKKCGSH(SEQ ID NO: 43)

[0058] The promoter of the cspB gene derived from the C. glutamicum ATCC13869 strain was ligated upstream of the nucleotide sequence described in TorAss-FBP-PAS, and a KpnI site was added to the 5'-side and an ApaI site was added to the 3'-side. An expression cassette of TorAss-FBP-PAS was designed and fully synthesized. The fully synthesized DNA fragment (expression cassette of TorAss-FBP-PAS) was inserted into the KpnI-ApaI site of pPK10 described in Reference Example 1 to construct pPK10_TorAss-FBP-PAS, a secretion expression plasmid for the FBP-PAS dimer peptide using the TorA signal sequence. As a result of nucleotide sequencing of the inserted fragment, it was confirmed that the expression cassette of TorAss-FBP-PAS was constructed as designed. Nucleotide sequencing was performed using the BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the 3130 Genetic Analyzer (Applied Biosystems).

[0059] (1-5) Secretion expression of the FBP-PAS dimer peptide in C. glutamicum Using the pPK10_TorAss-FBP-PAS constructed above, the C. glutamicum YDK0107 strain described in WO2016 / 171224 was transformed to obtain the YDK0107 / pPK10_TorAss-FBP-PAS strain. The obtained transformant was cultured at 30 °C for 72 hours in MMTG liquid medium (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, ferrous sulfate heptahydrate 0.03 g, manganese sulfate pentahydrate 0.03 g, thiamine hydrochloride 0.45 mg, biotin 0.45 mg, DL-methionine 0.15 g, soy hydrochloric acid hydrolyzate (total nitrogen content 0.2 g), calcium carbonate 50 g, adjusted to pH 7.0 with water to 1 L) containing 25 mg / L of kanamycin. After completion of the culture, 6.5 μL of the culture supernatant obtained by centrifuging each culture solution was subjected to reducing SDS-PAGE using NuPAGE (registered trademark) 12% Bis-Tirs Gel (Thermo Fisher Scientific), and then stained with Quick-CBB (Wako). As a result, a protein band presumed to be FBP-PAS was detected in the culture supernatant of the YDK0107 / pPK10_TorAss-FBP-PAS strain (Figure 1, lanes 2 to 5).

[0060] (1-6) Purification of FBP-PAS dimer peptide The culture supernatant containing FBP-PAS obtained above was purified by dialysis in PBS (Mg- / Ca-) buffer for 2 days using a dialysis membrane (MWCO = 3.5 kDa, Spectrum).

[0061] Example 2: Confirmation of the molecular weight and disulfide cyclic structure of FBP-PAS dimer peptide 5 mM of Tris(2-carboxyethyl)phosphine Hydrochloride (TCEP-HCl) was added to the FBP-PAS dimer peptide solution and incubated at room temperature for 48 hours to reduce the disulfide bonds. α-Cyano-4-hydroxycinnamic Acid (CHCA) was mixed with FBP-PAS and the FBP-PAS dimer peptide treated with TCEP-HCl, respectively, and the molecular weights were measured by MALDI-TOF-MS (Shimadzu, AXIMA-TOF2). As shown in Figure 2, a value close to the molecular weight (m / z 5621) of the FBP-PAS dimer peptide that forms two intramolecular disulfide bonds before reduction with TCEP-HCl was obtained. Also, a value close to the molecular weight (m / z 5625) after reduction with TCEP-HCl where all disulfide bonds were reduced was obtained. From these results, it was confirmed that the FBP-PAS dimer peptide expressed by Corynex (registered trademark) has the designed peptide sequence and further has a structure containing two intramolecular disulfide bonds.

[0062] Example 3: Evaluation of FGFR inhibitory activity of FBP-PAS dimer peptide The inhibitory activities of the FBP-PAS dimer peptide and the chemically synthesized FBP monomer peptide against FGFR were evaluated by a luciferase assay. To 25 μL of Opti-MEM medium (Thermo Fisher Scientific), 0.6 μL of Attractene Transfection Reagent (QIAGEN) was added and incubated at room temperature for 5 minutes. To the above mixed solution, a mixed solution of 25 μL of Opti-MEM and 1 μL of SRE reporter vector (QIAGEN) was added and incubated at room temperature for 20 minutes. This mixed solution was added to a 96-well plate, and 40,000 cells / well of HEK293E cells were seeded on it and incubated overnight in a 37 °C, 5% CO2 incubator. After transfection, all the culture supernatants were removed, and 100 μL of Opti-MEM medium (evaluation basal medium) containing 0.5% FBS (Thermo Fisher Scientific), 1% non-essential amino acid solution (Thermo Fisher Scientific), and penicillin-streptomycin (Nacalai Tesque) was added and cultured at 37 °C for 4 hours to make the cells in a starved state. 10 μM of the FBP-PAS dimer peptide or the FBP monomer peptide was added to the evaluation basal medium containing 0 - 10 ng / mL of bFGF, and 100 μL was added to the cells. The cells were stimulated by culturing overnight in a 37 °C, 5% CO2 incubator. For the detection of signal intensity, the Dual-Luciferase Reporter Assay System (Promega) was used. 100 μL of the culture supernatant was removed, 50 μL of Glo reagent was added, and the cells were lysed at room temperature for 10 minutes. The activation of the FGFR-Erk-SRE pathway was quantified by detecting the luminescence of Firefly luciferase emitted from the cell lysate using a plate reader. Subsequently, 50 μL of the Glo & Stop reagent solution was added, and after 10 minutes, the luminescence of Renilla luciferase, which is an internal standard, was detected to quantify the cell number. The signal activity of each sample was quantified as SRE activity = (luminescence intensity of Firefly luciferase) / (luminescence intensity of Renilla luciferase). The relative signal activity value with respect to the sample without the test compound was determined and defined as the Relative reporter activity. As shown in Fig. 3, it was revealed that the FBP-PAS dimer peptide strongly inhibits the FGFR signal by the same amount of bFGF compared to the FBP monomer peptide. From these results, it was clarified that the FBP-PAS dimer peptide inhibits FGFR and has higher inhibitory activity compared to the FBP monomer peptide.

[0063] Example 4: Design of HGF-PAS dimer peptide and its secretory expression in C. glutamicum (4-1) Outline of the design of HGF-PAS dimer peptide The peptide dimer molecule obtained by dimerizing the Met-binding cyclic peptide aMD4 reported by K. Ito, et al. in Nat. Commun. 6, 6373, 2015 with a PEG linker cannot be expressed in C. glutamicum as a single polypeptide chain for the following reasons. (1) aMD4 is cyclized by an intramolecular thioether bond formed by the reaction of a chloroacetyl group added to the amino group at the N-terminus and the sulfhydryl group of the cysteine side chain in the sequence. However, the thioether bond cannot be expressed in C. glutamicum. (2) Dimerization is achieved by binding the C-termini of the cyclic peptides using a Bis-Maleimide-PEG linker, which cannot be expressed in C. glutamicum. Therefore, the molecule was designed to be in an expressible form by applying the following conversions. Regarding (1) above, the thioether bond was converted to a disulfide bond formed by the sulfhydryl groups of two cysteine side chains. Although the cyclization site becomes two atoms longer when converted to a disulfide bond, it was designed so that the cyclization site becomes one atom shorter by deleting the residue next to the cysteine on the N-terminal side (tyrosine in the case of aMD4). Regarding (2) above, a PAS sequence was used instead of the PEG linker, and the peptide sequence was designed to be "First Disulfide-Converted aMD4" - "PAS Linker" - "Second Disulfide-Converted aMD4" so that it can be expressed as a single polypeptide chain. That is, the C-terminus of the first disulfide-converted aMD4 and the N-terminus of the PAS linker, and the N-terminus of the second disulfide-converted aMD4 and the C-terminus of the PAS linker are linked by amide bonds and can be expressed as a single polypeptide chain.

[0064] (4-2) Preparation of HGF-PAS Dimer Peptide The prepared HGF-PAS dimer peptide is as follows. (b) PAS-aMD4-PEG11 CYRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPAPAAPAAPGCYRQFNRRTHEVWNLDC (SEQ ID NO: 44) (c) PAS-aMD4dY-PEG11 CRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPAPAAPAAPGCRQFNRRTHEVWNLDC (SEQ ID NO: 45) (d)PAS-aMD4-PEG3 CYRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPGCYRQFNRRTHEVWNLDC (SEQ ID NO: 46) (e)AET-PAS-aMD4-PEG11 AETCYRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPAPAAPAAPGCYRQFNRRTHEVWNLDC (SEQ ID NO: 47) (f)AET-PAS-aMD4dY-PEG11 AETCRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPAPAAPAAPGCRQFNRRTHEVWNLDC (SEQ ID NO: 48) (g)AET-PAS-aMD4-PEG3 AETCYRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPGCYRQFNRRTHEVWNLDC (SEQ ID NO: 49)

[0065] (4-3) Expression of HGF-PAS dimer peptide The expression of these HGF-PAS dimer peptides was examined using Corynex (registered trademark). Hereinafter, examples of the expression examination using Corynex (registered trademark) will be described.

[0066] (4-4) Construction of secretion expression plasmids of PAS-aMD4-PEG11, PAS-aMD4dY-PEG11, and PAS-aMD4-PEG3 using TorA signal sequence As HGF-PAS dimer peptides, three types of amino acid sequences of (b) PAS-aMD4-PEG11 (hereinafter sometimes referred to as "PAS-aMD4-PEG11"), (c) PAS-aMD4dY-PEG11 (hereinafter sometimes referred to as "PAS-aMD4dY-PEG11"), and (d) PAS-aMD4-PEG3 (hereinafter sometimes referred to as "PAS-aMD4-PEG3") were designed respectively, and the nucleotide sequences encoding these proteins were designed considering the codon usage frequency of C. glutamicum. Furthermore, the following expression cassettes were designed respectively to enable secretion expression by C. glutamicum. PAS-aMD4-PEG11 was secreted and expressed as a fusion protein of a 39-amino acid residue signal peptide of TorA derived from E. coli and PAS-aMD4-PEG11 (hereinafter referred to as "TorAss-PAS-aMD4-PEG11"). The nucleotide sequence and amino acid sequence encoding the designed TorAss-PAS-aMD4-PEG11 are shown in SEQ ID NO: 50 and 51 respectively.

[0067] Nucleotide sequence encoding TorAss-PAS-aMD4-PEG11 atgaacaataacgatctctttcaggcatcacgtcggcgttttctggcacaactcggcggcttaaccgtcgccgggatgctggggccgtcattgttaacgccgcgacgtgcgactgcgtgttaccgtcaattcaaccgtcgcacccacgaagtttggaacctggactgtggcgcagcacccgcagccccagcccccgcagcacctgcagccccagcaccagctgcaccagccgctccaggctgctaccgtcagttcaaccgccgcacccacgaagtttggaatttggattgctaa (SEQ ID NO: 50) Amino acid sequence of TorAss-PAS-aMD4-PEG11 MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATACYRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPAPAAPAAPGCYRQFNRRTHEVWNLDC (SEQ ID NO: 51)

[0068] PAS-aMD4dY-PEG11 was secreted and expressed as a fusion protein of the 39 amino acid residues of the signal peptide of TorA derived from E. coli and PAS-aMD4dY-PEG11 (hereinafter referred to as "TorAss-PAS-aMD4dY-PEG11"). The nucleotide sequence and amino acid sequence encoding the designed TorAss-PAS-aMD4dY-PEG11 are shown in SEQ ID NO: 52 and SEQ ID NO: 53, respectively.

[0069] Nucleotide sequence encoding TorAss-PAS-aMD4dY-PEG11 atgaacaataacgatctctttcaggcatcacgtcggcgttttctggcacaactcggcggcttaaccgtcgccgggatgctggggccgtcattgttaacgccgcgacgtgcgactgcgtgccgtcagttcaaccgtcgcacccacgaagtttggaatctggactgtggcgcagcccccgcagcacccgctcccgcagcacccgcagctccagcaccagcagccccagcagcaccaggctgccgccagttcaaccgtcgcacccacgaagtttggaatttggattgttag (SEQ ID NO: 52) Amino acid sequence of TorAss-PAS-aMD4dY-PEG11 MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATACRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPAPAAPAAPGCRQFNRRTHEVWNLDC (SEQ ID NO: 53)

[0070] PAS-aMD4-PEG3 was secreted and expressed as a fusion protein of the 39 amino acid residues of the signal peptide of TorA derived from E. coli and PAS-aMD4-PEG3 (hereinafter referred to as "TorAss-PAS-aMD4-PEG3"). The nucleotide sequence and amino acid sequence encoding the designed TorAss-PAS-aMD4-PEG3 are shown in SEQ ID NO: 54 and SEQ ID NO: 55, respectively.

[0071] Nucleotide sequence encoding TorAss-PAS-aMD4-PEG3 atgaacaataacgatctctttcaggcatcacgtcggcgttttctggcacaactcggcggcttaaccgtcgccgggatgctggggccgtcattgttaacgccgcgacgtgcgactgcgtgttaccgtcagttcaaccgccgtacccacgaagtctggaacctggattgtggtgccgcccccgccgctcccgctcccgccgctccggcagcccctggctgctaccgtcagtttaaccgtcgcacccacgaagtctggaacctggattgctaa(SEQ ID NO: 54) Amino acid sequence of TorAss-PAS-aMD4-PEG3 MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATACYRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPGCYRQFNRRTHEVWNLDC(SEQ ID NO: 55)

[0072] Upstream of the nucleotide sequences described in TorAss-PAS-aMD4-PEG11, TorAss-PAS-aMD4dY-PEG11, and TorAss-PAS-aMD4-PEG3, a promoter of the cspB gene derived from the C. glutamicum ATCC13869 strain was linked, and a KpnI site was added to the 5'-side and an ApaI site was added to the 3'-side. Expression cassettes for each HGF-PAS dimer peptide were designed and fully synthesized. The fully synthesized DNA fragment (expression cassette for the HGF-PAS dimer peptide) was inserted into the KpnI-ApaI site of pPK10 described in Reference Example 1, whereby pPK10_TorAss-PAS-aMD4-PEG11, pPK10_TorAss-PAS-aMD4dY-PEG11, and pPK10_TorAss-PAS-aMD4-PEG3, which are secretion expression plasmids for each HGF-PAS dimer peptide utilizing the TorA signal sequence, were each constructed. As a result of nucleotide sequencing of the inserted fragment, it was confirmed that expression cassettes for the HGF-PAS dimer peptide as designed were constructed. Nucleotide sequencing was performed using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the 3130 Genetic Analyzer (Applied Biosystems).

[0073] (4-5) Secretion expression of each HGF-PAS dimer peptide in C. glutamicum Using pPK10_TorAss-PAS-aMD4-PEG11, pPK10_TorAss-PAS-aMD4dY-PEG11, and pPK10_TorAss-PAS-aMD4-PEG3 constructed above, the C. glutamicum YDK0107 strain described in WO2016 / 171224 was transformed to obtain the YDK0107 / pPK10_TorAss-PAS-aMD4-PEG11 strain, the YDK0107 / pPK10_TorAss-PAS-aMD4dY-PEG11 strain, and the YDK0107 / pPK10_TorAss-PAS-aMD4-PEG3 strain. Each of the obtained transformants was cultured at 30°C for 72 hours in MMTG liquid medium (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, ferrous sulfate heptahydrate 0.03 g, manganese sulfate pentahydrate 0.03 g, thiamine hydrochloride 0.45 mg, biotin 0.45 mg, DL-methionine 0.15 g, soy hydrochloric acid hydrolysate (total nitrogen content 0.2 g), calcium carbonate 50 g, adjusted to 1 L with water and pH 7.0) containing 25 mg / L of kanamycin. After completion of the culture, 6.5 μL of the culture supernatant obtained by centrifuging each culture solution was subjected to reducing SDS-PAGE using NuPAGE (registered trademark) 12% Bis-Tirs Gel (Thermo Fisher Scientific), and then stained with Quick-CBB (Wako). As a result, protein bands presumed to be PAS-aMD4-PEG11 were detected in the culture supernatant of the YDK0107 / pPK10_TorAss-PAS-aMD4-PEG11 strain (Figure 4, lanes 2-5), protein bands presumed to be PAS-aMD4dY-PEG11 were detected in the culture supernatant of the YDK0107 / pPK10_TorAss-PAS-aMD4dY-PEG11 strain (Figure 4, lanes 6-9), and protein bands presumed to be PAS-aMD4-PEG3 were detected in the culture supernatant of the YDK0107 / pPK10_TorAss-PAS-aMD4-PEG3 strain (Figure 4, lanes 10-13).

[0074] Construction of each secretion expression plasmid of AET-PAS-aMD4-PEG11, AET-PAS-aMD4dY-PEG11, and AET-PAS-aMD4-PEG3 using the (4-6)CspB signal sequence As HGF-PAS dimeric peptides, the three amino acid sequences of (e) AET-PAS-aMD4-PEG11 (hereinafter sometimes referred to as "AET-PAS-aMD4-PEG11"), (f) AET-PAS-aMD4dY-PEG11 (hereinafter sometimes referred to as "AET-PAS-aMD4dY-PEG11"), and (g) AET-PAS-aMD4-PEG3 (hereinafter sometimes referred to as "AET-PAS-aMD4-PEG3") were designed respectively, and the nucleotide sequences encoding these proteins were designed considering the codon usage frequency of C. glutamicum. Furthermore, the following expression cassettes were designed respectively to enable secretory expression by C. glutamicum. AET-PAS-aMD4-PEG11 was secreted and expressed as a fusion protein of the 30 amino acid residues of the signal peptide of CspB derived from C. glutamicum ATCC13869 strain and AET-PAS-aMD4-PEG11 (hereinafter referred to as "CspBss-AET-PAS-aMD4-PEG11"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AET-PAS-aMD4-PEG11 are shown in SEQ ID NO: 56 and 57 respectively.

[0075] Nucleotide sequence encoding CspBss-AET-PAS-aMD4-PEG11 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagacctgttaccgtcaattcaaccgtcgcacccacgaagtttggaacctggactgtggcgcagcacccgcagccccagcccccgcagcacctgcagccccagcaccagctgcaccagccgctccaggctgctaccgtcagttcaaccgccgcacccacgaagtttggaatttggattgctaa (SEQ ID NO: 56) Amino acid sequence of CspBss-AET-PAS-aMD4-PEG11 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETCYRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPAPAAPAAPGCYRQFNRRTHEVWNLDC (SEQ ID NO: 57)

[0076] AET-PAS-aMD4dY-PEG11 was secreted and expressed as a fusion protein of the 30 amino acid residues of the signal peptide of CspB derived from C. glutamicum ATCC13869 strain and AET-PAS-aMD4dY-PEG11 (hereinafter referred to as "CspBss-AET-PAS-aMD4dY-PEG11"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AET-PAS-aMD4dY-PEG11 are shown in SEQ ID NO: 58 and 59, respectively.

[0077] Nucleotide sequence encoding CspBss-AET-PAS-aMD4dY-PEG11 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagacctgccgtcagttcaaccgtcgcacccacgaagtttggaatctggactgtggcgcagcccccgcagcacccgctcccgcagcacccgcagctccagcaccagcagccccagcagcaccaggctgccgccagttcaaccgtcgcacccacgaagtttggaatttggattgttag (SEQ ID NO: 58) Amino acid sequence of CspBss-AET-PAS-aMD4dY-PEG11 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETCRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPAPAAPAAPGCRQFNRRTHEVWNLDC (SEQ ID NO: 59)

[0078] AET-PAS-aMD4-PEG3 was secreted and expressed as a fusion protein of the 30 amino acid residues of the signal peptide of CspB derived from the C. glutamicum ATCC13869 strain and AET-PAS-aMD4-PEG3 (hereinafter referred to as "CspBss-AET-PAS-aMD4-PEG3"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AET-PAS-aMD4-PEG3 are shown in SEQ ID NO: 60 and SEQ ID NO: 61, respectively.

[0079] Nucleotide sequence encoding CspBss-AET-PAS-aMD4-PEG3 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagacctgttaccgtcagttcaaccgccgtacccacgaagtctggaacctggattgtggtgccgcccccgccgctcccgctcccgccgctccggcagcccctggctgctaccgtcagtttaaccgtcgcacccacgaagtctggaacctggattgctaa (SEQ ID NO: 60) Amino acid sequence of CspBss-AET-PAS-aMD4-PEG3 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETCYRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPGCYRQFNRRTHEVWNLDC (SEQ ID NO: 61)

[0080] The promoter of the cspB gene derived from the C. glutamicum ATCC13869 strain was linked upstream of the nucleotide sequences described in AET-PAS-aMD4-PEG11, AET-PAS-aMD4dY-PEG11, and AET-PAS-aMD4-PEG3. Furthermore, a KpnI site was added to the 5'-side and a BamHI site was added to the 3'-side, and the expression cassettes of each HGF-PAS dimer peptide were designed and fully synthesized. The fully synthesized DNA fragment (expression cassette of the HGF-PAS dimer peptide) was inserted into the KpnI-BamHI site of pPK4 described in JP-A-9-322774, thereby constructing pPK4_CspBss-AET-PAS-aMD4-PEG11, pPK4_CspBss-AET-PAS-aMD4dY-PEG11, and pPK4_CspBss-AET-PAS-aMD4-PEG3, which are secretion expression plasmids of each HGF-PAS dimer peptide using the CspB signal sequence. As a result of nucleotide sequence determination of the inserted fragment, it was confirmed that the expression cassettes of the HGF-PAS dimer peptides as designed were constructed. The nucleotide sequence determination was performed using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the 3130 Genetic Analyzer (Applied Biosystems).

[0081] (4-7) Secretion expression of each HGF-PAS dimer peptide in C. glutamicum Using pPK4_CspBss-AET-PAS-aMD4-PEG11, pPK4_CspBss-AET-PAS-aMD4dY-PEG11, and pPK4_CspBss-AET-PAS-aMD4-PEG3 constructed above, the C. glutamicum YDK010::phoS(W302C) strain described in WO2016 / 171224 was transformed to obtain the YDK010::phoS(W302C) / pPK4_CspBss-AET-PAS-aMD4-PEG11 strain, the YDK010::phoS(W302C) / pPK4_CspBss-AET-PAS-aMD4dY-PEG11 strain, and the YDK010::phoS(W302C) / pPK4_CspBss-AET-PAS-aMD4-PEG3 strain. Each of the obtained transformants was cultured in MMTG liquid medium (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, iron (III) sulfate heptahydrate 0.03 g, manganese (II) sulfate pentahydrate 0.03 g, thiamine hydrochloride 0.45 mg, biotin 0.45 mg, DL-methionine 0.15 g, soybean hydrochloric acid hydrolysate (total nitrogen content 0.2 g), calcium carbonate 50 g, adjusted to pH 7.0 with water to 1 L) containing 25 mg / L kanamycin at 30°C for 72 hours. After completion of the culture, 6.5 μL of the culture supernatant obtained by centrifuging each culture solution was subjected to reducing SDS-PAGE using NuPAGE (registered trademark) 12% Bis-Tirs Gel (Thermo Fisher Scientific), followed by staining with Quick-CBB (Wako). As a result, protein bands presumably corresponding to AET-PAS-aMD4-PEG11 were detected in the culture supernatant of the YDK010::phoS(W302C) / pPK4_CspBss-AET-PAS-aMD4-PEG11 strain (Figure 5, lanes 2-5), protein bands presumably corresponding to AET-PAS-aMD4dY-PEG11 were detected in the culture supernatant of the YDK010::phoS(W302C) / pPK4_CspBss-AET-PAS-aMD4dY-PEG11 strain (Figure 5, lanes 6-9), and protein bands presumably corresponding to AET-PAS-aMD4-PEG3 were detected in the culture supernatant of the YDK010::phoS(W302C) / pPK4_CspBss-AET-PAS-aMD4-PEG3 strain (Figure 5, lanes 10-13).

[0082] (4-8) Purification of HGF-PAS dimer peptide Purification of the HGF-PAS dimer peptide obtained above was performed using an AKTA explorer (GE Healthcare) and a Superdex 75 10 / 300 GL column (GE Healthcare). 1 mL of the culture supernatant was injected into the AKTA explorer, and the protein was fractionated by flowing PBS (10 mM phosphate buffer, 140 mM NaCl, pH 7.4) at a flow rate of 0.5 mL / min to obtain the target HGF-PAS dimer peptide fraction.

[0083] Example 5: Molecular weight analysis of HGF-PAS dimer peptide AET-PAS-aMD4dY-PEG11, AET-PAS-aMD4-PEG11, and AET-PAS-aMD4-PEG3 were each mixed with α-Cyano-4-hydroxycinnamic Acid (CHCA), and the molecular weights were measured using MALDI-TOF-MS (Shimadzu, AXIMA-TOF2). As shown in Fig. 6, values almost identical to the theoretical molecular weights of the oxidized dimeric peptides each forming two disulfide bonds within the molecule (AET-PAS-aMD4dY-PEG11: m / z 6320, AET-PAS-aMD4-PEG11: m / z 6646, AET-PAS-aMD4-PEG3: m / z 6000) were obtained. It was confirmed that peptides with the correct amino acid sequences containing two disulfide bonds were expressed.

[0084] Example 6: Activity Evaluation of HGF-PAS Dimeric Peptide The Met activation ability of the HGF-PAS dimeric peptide was evaluated by a luciferase assay. To 25 μL of Opti-MEM medium (Thermo Fisher Scientific), 0.6 μL of Attractene Transfection Reagent (QIAGEN) was added and incubated at room temperature for 5 minutes. To the above mixed solution, a mixed solution of 25 μL of Opti-MEM and 1 μL of SRE reporter vector (QIAGEN) was added and incubated at room temperature for 20 minutes. This mixed solution was added to a 96-well plate, and 40,000 cells / well of HEK293E cells were seeded on it and incubated overnight in a 37 °C, 5% CO2 incubator. After transfection, all the culture supernatants were removed, and 100 μL of Opti-MEM medium (evaluation basal medium) containing 0.5% FBS (Thermo Fisher Scientific), 1% non-essential amino acid solution (Thermo Fisher Scientific), and penicillin-streptomycin (Nacalai Tesque) was added and cultured at 37 °C for 4 hours to make the cells in a starvation state. To the evaluation basal medium, 0 - 200 ng / mL of HGF, or 0 - 800 ng / mL of AET-PAS-aMD4dY-PEG11, AET-PAS-aMD4-PEG11, or AET-PAS-aMD4-PEG3, or culture supernatant containing PAS-aMD4dY-PEG11, PAS-aMD4-PEG11, or PAS-aMD4-PEG3 diluted 10-fold with respect to the medium was added, and 100 μL of the evaluation basal medium was added to the cells. Stimulation of the cells was performed by culturing overnight in a 37 °C, 5% CO2 incubator. For the detection of signal intensity, the Dual-Luciferase Reporter Assay System (Promega) was used. 100 μL of the culture supernatant was removed, 50 μL of Glo reagent was added, and the cells were lysed at room temperature for 10 minutes. Activation of the HGF-Erk-SRE pathway was quantified by detecting the luminescence of Firefly luciferase emitted from the cell lysate using a plate reader. Subsequently, 50 μL of the Glo & Stop reagent solution was added, and after 10 minutes, the luminescence of Renilla luciferase, which is an internal standard, was detected to quantify the number of cells. The signal activity of each sample was quantified as SRE activity = (luminescence intensity of Firefly luciferase) / (luminescence intensity of Renilla luciferase). The relative signal activity value with respect to the sample without the evaluation compound was determined and used as the relative receptor activity (Relative reporter activity). As shown in Figures 7 - 9, it was revealed that all HGF-PAS dimer peptides have the ability to activate signals. In particular, it was revealed that AET-PAS-aMD4dY-PEG11 and PAS-aMD4dY-PEG11 have maximum activity comparable to 100 ng / mL HGF.

[0085] Example 7: Specificity Evaluation of HGF-PAS Dimer Peptides The receptor tyrosine kinase (RTK) specificity of the HGF-PAS dimer peptide was evaluated using the Proteome Profiler Human Phospho-RTK Array Kit (R&D Systems). Confluent HEK293E cells in a 10 cm cell culture dish were starved by culturing them for 24 hours in D-MEM medium containing 0.5% FBS (Thermo Fisher Scientific) and penicillin-streptomycin (Nacalai Tesque). The starved HEK293E cells were stimulated with 200 ng / mL of AET-PAS-aMD4dY-PEG11 for 10 minutes. As a positive control, stimulation was similarly performed with 100 ng / mL of HGF for 10 minutes. As a negative control, stimulation was performed by adding PBS for 10 minutes (Mock group). After removing the medium and washing the cells once with PBS, the cells were lysed by adding 1 mL of Lysis buffer 17 supplemented with Protease inhibitor cocktail (Nacalai Tesque). The lysate was centrifuged at 14,000 G for 5 minutes, and the supernatant was obtained as the lysate. The lysate was analyzed using the Proteome Profiler Human Phospho-RTK Array Kit. The Molecular Imager ChemiDoc XRS System (Bio-Rad) was used for chemiluminescence detection. As shown in Fig. 10, it was confirmed that all RTKs in the cells stimulated with PBS (Mock group) were in the dephosphorylated state. Also, when stimulated with HGF, it was confirmed that Met, the receptor for HGF, was specifically phosphorylated. When stimulation was applied with AET-PAS-aMD4dY-PEG11, Met was specifically phosphorylated, similar to HGF. From these results, it became clear that AET-PAS-aMD4dY-PEG11 performs Met-specific activation. Note that Fig. 11 shows each array of the Proteome Profiler Human Phospho-RTK Array Kit (R&D Systems).

[0086] Example 8: Evaluation of the cell growth promoting activity of the HGF-PAS dimer peptide It has been reported that HGF promotes cell proliferation by activating Met (K. Ito, et al. Nat. Commun. 6, 6373, 2015). Therefore, an evaluation was performed using HuCCT1 cell culture to determine whether the HGF-PAS dimer peptide has an activity to promote cell proliferation similar to that of HGF. RPMI1640 medium (Nacalai Tesque) supplemented with 5% FBS was added to a 24-well cell culture plate, and HuCCT1 cells were seeded at 5,000 cells / well. 35 ng / mL HGF or 40 ng / mL AET-PAS-aMD4dY-PEG11 was added, and the cells were cultured for 5 days under the conditions of 37 °C and 5% CO2. The cells were detached with 0.25% Trypsin / EDTA (Thermo Fisher Scientific), and the cell count was quantified using a Countess Automated Cell Counter (Thermo Fisher Scientific) (N = 3). As shown in Fig. 12, the cells cultured with the addition of HGF or AET-PAS-aMD4dY-PEG11 both showed a proliferation ability about 2.5 times higher than that of the non-addition group (Mock). From this, it was clarified that AET-PAS-aMD4dY-PEG11, like HGF, promotes cell proliferation.

[0087] Example 9: Evaluation of the migration activity of the HGF-PAS dimer peptide It has been reported that HGF promotes cell migration ability by activating Met (K. Ito, et al. Nat. Commun. 6, 6373, 2015). Therefore, an evaluation was performed using HuCCT1 cell culture to determine whether the HGF-PAS dimer peptide has an activity to promote cell migration similar to that of HGF. 20,000 HuCCT1 cells suspended in low-serum medium (RPMI 1640 medium supplemented with 0.5% FBS) were added to a 24-well Transwell insert (pore size 3.0 μm, Corning). Low-serum medium supplemented with 100 ng / mL HGF or 200 ng / mL AET-PAS-aMD4dY-PEG11 was added to the lower layer. After culturing for 24 hours under the conditions of 37 °C and 5% CO2, the Transwell insert was washed three times with PBS, and the cells were fixed and stained with methanol containing 0.5% Crystal Violet (Nacalai Tesque) for 10 minutes. The fixed and stained Transwell insert was washed with distilled water. The cells inside the insert were removed with a scraper. The cells that had migrated outside the insert and were stained were photographed in the bright-field observation mode of a BZ-X microscope (Keyence). Figure 13 shows the bright-field image of the lower part of the Transwell insert stained with Crystal Violet and the quantitative value of the migration ability. The migration ability was calculated as (the area of the stained region) / (the insert area), and the relative value (Relative migration efficiency) was calculated with respect to the value of the non-added group (Mock) (N = 2). The results are shown in Figure 14. When cultured with the addition of HGF or AET-PAS-aMD4dY-PEG11, the proportion of cells migrating to the lower part of the Transwell insert increased, indicating that AET-PAS-aMD4dY-PEG11 promotes cell migration in the same way as HGF.

[0088] Example 10: Evaluation of the wound healing effect of the HGF-PAS dimer peptide It has been reported that HGF promotes the wound healing ability of cells by activating Met (K. Ito, et al. Nat. Commun. 6, 6373, 2015). Therefore, it was evaluated using HuCCT1 cell culture whether the HGF-PAS dimer peptide promotes wound healing in the same way as HGF. HuCCT1 cells suspended in RPMI1640 medium supplemented with 10% FBS were added to each well of a 96-well cell culture plate at a density of 50,000 cells per well and incubated overnight at 37 °C under 5% CO2. Cells at the center of the culture well were scraped off using a 1000 μL Thermo Scientific ART tip (Thermo Fisher Scientific) and washed with low serum medium (RPMI1640 medium supplemented with 0.5% FBS). Low serum medium supplemented with 33 ng / mL of HGF or 100 ng / mL of AET-PAS-aMD4dY-PEG11 was added, and the cells were cultured at 37 °C under 5% CO2. For cell observation, BioStudio-T (Nikon Corporation) was used to automatically acquire cell images every hour. The wound healing rate (μm / h) was calculated as ((the width of the wound 1 hour after wound creation) - (the width of the wound 7 hours after wound creation)) / 6 (N = 4). Figure 15 shows the phase-contrast images of the cells 1 hour and 9 hours after wound creation, and Figure 16 shows the wound healing rate. Since the wound healing rate of the cells treated with HGF or AET-PAS-aMD4dY-PEG11 was improved by about 1.6 times, it was revealed that AET-PAS-aMD4dY-PEG11, like HGF, improves the early morning healing ability of cells.

[0089] Example 11: Evaluation of the effect of HGF-PAS dimer peptide on tubular structure formation It is known that HGF can induce the formation of tubular structures by acting on HUVEC cells and HuCCT1 cells encapsulated in collagen gel (K. Ito, et al. Nat. Commun. 6, 6373, 2015). In addition, while Met full agonists having the same function as HGF can induce the formation of tubular structures in the above cells, it has been reported that partial agonists having only some functions cannot promote the formation of tubular structures (W. Miao, et al. Int. J. Mol. Sci. 19, 3141, 2018). Therefore, it was decided to confirm whether the HGF-PAS dimer peptide is a full agonist or a partial agonist using a tubular structure formation experiment with HuCCT1 cells. To 8 volumes of CellMatrix type IA (Shinden Gelatin), 1 volume of 10-fold concentrated D-MEM / F12 (Thermo Fisher Scientific) and 1 volume of reconstitution buffer (22 g / L sodium bicarbonate, 47.7 g / L HEPES, 0.05N aqueous sodium hydroxide solution) were sequentially mixed on ice. HuCCT1 cells at 25,000 cells per 1 mL of the mixed solution were added and mixed on ice. 400 μL of the above mixed solution was added to a 48-well cell culture plate and allowed to stand at 37 °C under 5% CO2 for 4 hours to gelate. 500 μL of RPMI1640 medium supplemented with 10% FBS was added onto the formed cell-encapsulating collagen gel. The group of cells to be stimulated was cultured with the addition of 35 ng / mL of HGF or 40 ng / mL of AET-PAS-aMD4dY-PEG11. The medium was replaced in its entirety at a frequency not exceeding 3 days. On the 7th day of culture, the morphology of the cells in the collagen gel was observed with an inverted microscope. Figure 17 shows the morphology of HuCCT1 cells on the 7th day of culture (scale bar: 500 μm). The cells to which HGF or AET-PAS-aMD4dY-PEG11 was added formed tubular structures about 500 - 1000 μm in size, while the non-added cells showed a spherical morphology. From these results, it was revealed that AET-PAS-aMD4dY-PEG11 can promote the formation of tubular structures in HuCCT1 cells, similar to rhHGF, and is a full agonist for Met.

[0090] Example 12: Design of HGF-PAS heterodimer peptide and its secretory expression in C. glutamicum (12-1) Outline of the design of HGF-PAS heterodimer peptide The aMD4 and aMD5 of the Met-binding cyclic peptides reported in K. Ito, et al. Nat. Commun. 6, 6373, 2015 were expressed as heterodimer molecules. In the dimer peptide synthesis method in K. Ito, et al. Nat. Commun. 6, 6373, 2015, it is difficult to selectively synthesize a dimer peptide of "aMD4"-"linker"-"aMD5". That is, when two types of peptides are linked via a PEG molecule, four types of dimer peptides, namely "aMD4"-"linker"-"aMD4", "aMD4"-"linker"-"aMD5", "aMD5"-"linker"-"aMD4", and "aMD5"-"linker"-"aMD5", will be produced. Therefore, in this example, by implementing the following design, it becomes possible to express a heterodimer peptide dimerized by a PAS linker as a single polypeptide chain in C. glutamicum. (1) Since aMD4 and aMD5 are cyclized by an intramolecular thioether bond formed by the reaction between the chloroacetyl group added to the amino group at the N-terminus and the sulfhydryl group of the cysteine side chain in the sequence, they are cyclized by a disulfide bond formed by the sulfhydryl groups of two cysteine side chains. (2) Dimerization is achieved by binding the C-termini of the cyclic peptides using a Bis-Maleimide-PEG linker. Since it cannot be expressed in C. glutamicum, the C-terminus of aMD4 and the N-terminus of aMD5 are bound with a PAS linker peptide. With the above design, the "aMD4"-"PAS linker"-"aMD5" peptide is selectively expressed.

[0091] (12-2) Preparation of HGF-PAS heterodimer peptide The prepared HGF-PAS heterodimer peptide is as follows. (h) AET-PAS-aMD4_aMD5-PEG11 AETCYRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPAPAAPAAPGCYWYYAWDQTYKAFPC (SEQ ID NO: 62)

[0092] (12-3) Expression of HGF-PAS heterodimer peptide The expression of HGF-PAS heterodimer peptide was examined using Corynex (registered trademark). Hereinafter, examples of the examination of expression by Corynex (registered trademark) will be described.

[0093] (12-4) Construction of a secretion expression plasmid of AET-PAS-aMD4_aMD5-PEG11 using the CspB signal sequence As the HGF-PAS heterodimer peptide, the amino acid sequence of the above (h) AET-PAS-aMD4_aMD5-PEG11 (hereinafter, may be referred to as "AET-PAS-aMD4_aMD5-PEG11") was designed, and the nucleotide sequence encoding this protein was designed in consideration of the codon usage frequency of C. glutamicum. Furthermore, the following expression cassette was designed to enable secretion expression by C. glutamicum. AET-PAS-aMD4_aMD5-PEG11 was secreted and expressed as a fusion protein of 30 amino acid residues of the signal peptide of CspB derived from C. glutamicum ATCC13869 strain and AET-PAS-aMD4_aMD5-PEG11 (hereinafter, referred to as "CspBss-AET-PAS-aMD4_aMD5-PEG11"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AET-PAS-aMD4_aMD5-PEG11 are shown in SEQ ID NO: 63 and SEQ ID NO: 64, respectively.

[0094] Nucleotide sequence encoding CspBss-AET-PAS-aMD4_aMD5-PEG11 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagacctgttaccgtcagttcaaccgtcgcacccacgaagtttggaacctggattgtggcgcagcccccgcagcccccgcacccgcagcccctgcagccccagccccagctgcaccagcagcacctggctgttactggtattatgcgtgggatcagacctacaaagcgtttccgtgttag(SEQ ID NO: 63) Amino acid sequence of CspBss-AET-PAS-aMD4_aMD5-PEG11 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETCYRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPAPAAPAPAAPGCYWYYAWDQTYKAFPC(SEQ ID NO: 64)

[0095] The promoter of the cspB gene derived from C. glutamicum ATCC13869 strain was linked upstream of the nucleotide sequence described in AET-PAS-aMD4_aMD5-PEG11, and a KpnI site was added to the 5'-side and a BamHI site was added to the 3'-side to design and fully synthesize the expression cassette of the HGF-PAS heterodimer peptide. The fully synthesized DNA fragment (expression cassette of the HGF-PAS heterodimer peptide) was inserted into the KpnI-BamHI site of pPK4 described in JP-A-9-322774 to construct pPK4_CspBss-AET-PAS-aMD4_aMD5-PEG1, a secretion expression plasmid of the HGF-PAS heterodimer peptide using the CspB signal sequence. As a result of nucleotide sequence determination of the inserted fragment, it was confirmed that the expression cassette of the HGF-PAS heterodimer peptide as designed was constructed. Nucleotide sequence determination was performed using the BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the 3130 Genetic Analyzer (Applied Biosystems).

[0096] (12-5) Secretory expression of the HGF-PAS heterodimer peptide in C. glutamicum Using pPK4_CspBss-AET-PAS-aMD4_aMD5-PEG11 constructed above, the C. glutamicum YDK010::phoS(W302C) strain described in WO2016 / 171224 was transformed to obtain the YDK010::phoS(W302C) / pPK4_CspBss-AET-PAS-aMD4_aMD5-PEG11 strain. The obtained transformants were cultured at 30 °C for 72 hours in MMTG liquid medium (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, ferrous sulfate heptahydrate 0.03 g, manganese sulfate pentahydrate 0.03 g, thiamine hydrochloride 0.45 mg, biotin 0.45 mg, DL-methionine 0.15 g, soybean hydrochloride hydrolyzate (total nitrogen content 0.2 g), calcium carbonate 50 g, adjusted to pH 7.0 with water to 1 L) containing 25 mg / L of kanamycin. After the culture was completed, 6.5 μL of the culture supernatant obtained by centrifuging each culture solution was subjected to reducing SDS-PAGE using NuPAGE (registered trademark) 12% Bis-Tirs Gel (Thermo Fisher Scientific), and then stained with Quick-CBB (Wako). As a result, a protein band presumed to be AET-PAS-aMD4_aMD5-PEG11 was detected in the culture supernatant of the YDK010::phoS(W302C) / pPK4_CspBss-AET-PAS-aMD4_aMD5-PEG11 strain (Figure 18, lanes 2-5).

[0097] Example 13: Molecular weight analysis of the HGF-PAS heterodimer peptide The AET-PAS-aMD4_aMD5-PEG11 peptide expression supernatant and α-Cyano-4-hydroxycinnamic Acid (CHCA) were mixed, and the molecular weight was measured using MALDI-TOF-MS (Shimadzu, AXIMA-TOF2). As shown in Fig. 19, a value almost identical to the theoretical molecular weight of the oxidized HGF-PAS heterodimer peptide that forms two disulfide bonds within the molecule (AET-PAS-aMD4_aMD5-PEG11: m / z 6514) was obtained. It was confirmed that a peptide with the correct amino acid sequence containing two disulfide bonds was expressed.

[0098] Example 14: Design of HGF-PAS dimer peptide linker variants and their secretory expression in C. glutamicum (14-1) Outline of the design of HGF-PAS dimer peptide linker variants AET-PAS-aMD4dY-PEG11 having HGF-like activity has a structure in which the first aMD4dY cyclic peptide (represented by SEQ ID NO: 2) and the second aMD4dY cyclic peptide are linked by a 22-amino acid PAS linker. By changing the length of this PAS linker, it is expected that the HGF-like activity will change. Therefore, linker variants were designed in which the PAS linker site was changed to a PAS repeat sequence from 8 amino acids to 200 amino acids. In addition, as a flexible linker, in addition to the PAS linker, a GS linker, which is a repeating sequence of glycine and serine, is also known. Therefore, a linker variant in which aMD4dY is bound to the N-terminus and C-terminus of a GS linker consisting of a repeating sequence of 4 glycine residues and 1 serine residue (GGGGS), that is, a peptide sequence was designed so that it would be "the first aMD4dY cyclic peptide" - "GS linker" - "the second aMD4dY cyclic peptide".

[0099] (14-2) Preparation of HGF-PAS dimer peptide linker variants The prepared HGF-PAS dimer peptide linker variants are as follows. (i) AET-aMD4dY-PAS8 AETCRQFNRRTHEVWNLDCGAAPAAPAPGCRQFNRRTHEVWNLDC (SEQ ID NO: 81) (j) AET-aMD4dY-PAS49 AETCRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAGCRQFNRRTHEVWNLDC (SEQ ID NO: 82) (k) AET-aMD4dY-PAS100 AETCRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAGCRQFNRRTHEVWNLDC (SEQ ID NO: 83) (l) AET-aMD4dY-PAS200 AETCRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPGCRQFNRRTHEVWNLDC (SEQ ID NO: 84) (m) AET-aMD4dY-GS15 AETCRQFNRRTHEVWNLDCGGGGGSGGGGSGGGGSGCRQFNRRTHEVWNLDC (SEQ ID NO: 85) (n) AET-aMD4dY-GS22 AETCRQFNRRTHEVWNLDCGGGGGSGGGGSGGGGSGGGGSGGGCRQFNRRTHEVWNLDC (SEQ ID NO: 86)

[0100] (14-3) Expression of HGF-PAS dimer peptide linker variants The expression of these HGF-PAS dimer peptide linker variants was examined using Corynex (registered trademark). Hereinafter, examples of the expression examination using Corynex (registered trademark) will be described.

[0101] Construction of Secretion Expression Plasmids of AET-aMD4dY-PAS8, AET-aMD4dY-PAS49, AET-aMD4dY-PAS100, AET-aMD4dY-PAS200, AET-aMD4dY-GS15, and AET-aMD4dY-GS22 Using the (14-4)CspB Signal Sequence As HGF-PAS dimer peptide linker variants, the six amino acid sequences of (i) AET-aMD4dY-PAS8 (hereinafter sometimes referred to as "AET-aMD4dY-PAS8"), (j) AET-aMD4dY-PAS49 (hereinafter sometimes referred to as "AET-aMD4dY-PAS49"), (k) AET-aMD4dY-PAS100 (hereinafter sometimes referred to as "AET-aMD4dY-PAS100"), (l) AET-aMD4dY-PAS200 (hereinafter sometimes referred to as "AET-aMD4dY-PAS200"), (m) AET-aMD4dY-GS15 (hereinafter sometimes referred to as "AET-aMD4dY-GS15"), and (n) AET-aMD4dY-GS22 (hereinafter sometimes referred to as "AET-aMD4dY-GS22") were designed respectively, and the base sequences encoding these proteins were designed considering the codon usage frequency of C. glutamicum. Furthermore, the following expression cassettes were designed respectively to enable secretion expression by C. glutamicum. AET-aMD4dY-PAS8 was secreted and expressed as a fusion protein of the 30 amino acid residues of the signal peptide of CspB derived from C. glutamicum ATCC13869 strain and AET-aMD4dY-PAS8 (hereinafter referred to as "CspBss-AET-aMD4dY-PAS8"). The base sequence and amino acid sequence encoding the designed CspBss-AET-aMD4dY-PAS8 are shown in SEQ ID NO: 87 and 88 respectively.

[0102] Base Sequence Encoding CspBss-AET-aMD4dY-PAS8 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagacctgccgccagttcaaccgccgcacccacgaagtgtggaacctggattgcggcgctgcaccagccgctccagcaccaggctgccgccagttcaaccgccgcacccacgaagtgtggaacctggattgctaa(SEQ ID NO: 87) Amino acid sequence of CspBss-AET-aMD4dY-PAS8 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETCRQFNRRTHEVWNLDCGAAPAAPAPGCRQFNRRTHEVWNLDC(SEQ ID NO: 88)

[0103] AET-aMD4dY-PAS49 was secreted and expressed as a fusion protein of the 30 amino acid residues of the signal peptide of CspB derived from C. glutamicum ATCC13869 strain and AET-aMD4dY-PAS49 (hereinafter referred to as "CspBss-AET-aMD4dY-PAS49"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AET-aMD4dY-PAS49 are shown in SEQ ID NOs: 89 and 90, respectively.

[0104] Nucleotide sequence encoding CspBss-AET-aMD4dY-PAS49 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagacctgtcgtcagttcaaccgtcgcacccacgaagtttggaatttggactgtggcgcagcaccagcggcaccagcaccagcagccccagcggcaccagccccagctgcaccagccgctccagcaccagcagcacccgcagcccccgctcccgcagcccccgccgcaccagcacccgcagcaccagcagcacccgctccagcaggttgtcgccaattcaaccgccgcacccacgaagtgtggaacctggattgctaa(SEQ ID NO:89) Amino acid sequence of CspBss-AET-aMD4dY-PAS49 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETCRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAGCRQFNRRTHEVWNLDC(SEQ ID NO:90)

[0105] AET-aMD4dY-PAS100 was secreted and expressed as a fusion protein of the 30 amino acid residues of the signal peptide of CspB derived from C. glutamicum ATCC13869 strain and AET-aMD4dY-PAS100 (hereinafter referred to as "CspBss-AET-aMD4dY-PAS100"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AET-aMD4dY-PAS100 are shown in SEQ ID NO:91 and SEQ ID NO:92, respectively.

[0106] Nucleotide sequence encoding CspBss-AET-aMD4dY-PAS100 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagacctgtcgtcagttcaaccgtcgcacccacgaagtttggaatttggactgtggcgcagcaccagcggcaccagcaccagcagccccagcggcaccagccccagctgcaccagccgctccagcaccagcagccccagcagcccccgctccagcagcacccgcagcaccagctcccgcagccccagcagccccagccccagcagcaccagcagccccagctccagcagccccagctgcaccagctccagccgcaccagcagctccagccccagccgctcccgccgccccagcccccgccgcaccagccgcccctgctcccgccgccccagccgctcccgctccagccgctcctgccggttgccgccagttcaaccgccgcacccacgaagtgtggaacctggattgctaa(SEQ ID NO: 91) Amino acid sequence of CspBss-AET-aMD4dY-PAS100 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETCRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAGCRQFNRRTHEVWNLDC(SEQ ID NO: 92)

[0107] AET-aMD4dY-PAS200 was secreted and expressed as a fusion protein of the 30 amino acid residues of the signal peptide of CspB derived from C. glutamicum ATCC13869 strain and AET-aMD4dY-PAS200 (hereinafter referred to as "CspBss-AET-aMD4dY-PAS200"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AET-aMD4dY-PAS200 are shown in SEQ ID NOs: 93 and 94, respectively.

[0108] The nucleotide sequence encoding CspBss-AET-aMD4dY-PAS200 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagacctgtcgtcagttcaaccgtcgcacccacgaagtttggaatttggactgtggcgcagcaccagcggcaccagcaccagcagccccagcggcaccagccccagctgcaccagccgctccagcaccagccgcccccgccgcccccgcccctgctgctcctgctgctcccgctcccgctgctcccgctgcccctgctcctgctgctcccgctgcgccagccccagcagcaccagctgccccagcaccagctgcaccagccgcaccagctccagcagccccagctgctccagcaccagcagcaccagcagcaccagctccagcggcaccagcagcaccagcacctgcggcaccagcggctccagccccagcagcaccagcagcgccagcaccagcagctccagcagcaccagcaccagcagcaccagcggcaccagccccagctgcaccagccgctccagcaccagctgccccagcagctccagccccagccgcacctgccgcacctgcccctgccgctcctgctgcccccgctcctgccgctcctgctgcccctgcccctgctgctcctgctgctccagctccagccgcaccagccgctccagcaccagctgcaccagcagctccagccccagctgccccagcagctccagcccctggttgccgccaatttaatcgccgcacccacgaggtctggaatcttgactgctaa(SEQ ID NO: 93) The amino acid sequence of CspBss-AET-aMD4dY-PAS200 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETCRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPGCRQFNRRTHEVWNLDC (SEQ ID NO: 94)

[0109] AET-aMD4dY-GS15 was secreted and expressed as a fusion protein of the 30 amino acid residues of the signal peptide of CspB derived from C. glutamicum ATCC13869 strain and AET-aMD4dY-GS15 (hereinafter referred to as "CspBss-AET-aMD4dY-GS15"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AET-aMD4dY-GS15 are shown in SEQ ID NO: 95 and 96, respectively.

[0110] Nucleotide sequence encoding CspBss-AET-aMD4dY-GS15 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagacctgccgccagttcaaccgccgcacccacgaagtgtggaacctggattgcggcggcggcggcggctccggcggcggcggctccggcggcggcggctccggctgccgccagttcaaccgccgcacccacgaagtgtggaacctggattgctaa (SEQ ID NO: 95) Amino acid sequence of CspBss-AET-aMD4dY-GS15 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETCRQFNRRTHEVWNLDCGGGGGSGGGGSGGGGSGCRQFNRRTHEVWNLDC (SEQ ID NO: 96)

[0111] AET-aMD4dY-GS22 was secreted and expressed as a fusion protein of the 30 amino acid residues of the signal peptide of CspB derived from C. glutamicum ATCC13869 strain and AET-aMD4dY-GS22 (hereinafter referred to as "CspBss-AET-aMD4dY-GS22"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AET-aMD4dY-GS22 are shown in SEQ ID NO: 97 and 98, respectively.

[0112] Nucleotide sequence encoding CspBss-AET-aMD4dY-GS22 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagacctgccgccagttcaaccgccgcacccacgaagtgtggaacctggattgcggcggcggcggcggctccggcggcggcggttctggcggtggcggttctggcggcggcggttctggcggcggttgtcgtcagttcaatcgtcgcacccacgaagtctggaatctcgactgctaa (SEQ ID NO: 97) Amino acid sequence of CspBss-AET-aMD4dY-GS22 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETCRQFNRRTHEVWNLDCGGGGGSGGGGSGGGGSGGGGSGGGCRQFNRRTHEVWNLDC (SEQ ID NO: 98)

[0113] The promoter of the cspB gene derived from the C. glutamicum ATCC13869 strain was ligated upstream of the nucleotide sequences described in CspBss-AET-aMD4dY-PAS8, CspBss-AET-aMD4dY-PAS49, CspBss-AET-aMD4dY-PAS100, CspBss-AET-aMD4dY-PAS200, CspBss-AET-aMD4dY-GS15, and CspBss-AET-aMD4dY-GS22. Furthermore, a KpnI site was added to the 5'-side and a BamHI site was added to the 3'-side, and the expression cassettes of each HGF-PAS dimer peptide linker variant were designed and fully synthesized. The fully synthesized DNA fragment (the expression cassette of the HGF-PAS dimer peptide linker variant) was inserted into the KpnI-BamHI site of pPK4 described in JP-A-9-322774 to obtain pPK4_CspBss-AET-aMD4dY-PAS8, pPK4_CspBss-AET-aMD4dY-PAS49, pPK4_CspBss-AET-aMD4dY-PAS100, pPK4_CspBss-AET-aMD4dY-PAS200, pPK4_CspBss-AET-aMD4dY-GS15, and pPK4_CspBss-AET-aMD4dY-GS22, which are secretion expression plasmids of each HGF-PAS dimer peptide linker variant using the CspB signal sequence. As a result of determining the nucleotide sequence of the inserted fragment, it was confirmed that the expression cassettes of the HGF-PAS dimer peptide linker variants were constructed as designed. The nucleotide sequence determination was performed using the BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the 3500xL Genetic Analyzer (Applied Biosystems). Similarly, pPK4_CspBss-AET-PAS-aMD4dY-PEG11 was constructed and its construction was confirmed. AET-PAS-aMD4dY-PEG11 is as described in Example 4 and has a PAS linker consisting of 22 amino acids.

[0114] (14-5) Secretory expression of each HGF-PAS dimer peptide in C. glutamicum Using pPK4_CspBss-AET-aMD4dY-PAS8, pPK4_CspBss-AET-aMD4dY-PAS49, pPK4_CspBss-AET-aMD4dY-PAS100, pPK4_CspBss-AET-aMD4dY-PAS200, pPK4_CspBss-AET-aMD4dY-GS15, and pPK4_CspBss-AET-aMD4dY-GS22 constructed above, the C. glutamicum YDK010::phoS(W302C) strain described in WO2016 / 171224 was transformed to obtain the YDK010::phoS(W302C) / pPK4_CspBss-AET-aMD4dY-PAS8 strain, YDK010::phoS(W302C) / pPK4_CspBss-AET-aMD4dY-PAS49 strain, YDK010::phoS(W302C) / pPK4_CspBss-AET-aMD4dY-PAS100 strain, YDK010::phoS(W302C) / pPK4_CspBss-AET-aMD4dY-PAS200 strain, YDK010::phoS(W302C) / pPK4_CspBss-AET-aMD4dY-GS15 strain, and YDK010::phoS(W302C) / pPK4_CspBss-AET-aMD4dY-GS22 strain. Each of the obtained transformants was cultured at 30 °C for 72 hours in MMTG liquid medium (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, ferrous sulfate heptahydrate 0.03 g, manganese sulfate pentahydrate 0.03 g, thiamine hydrochloride 0.45 mg, biotin 0.45 mg, DL-methionine 0.15 g, soybean hydrochloride hydrolysate (total nitrogen content 0.2 g), calcium carbonate 50 g, adjusted to 1 L with water and pH 7.0) containing 25 mg / L of kanamycin. After the cultivation was completed, 6.5 μL of the culture supernatant obtained by centrifuging each culture solution was subjected to reducing SDS-PAGE using NuPAGE (registered trademark) 12% Bis-Tirs Gel (Thermo Fisher Scientific), and then stained with Quick-CBB (Wako). As a result, a protein band presumed to be AET-aMD4dY-PAS8 in the culture supernatant of the YDK010::phoS(W302C) / pPK4_CspBss-AET-aMD4dY-PAS8 strain (Figure 20, lane 4), a protein band presumed to be AET-aMD4dY-PAS49 in the culture supernatant of the YDK010::phoS(W302C) / pPK4_CspBss-AET-aMD4dY-PAS49 strain (Figure 20, lane 6), a protein band presumed to be AET-aMD4dY-PAS100 in the culture supernatant of the YDK010::phoS(W302C) / pPK4_CspBss-AET-aMD4dY-PAS100 strain (Figure 20, lane 7), a protein band presumed to be AET-aMD4dY-PAS200 in the culture supernatant of the YDK010::phoS(W302C) / pPK4_CspBss-AET-aMD4dY-PAS200 strain (Figure 20, lane 8), a protein band presumed to be AET-aMD4dY-GS15 in the culture supernatant of the YDK010::phoS(W302C) / pPK4_CspBss-AET-aMD4dY-GS15 strain (Figure 20, lane 2), and a protein band presumed to be AET-aMD4dY-GS22 in the culture supernatant of the YDK010::phoS(W302C) / pPK4_CspBss-AET-aMD4dY-GS22 strain (Figure 20, lane 3) were each detected. Also, the YDK010::phoS(W302C) / pPK4_CspBss-AET-PAS-aMD4dY-PEG11 strain obtained by the same method as above was cultured in the same manner as above, and a protein band presumed to be AET-PAS-aMD4dY-PEG11 (hereinafter also referred to as "AET-aMD4dY-PAS22") in the culture supernatant thereof was detected (Figure 20, lane 5). When comparing 22-residue linkers of the same length, AET-aMD4dY-PAS22 (Figure 20, lane 5: the same molecule as AET-PAS-aMD4dY-PEG11) showed higher secretion than AET-aMD4dY-GS22 (Figure 20, lane 3), that is, higher secretion was achieved when using a PAS linker than a GS linker.

[0115] Example 15: Molecular weight analysis of HGF-PAS dimer peptides For the cell filtrates of each HGF-PAS dimer peptide after culturing in Example 14, the molecular weight was measured using LC-MS (Waters, ACQUITY UPLC H-Class / SQD2). The analysis conditions are shown in Table 2.

[0116] [Table 2]

[0117] As shown in Figures 21 to 26, values almost identical to the theoretical values of the molecular weights of the oxidized dimer peptides that form two disulfide bonds within the molecule were obtained. From this, it was confirmed that peptides with the correct amino acid sequence containing two disulfide bonds were successfully expressed.

[0118] Example 16: Activity evaluation of HGF-PAS dimer peptide linker variants The Met activation ability of the HGF-PAS dimer peptide linker variants was evaluated by a luciferase assay. To 25 μL of Opti-MEM medium (Thermo Fisher Scientific), 0.6 μL of Attractene Transfection Reagent (QIAGEN) was added and incubated at room temperature for 5 minutes. To the above mixed solution, a mixed solution of 25 μL of Opti-MEM and 1 μL of SRE reporter vector (QIAGEN) was added and incubated at room temperature for 20 minutes. This mixed solution was added to a 96-well plate, and 40,000 cells / well of HEK293E cells were seeded on it and incubated overnight in a 37 °C, 5% CO2 incubator. After transfection, all the culture supernatant was removed, and 100 μL of Opti-MEM medium (evaluation basal medium) containing 0.5% FBS (Thermo Fisher Scientific), 1% non-essential amino acid solution (Thermo Fisher Scientific), and penicillin-streptomycin (Nacalai Tesque) was added and cultured at 37 °C for 4 hours to make the cells in a starved state. To the evaluation basal medium, HGF at 0 - 100 ng / mL was added, or culture supernatants containing AET-aMD4dY-PAS8, AET-aMD4dY-PAS22 (the same molecule as AET-PAS-aMD4dY-PEG11), AET-aMD4dY-PAS49, AET-aMD4dY-PAS100, AET-aMD4dY-PAS200, AET-aMD4dY-GS15, or AET-aMD4dY-GS22 were added so as to be diluted at a predetermined ratio with respect to the medium, and 100 μL of the evaluation basal medium was added to the cells. The cells were stimulated by culturing overnight in a 37 °C, 5% CO2 incubator. For the detection of signal intensity, the Dual-Luciferase Reporter Assay System (Promega) was used. 100 μL of the culture supernatant was removed, 50 μL of Glo reagent was added, and the cells were lysed at room temperature for 10 minutes. The activation of the HGF-Erk-SRE pathway was quantified by detecting the luminescence of Firefly luciferase emitted from the cell lysate using a plate reader. Subsequently, 50 μL of the Glo & Stop reagent solution was added, and after 10 minutes, the luminescence of Renilla luciferase, which is an internal standard, was detected to quantify the number of cells. The signal activity of each sample was quantified as SRE activity = (luminescence intensity of Firefly luciferase) / (luminescence intensity of Renilla luciferase). The relative signal activity value with respect to the sample without the evaluation compound was determined and used as the relative reporter activity. As shown in Fig. 27, it was revealed that all HGF-PAS dimer peptides with different PAS linker lengths activate the HGF signal. The highest efficiency was exhibited when the length of the PAS linker was 22 amino acids, and as shown in Table 3 below, it was revealed that when it exceeded 100 amino acids, the maximum activity (EC50) was halved. Also, as shown in Fig. 28, activity was obtained even when dimerized with 15-amino acid and 22-amino acid GS linkers, but their maximum activity (EC50) was less than half that of the PAS linker as shown in Table 3 below, indicating that a dimer peptide with high activity can be prepared by using the PAS linker.

[0119]

Table 3

[0120] Example 17: Design of a stabilized HGF-PAS dimer peptide and its secretory expression in C. glutamicum

[0121] (17-1) Outline of the design The HGF-PAS peptide with enhanced stability was designed as follows. As a method for enhancing the stability of peptides and proteins, a method of adding a long-chain PAS sequence has been reported (Special Table 2013-531480 and Special Table 2010-531139). As the HGF-PAS peptide with enhanced stability, a peptide was designed in which a PAS sequence (PAS200) consisting of 200 amino acids was bound to the C-terminus of AET-aMD4dY-PAS22 (the same molecule as AET-PAS-aMD4dY-PEG11). That is, the peptide sequence was designed to be "the first aMD4dY cyclic peptide" - "PAS linker" - "the second aMD4dY cyclic peptide" - "long-chain PAS sequence (PAS200)".

[0122] (17-2) Preparation of Stabilized HGF-PAS Dimer Peptide The prepared stabilized HGF-PAS dimer peptide is as follows. (o) AET-aMD4dY-PAS22-PAS200 AETCRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPAPAAPAAPGCRQFNRRTHEVWNLDCAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAP (SEQ ID NO: 99)

[0123] (17-3) Expression of Stabilized HGF-PAS Dimer Peptide The expression of the stabilized HGF-PAS dimer peptide was examined using Corynex (registered trademark). Hereinafter, examples of the expression examination using Corynex (registered trademark) will be described.

[0124] (17-4) Construction of AET-aMD4dY-PAS22-PAS200 Secretory Expression Plasmid Using CspB Signal Sequence As a stabilized HGF-PAS dimer peptide, the amino acid sequence of the above (o) AET-aMD4dY-PAS22-PAS200 (hereinafter sometimes referred to as "AET-aMD4dY-PAS22-PAS200") was designed, and the nucleotide sequence encoding this protein was designed in consideration of the codon usage frequency of C. glutamicum. Furthermore, the following expression cassette was designed to enable secretion expression by C. glutamicum. AET-aMD4dY-PAS22-PAS200 was secreted and expressed as a fusion protein of the signal peptide of 30 amino acid residues of CspB derived from C. glutamicum ATCC13869 strain and AET-aMD4dY-PAS22-PAS200 (hereinafter referred to as "CspBss-AET-aMD4dY-PAS22-PAS200"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AET-aMD4dY-PAS22-PAS200 are shown in SEQ ID NO: 100 and SEQ ID NO: 101, respectively.

[0125] Nucleotide sequence encoding CspBss-AET-aMD4dY-PAS22-PAS200 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagacctgccgtcagttcaaccgccgtacccacgaagtttggaatctcgactgtggtgccgcccctgccgctcctgcccctgccgccccagccgctccagcaccagccgctccagcagccccaggctgccgccagttcaaccgccgcacccacgaagtgtggaacctggattgcgcagctccagcagcccctgcccccgccgccccagccgcccctgcccccgccgcccccgccgcacccgcccctgccgcccccgccgcccccgcccctgctgctcctgctgctcccgctcccgctgctcccgctgcccctgctcctgctgctcccgctgcgccagccccagcagcaccagctgccccagcaccagctgcaccagccgcaccagctccagcagccccagctgctccagcaccagcagcaccagcagcaccagctccagcggcaccagcagcaccagcacctgcggcaccagcggctccagccccagcagcaccagcagcgccagcaccagcagctccagcagcaccagcaccagcagcaccagcggcaccagccccagctgcaccagccgctccagcaccagctgccccagcagctccagccccagctgcccctgctgcccccgctcctgccgctcccgctgcccctgcccctgctgctcctgctgctcccgctcctgccgctcccgctgcgccagctccagcagccccagctgcaccagccccagccgcaccagccgccccagcccccgcagcacccgcagcaccagccccataa(SEQ ID NO: 100) Amino acid sequence of CspBss-AET-aMD4dY-PAS22-PAS200 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETCRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPAPAAPAAPGCRQFNRRTHEVWNLDCAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAP(Sequence number: 101)

[0126] The promoter of the cspB gene derived from the C. glutamicum ATCC13869 strain was connected upstream of the nucleotide sequence described in CspBss-AET-aMD4dY-PAS22-PAS200, and a KpnI site was added to the 5'-side and a BamHI site was added to the 3'-side. An expression cassette for a stabilized HGF-PAS dimer peptide was designed and fully synthesized. The fully synthesized DNA fragment (expression cassette for a stabilized HGF-PAS dimer peptide) was inserted into the KpnI-BamHI site of pPK4 described in JP-A-9-322774 to construct pPK4_CspBss-AET-aMD4dY-PAS22-PAS200, a secretion expression plasmid for a stabilized HGF-PAS dimer peptide that utilizes the CspB signal sequence. As a result of nucleotide sequencing of the inserted fragment, it was confirmed that an expression cassette for a stabilized HGF-PAS dimer peptide as designed was constructed. Nucleotide sequencing was performed using the BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the 3500xL Genetic Analyzer (Applied Biosystems).

[0127] (17-5) Secretion expression of stabilized HGF-PAS dimer peptide in C. glutamicum Using pPK4_CspBss-AET-aMD4dY-PAS22-PAS200 constructed above, the C. glutamicum YDK010::phoS(W302C) strain described in WO2016 / 171224 was transformed to obtain the YDK010::phoS(W302C) / pPK4_CspBss-AET-aMD4dY-PAS22-PAS200 strain. Each of the obtained transformants was cultured at 30 °C for 72 hours in MMTG liquid medium (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, iron (III) sulfate heptahydrate 0.03 g, manganese (II) sulfate pentahydrate 0.03 g, thiamine hydrochloride 0.45 mg, biotin 0.45 mg, DL-methionine 0.15 g, soy peptone (total nitrogen content 0.2 g), calcium carbonate 50 g, adjusted to pH 7.0 with water to 1 L) containing 25 mg / L of kanamycin. After completion of the culture, 6.5 μL of the culture supernatant obtained by centrifuging each culture solution was subjected to reducing SDS-PAGE using NuPAGE® 4-12% Bis-Tirs Gel (Thermo Fisher Scientific) and then stained with Quick-CBB (Wako). As a result, a protein band presumed to be AET-aMD4dY-PAS22-PAS200 was detected in the culture supernatant of the YDK010::phoS(W302C) / pPK4_CspBss-AET-aMD4dY-PAS22-PAS200 strain (Figure 29, lanes 2-5).

[0128] Example 18: Molecular weight analysis of stabilized HGF-PAS dimer peptide Regarding the cell filtrate of the stabilized HGF-PAS dimer peptide after the culture in Example 17, the molecular weight was measured using LC-MS (Waters, ACQUITY UPLC / SQD2). The analysis conditions were as described in Table 2. As shown in Figure 30, values almost identical to the theoretical values of the molecular weights of the oxidized dimer peptides each forming two disulfide bonds in the molecule were obtained. From this, it was confirmed that a peptide with the correct amino acid sequence containing two disulfide bonds was successfully expressed.

[0129] Example 19: Activity Evaluation of Stabilized HGF-PAS Dimer Peptide The Met activation ability of the stabilized HGF-PAS dimer peptide was evaluated by a luciferase assay. To 25 μL of Opti-MEM medium (Thermo Fisher Scientific), 0.6 μL of Attractene Transfection Reagent (QIAGEN) was added and incubated at room temperature for 5 minutes. To the above mixed solution, a mixed solution of 25 μL of Opti-MEM and 1 μL of SRE reporter vector (QIAGEN) was added and incubated at room temperature for 20 minutes. This mixed solution was added to a 96-well plate, and 40,000 cells / well of HEK293E cells were seeded on it and incubated overnight in a 37 °C, 5% CO2 incubator. After transfection, all the culture supernatants were removed, and 100 μL of Opti-MEM medium (evaluation basal medium) containing 0.5% FBS (Thermo Fisher Scientific), 1% non-essential amino acid solution (Thermo Fisher Scientific), and penicillin-streptomycin (Nacalai Tesque) was added and cultured at 37 °C for 4 hours to make the cells in a starved state. To the evaluation basal medium, HGF at 0 - 100 ng / mL was added, or the culture supernatant containing AET-aMD4dY-PAS22-PAS200 was added so as to be diluted at a predetermined ratio with respect to the medium, and 100 μL of the evaluation basal medium was added to the cells. The cells were stimulated by culturing overnight in a 37 °C, 5% CO2 incubator. The Dual-Luciferase Reporter Assay System (Promega) was used for detecting signal intensity. 100 μL of the culture supernatant was removed, 50 μL of Glo reagent was added, and the cells were lysed at room temperature for 10 minutes. The activation of the HGF-Erk-SRE pathway was quantified by detecting the luminescence of Firefly luciferase emitted from the cell lysate using a plate reader. Subsequently, 50 μL of the Glo & Stop reagent solution was added, and after 10 minutes, the luminescence of Renilla luciferase, which is an internal standard, was detected to quantify the number of cells. The signal activity of each sample was quantified as SRE activity = (luminescence intensity of Firefly luciferase) / (luminescence intensity of Renilla luciferase). The relative signal activity value with respect to the sample without the test compound was determined and defined as the relative reporter activity. As shown in Fig. 31, it was revealed that the stabilized HGF-PAS dimer peptide has signal activation ability and its maximum activity is comparable to that of 100 ng / mL HGF.

[0130] Example 20: Design of EPO-PAS dimer peptide and its secretory expression in C. glutamicum (20-1) Outline of design Peginesatide has been reported as an erythropoietin-mimicking peptide. Peginesatide has a structure in which a lysine is added to the C-terminus of the erythropoietin receptor-binding peptide, and the ε-amino groups are linked by a dicarboxylic acid linker linked by a long-chain PEG. However, expressing Peginesatide as a single polypeptide chain in C. glutamicum cannot be done for the following reasons. (1) Peginesatide contains non-natural amino acids, and peptides containing non-natural amino acids cannot be expressed in C. glutamicum. (2) Peptides containing a dicarboxylic acid linker cannot be expressed in C. glutamicum. (3) Dimerization is achieved by binding the C-terminals of two cyclic peptides with the ε-amino group of lysine, but structures in which the C-terminals are bound to each other and structures in which the ε-amino group of lysine forms an amide bond with another amino acid cannot be expressed in C. glutamicum. Therefore, the molecule was designed to be expressible by adding the following conversions. Regarding (1) above, it is possible to express using an erythropoietin receptor-binding cyclic peptide (US5773569A) that does not contain unnatural amino acids. Regarding (2) above, it is possible to express using a peptide linker. Regarding (3) above, instead of binding the C-terminals of the cyclic peptides to each other, the N-terminal of one cyclic peptide and the C-terminal of the other peptide were bound using a PAS sequence. That is, the peptide sequence was designed to be "first erythropoietin receptor-binding cyclic peptide" - "PAS linker" - "second erythropoietin receptor-binding cyclic peptide". That is, the C-terminal of the first erythropoietin receptor-binding cyclic peptide and the N-terminal of the PAS linker, and the N-terminal of the second erythropoietin receptor-binding cyclic peptide and the C-terminal of the PAS linker are each bound by an amide bond and can be expressed as a single polypeptide chain.

[0131] (20-2) Preparation of EPO-PAS dimer peptide The prepared EPO-PAS dimer peptide is as follows. (p) EPO-PAS8 GGLYACHMGPMTWVCQPLRGAAPAAPAPGGLYACHMGPMTWVCQPLRG (SEQ ID NO: 102) (q) EPO-PAS22 GGLYACHMGPMTWVCQPLRGAAPAAPAPAAPAAPAPAAPAAPGGLYACHMGPMTWVCQPLRG (SEQ ID NO: 103) (r) AET-EPO-PAS8 AETGGLYACHMGPMTWVCQPLRGAAPAAPAPGGLYACHMGPMTWVCQPLRG (SEQ ID NO: 104) (s)AET-EPO-PAS22 AETGGLYACHMGPMTWVCQPLRGAAPAAPAPAAPAAPAPAAPAAPGGLYACHMGPMTWVCQPLRG (SEQ ID NO: 105)

[0132] (20-3) Expression of EPO-PAS dimer peptide The expression of these EPO-PAS dimer peptides was examined using Corynex®. Hereinafter, examples of the expression examination using Corynex® will be described.

[0133] (20-4) Construction of each secretion expression plasmid of EPO-PAS8 and EPO-PAS22 using CspA signal sequence As EPO-PAS dimer peptides, two kinds of amino acid sequences of the above (p)EPO-PAS8 (hereinafter sometimes referred to as "EPO-PAS8") and (q)EPO-PAS22 (hereinafter sometimes referred to as "EPO-PAS22") were designed respectively, and the base sequences encoding these proteins were designed considering the codon usage frequency of C. glutamicum. Furthermore, the following expression cassettes were designed respectively to enable secretion expression by C. glutamicum. EPO-PAS8 was secreted and expressed as a fusion protein of 25 amino acid residues of the signal peptide of CspA derived from C. ammoniagenes ATCC6872 strain and EPO-PAS8 (hereinafter referred to as "CspAss-EPO-PAS8"). The base sequence and amino acid sequence encoding the designed CspAss-EPO-PAS8 are shown in SEQ ID NO: 106 and 107 respectively.

[0134] Base sequence encoding CspAss-EPO-PAS8 atgaaacgcatgaaatcgctggctgcggcgctcaccgtcgctggggccatgctggccgcacctgtggcaacggcaggtggcctttacgcttgtcacatgggtccaatgacctgggtttgtcagcccctccgtggtgccgctcctgccgcccccgctcctggtggcctgtacgcatgccatatgggtccaatgacttgggtgtgccagccactccgcggctaa(SEQ ID NO: 106) Amino acid sequence of CspAss-EPO-PAS8 MKRMKSLAAALTVAGAMLAAPVATAGGLYACHMGPMTWVCQPLRGAAPAAPAPGGLYACHMGPMTWVCQPLRG(SEQ ID NO: 107)

[0135] EPO-PAS22 was secreted and expressed as a fusion protein (hereinafter referred to as "CspAss-EPO-PAS22") of 25 amino acid residues of the signal peptide of CspA derived from C. ammoniagenes ATCC6872 strain and EPO-PAS22. The nucleotide sequence and amino acid sequence encoding the designed CspAss-EPO-PAS22 are shown in SEQ ID NOs: 108 and 109, respectively.

[0136] Nucleotide sequence encoding CspAss-EPO-PAS22 atgaaacgcatgaaatcgctggctgcggcgctcaccgtcgctggggccatgctggccgcacctgtggcaacggcaggcggcctttacgcttgtcacatgggtccaatgacctgggtctgtcaacctctccgtggtgcagccccggcagcccccgctccagccgcaccagcagccccagccccagctgcaccagccgctccaggcggcctctacgcctgccacatgggtcctatgacctgggtgtgccaaccacttcgcggctaa(SEQ ID NO: 108) Amino acid sequence of CspAss-EPO-PAS22 MKRMKSLAAALTVAGAMLAAPVATAGGLYACHMGPMTWVCQPLRGAAPAAPAPAAPAAPAPAAPAAPGGLYACHMGPMTWVCQPLRG (SEQ ID NO: 109)

[0137] The promoter of the cspB gene derived from the C. glutamicum ATCC13869 strain was ligated upstream of the nucleotide sequences described in CspAss-EPO-PAS8 and CspAss-EPO-PAS22, and a KpnI site was added to the 5'-side and a BamHI site was added to the 3'-side. Expression cassettes for each EPO-PAS dimer peptide were designed and fully synthesized. The fully synthesized DNA fragment (expression cassette for the EPO-PAS dimer peptide) was inserted into the KpnI-BamHI site of pPK4 described in JP-A-9-322774 to construct pPK4_CspAss-EPO-PAS8 and pPK4_CspAss-EPO-PAS22, which are secretion expression plasmids for each EPO-PAS dimer peptide using the CspA signal sequence. As a result of nucleotide sequence determination of the inserted fragment, it was confirmed that the expression cassettes for the EPO-PAS dimer peptide were constructed as designed. Nucleotide sequence determination was performed using the BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the 3500xL Genetic Analyzer (Applied Biosystems).

[0138] (20-5) Construction of Secretion Expression Plasmids for AET-EPO-PAS8 and AET-EPO-PAS22 Using the CspB Signal Sequence As EPO-PAS dimer peptides, two types of amino acid sequences of the above (r)AET-EPO-PAS8 (hereinafter sometimes referred to as "AET-EPO-PAS8") and (s)AET-EPO-PAS22 (hereinafter sometimes referred to as "AET-EPO-PAS22") were designed respectively, and the nucleotide sequences encoding these proteins were designed considering the codon usage frequency of C. glutamicum. Furthermore, the following expression cassettes were designed respectively to enable secretory expression by C. glutamicum. AET-EPO-PAS8 was secreted and expressed as a fusion protein of the signal peptide of 30 amino acid residues of CspB derived from C. glutamicum ATCC13869 strain and AET-EPO-PAS8 (hereinafter referred to as "CspBss-AET-EPO-PAS8"). The nucleotide sequence and amino acid sequence of the designed CspBss-AET-EPO-PAS8 are shown in SEQ ID NO: 110 and SEQ ID NO: 111 respectively.

[0139] Nucleotide sequence encoding CspBss-AET-EPO-PAS8 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagaccggtggcctttacgcttgtcacatgggtccaatgacctgggtttgtcagcccctccgtggtgccgctcctgccgcccccgctcctggtggcctgtacgcatgccatatgggtccaatgacttgggtgtgccagccactccgcggctaa (SEQ ID NO: 110) Amino acid sequence of CspBss-AET-EPO-PAS8 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETGGLYACHMGPMTWVCQPLRGAAPAAPAPGGLYACHMGPMTWVCQPLRG (SEQ ID NO: 111)

[0140] AET-EPO-PAS22 was secreted and expressed as a fusion protein of the 30 amino acid residues of the signal peptide of CspB derived from the C. glutamicum ATCC13869 strain and AET-EPO-PAS22 (hereinafter referred to as "CspBss-AET-EPO-PAS22"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AET-EPO-PAS22 are shown in SEQ ID NO: 112 and SEQ ID NO: 113, respectively.

[0141] Nucleotide sequence encoding CspBss-AET-EPO-PAS22 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagaccggcggcctttacgcttgtcacatgggtccaatgacctgggtctgtcaacctctccgtggtgcagccccggcagcccccgctccagccgcaccagcagccccagccccagctgcaccagccgctccaggcggcctctacgcctgccacatgggtcctatgacctgggtgtgccaaccacttcgcggctaa(SEQ ID NO: 112) Amino acid sequence of CspBss-AET-EPO-PAS22 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETGGLYACHMGPMTWVCQPLRGAAPAAPAPAAPAAPAPAAPAAPGGLYACHMGPMTWVCQPLRG(SEQ ID NO: 113)

[0142] The promoter of the cspB gene derived from the C. glutamicum ATCC13869 strain was ligated upstream of the nucleotide sequences described in CspBss-AET-EPO-PAS8 and CspBss-AET-EPO-PAS22, and a KpnI site was added to the 5'-side and a BamHI site was added to the 3'-side. The expression cassettes of each EPO-PAS dimer peptide were designed and fully synthesized. The fully synthesized DNA fragment (expression cassette of the EPO-PAS dimer peptide) was inserted into the KpnI-BamHI site of pPK4 described in JP-A-9-322774 to construct pPK4_CspBss-AET-EPO-PAS8 and pPK4_CspBss-AET-EPO-PAS22, which are secretion expression plasmids of each EPO-PAS dimer peptide using the CspB signal sequence. As a result of nucleotide sequence determination of the inserted fragment, it was confirmed that the expression cassettes of the EPO-PAS dimer peptides as designed were constructed. The nucleotide sequence determination was performed using the BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the 3500xL Genetic Analyzer (Applied Biosystems).

[0143] (20-6) Secretion expression of each EPO-PAS dimer peptide in C. glutamicum Using pPK4_CspAss-EPO-PAS8, pPK4_CspAss-EPO-PAS22, pPK4_CspBss-AET-EPO-PAS8, and pPK4_CspBss-AET-EPO-PAS22 constructed above, the C. glutamicum YDK0107 strain described in WO2016 / 171224 was transformed to obtain the YDK0107 / pPK4_CspAss-EPO-PAS8 strain, YDK0107 / pPK4_CspAss-EPO-PAS22 strain, YDK0107 / pPK4_CspBss-AET-EPO-PAS8 strain, and YDK0107 / pPK4_CspBss-AET-EPO-PAS22 strain. Each of the obtained transformants was cultured at 30 °C for 72 hours in MMTG liquid medium (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, ferrous sulfate heptahydrate 0.03 g, manganese sulfate pentahydrate 0.03 g, thiamine hydrochloride 0.45 mg, biotin 0.45 mg, DL-methionine 0.15 g, soybean hydrochloride hydrolysate (total nitrogen content 0.2 g), calcium carbonate 50 g, adjusted to 1 L with water and pH 7.0) containing 25 mg / L of kanamycin. After completion of the culture, 6.5 μL of the culture supernatant obtained by centrifuging each culture solution was subjected to reducing SDS-PAGE using NuPAGE (registered trademark) 12% Bis-Tirs Gel (Thermo Fisher Scientific), and then stained with Quick-CBB (Wako). As a result, protein bands presumed to be EPO-PAS8 in the culture supernatant of the YDK0107 / pPK4_CspAss-EPO-PAS8 strain (Figure 32, lanes 2 to 5), protein bands presumed to be EPO-PAS22 in the culture supernatant of the YDK0107 / pPK4_CspAss-EPO-PAS22 strain (Figure 32, lanes 6 to 9), protein bands presumed to be AET-EPO-PAS8 in the culture supernatant of the YDK0107 / pPK4_CspBss-AET-EPO-PAS8 strain (Figure 32, lanes 10 to 13), and protein bands presumed to be AET-EPO-PAS22 in the culture supernatant of the YDK0107 / pPK4_CspBss-AET-EPO-PAS22 strain (Figure 32, lanes 14 to 17) were detected, respectively.

[0144] Example 21: Molecular weight analysis of EPO-PAS dimer peptide Regarding the cell filtrates of AET-EPO-PAS8 and AET-EPO-PAS22 after culturing in Example 20, the molecular weights were measured using LC-MS (Waters, ACQUITY UPLC / SQD2). The analysis conditions were as described in Table 2. As shown in FIGS. 33 and 34, values almost identical to the theoretical values of the molecular weights of the oxidized dimer peptides each forming two disulfide bonds within the molecule were obtained. From this, it was confirmed that peptides with the correct amino acid sequences containing two disulfide bonds were successfully expressed.

[0145] Example 22: Activity Evaluation of EPO-PAS Dimer Peptide The EpoR activation ability of the EPO-PAS dimer peptide was evaluated using the PathHunter® eXpress EpoR-JAK2 Functional Assay Kit (DiscoverX). Cells for EPO activity evaluation included in the PathHunter® eXpress EpoR-JAK2 Functional Assay Kit were seeded in a 96-well plate and cultured at 37 °C under 5% CO2 for 24 hours. To the cells, recombinant human erythropoietin (rhEPO, Peprotech) at 860 pg / L - 900 μg / L, or culture supernatants containing AET-EPO-PAS8 or AET-EPO-PAS22 diluted to a predetermined concentration (culturing was as described in Example 20), or the monomer peptide EMP35 (GGLYACHMGPMTWVCQPLRG, synthesized by GenScript Japan K.K.) were added. As a negative control, a culture supernatant obtained by culturing as described in Example 20 except using a vector not containing the gene for expressing the EPO-PAS dimer peptide (FIG. 32, lane 18), diluted at the same magnification as the culture supernatants containing AET-EPO-PAS8 or AET-EPO-PAS22, was added. To the cells stimulated at room temperature for 3 hours, the prepared Substrate Reagent was added and incubated for 60 minutes. The chemiluminescence intensity was quantified using a Nivo plate reader (Perkin Elmer). As shown in Fig. 35, it was revealed that AET-EPO-PAS8 and AET-EPO-PAS22 activate EpoR in a concentration-dependent manner. Their maximum activation ability was almost equivalent to that of rhEPO. Also, as shown in Table 4 below, the EC50 value of rhEPO is 1.1 x 10 -6 g / L, while that of AET-EPO-PAS8 is 1.9 x 10 -7 g / L and that of AET-EPO-PAS22 is 3.4 x 10 -7 g / L, indicating that they have high activity. Also, the monomeric peptide EMP35 shows an activity about 1000 times lower than that of the EPO-PAS dimeric peptide at 1.1 x 10 -4 g / L (Fig. 36, Table 4), revealing that higher agonist activity can be obtained by dimerization.

[0146]

Table 4

[0147] Example 23: Design of TPO-PAS Dimeric Peptide and Its Secretory Expression in C. glutamicum (23-1) Outline of Design Peptide dimers that bind to the thrombopoietin receptor c-Mpl cannot be expressed in C. glutamicum for the following reasons. Specifically, expressing the peptide dimer molecule (AF13948) obtained by dimerizing the linear c-Mpl-binding peptide AF12505 reported by S. E. Cwirla, et al. in Science, 276, 1696-1699, 1997 as a single polypeptide chain in C. glutamicum cannot be done for the following reasons. (1) Dimerization is achieved by binding the C-termini of the linear peptide AF12505 via the α- and ε-amino groups of lysine and cannot be expressed in C. glutamicum. (2) β-alanine, a non-proteinogenic amino acid, is bound to the C-terminus of AF12505 bound to the α-amino group of lysine, and peptides containing non-proteinogenic amino acids cannot be expressed in C. glutamicum. Therefore, the molecule was designed to be in an expressible form by making the following conversions. Instead of lysine and β-alanine, a PAS sequence was used, and the peptide sequence was designed to be "first AF12505" - "PAS linker" - "second AF12505" so that it could be expressed as a single polypeptide chain. That is, the C-terminus of the first AF12505 and the N-terminus of the PAS linker, and the N-terminus of the second AF12505 and the C-terminus of the PAS linker are each linked by an amide bond and can be expressed as a single polypeptide chain. Also, adding biotin to the C-terminus of the cyclic c-Mpl-binding peptide AF12285 reported in WO9640750 and multimerizing the peptide with streptavidin cannot be done in C. glutamicum as a single polypeptide chain for the following reasons. (1) Biotin is modified via the ε-amino group of lysine at the C-terminus of the cyclic peptide AF12285. Peptides containing biotin cannot be expressed in C. glutamicum. (2) The biotinylated cyclic peptide is multimerized with streptavidin. It is not possible to express streptavidin simultaneously with the peptide in C. glutamicum. Therefore, by adding the following molecular conversions, it was designed to be in an expressible form. Instead of multimerization with biotin-streptavidin, multimerization was performed using the PAS sequence, and the peptide sequence was designed to be "the first AF12285" - "PAS linker" - "the second AF12285" so that it can be expressed as a single polypeptide chain. That is, the C-terminus of the first AF12505 and the N-terminus of the PAS linker, and the N-terminus of the second AF12505 and the C-terminus of the PAS linker are each linked by an amide bond and can be expressed as a single polypeptide chain.

[0148] (23-2) Preparation of TPO-PAS dimer peptide The prepared TPO-PAS dimer peptide is as follows. (t) TPO2-PAS8 GGCADGPTLREWISFCGGAAPAAPAPGGCADGPTLREWISFCGG (SEQ ID NO: 114) (u) AET-TPO1-PAS8 AETIEGPTLRQWLAARAAAPAAPAPIEGPTLRQWLAARA (SEQ ID NO: 115) (v) AET-TPO2-PAS8 AETGGCADGPTLREWISFCGGAAPAAPAPGGCADGPTLREWISFCGG (SEQ ID NO: 116)

[0149] (23-3) Expression of TPO-PAS dimer peptide The expression of these TPO-PAS dimer peptides was examined using Corynex (registered trademark). Hereinafter, examples of the expression examination using Corynex (registered trademark) will be described.

[0150] (23-4) Construction of a secretion expression plasmid of TPO2-PAS8 using the CspA signal sequence As a TPO-PAS dimer peptide, the amino acid sequence of the above (t)TPO2-PAS8 (hereinafter sometimes referred to as "TPO2-PAS8") was designed, and the nucleotide sequence encoding this protein was designed in consideration of the codon usage frequency of C. glutamicum. Furthermore, the following expression cassette was designed to enable secretion expression by C. glutamicum. TPO2-PAS8 was secreted and expressed as a fusion protein (hereinafter referred to as "CspAss-TPO2-PAS8") of the 25 amino acid residues of the signal peptide of CspA derived from C. ammoniagenes ATCC6872 strain and TPO2-PAS8. The nucleotide sequence and amino acid sequence encoding the designed CspAss-TPO2-PAS8 are shown in SEQ ID NO: 117 and SEQ ID NO: 118, respectively.

[0151] Nucleotide sequence encoding CspAss-TPO2-PAS8 atgaaacgcatgaaatcgctggctgcggcgctcaccgtcgctggggccatgctggccgcacctgtggcaacggcaggtggttgtgctgatggtcctaccttgcgtgaatggatttccttctgcggcggcgctgctccggctgcccctgcccctggcggttgcgcagatggcccaaccctgcgcgaatggatctccttctgcggcggctaa(SEQ ID NO: 117) Amino acid sequence of CspAss-TPO2-PAS8 MKRMKSLAAALTVAGAMLAAPVATAGGCADGPTLREWISFCGGAAPAAPAPGGCADGPTLREWISFCGG(SEQ ID NO: 118)

[0152] The promoter of the cspB gene derived from the C. glutamicum ATCC13869 strain was ligated upstream of the nucleotide sequence described in CspAss-TPO2-PAS8, and a KpnI site was added to the 5'-side and a BamHI site was added to the 3'-side to design and fully synthesize an expression cassette for the TPO-PAS dimer peptide. The fully synthesized DNA fragment (expression cassette for the TPO-PAS dimer peptide) was inserted into the KpnI-BamHI site of pPK4 described in JP-A-9-322774 to construct pPK4_CspAss-TPO2-PAS8, a secretion expression plasmid for the TPO-PAS dimer peptide that utilizes the CspA signal sequence. As a result of nucleotide sequence determination of the inserted fragment, it was confirmed that an expression cassette for the TPO-PAS dimer peptide as designed was constructed. Nucleotide sequence determination was performed using the BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the 3500xL Genetic Analyzer (Applied Biosystems).

[0153] (23-5) Construction of Secretion Expression Plasmids for AET-TPO1-PAS8 and AET-TPO2-PAS8 Utilizing the CspB Signal Sequence As the TPO-PAS dimer peptide, two types of amino acid sequences, namely (u) AET-TPO1-PAS8 (hereinafter sometimes referred to as "AET-TPO1-PAS8") and (v) AET-TPO2-PAS8 (hereinafter sometimes referred to as "AET-EPO2-PAS8"), were designed respectively, and the nucleotide sequences encoding these proteins were designed in consideration of the codon usage frequency of C. glutamicum. Furthermore, the following expression cassettes were designed respectively to enable secretion expression by C. glutamicum. AET-TPO1-PAS8 was secreted and expressed as a fusion protein of the 30 amino acid residues of the signal peptide of CspB derived from C. glutamicum ATCC13869 strain and AET-TPO1-PAS8 (hereinafter referred to as "CspBss-AET-TPO1-PAS8"). The nucleotide sequence and amino acid sequence designed to encode CspBss-AET-TPO1-PAS8 are shown in SEQ ID NO: 119 and SEQ ID NO: 120, respectively.

[0154] Nucleotide sequence encoding CspBss-AET-TPO1-PAS8 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagaccatcgaaggtcctaccctccgtcaatggctggctgctcgtgccgccgctcccgccgctcctgctccaatcgaaggtcccaccctccgtcagtggctggcagcccgcgcataa (SEQ ID NO: 119) Amino acid sequence of CspBss-AET-TPO1-PAS8 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETIEGPTLRQWLAARAAAPAAPAPIEGPTLRQWLAARA (SEQ ID NO: 120)

[0155] AET-TPO2-PAS8 was secreted and expressed as a fusion protein of the 30 amino acid residues of the signal peptide of CspB derived from C. glutamicum ATCC13869 strain and AET-TPO2-PAS8 (hereinafter referred to as "CspBss-AET-TPO2-PAS8"). The nucleotide sequence and amino acid sequence designed to encode CspBss-AET-TPO2-PAS8 are shown in SEQ ID NO: 121 and SEQ ID NO: 122, respectively.

[0156] Nucleotide sequence encoding CspBss-AET-TPO2-PAS8 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagaccggtggttgtgctgatggtcctaccttgcgtgaatggatttccttctgcggcggcgctgctccggctgcccctgcccctggcggttgcgcagatggcccaaccctgcgcgaatggatctccttctgcggcggctaa(SEQ ID NO: 121) Amino acid sequence of CspBss-AET-TPO2-PAS8 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETGGCADGPTLREWISFCGGAAPAAPAPGGCADGPTLREWISFCGG(SEQ ID NO: 122)

[0157] The promoter of the cspB gene derived from the C. glutamicum ATCC13869 strain was connected upstream of the nucleotide sequences described in CspBss-AET-TPO1-PAS8 and CspBss-AET-TPO2-PAS8, and a KpnI site was added to the 5'-side and a BamHI site was added to the 3'-side. The expression cassettes of each TPO-PAS dimer peptide were designed and fully synthesized. The fully synthesized DNA fragment (expression cassette of the TPO-PAS dimer peptide) was inserted into the KpnI-BamHI site of pPK4 described in JP-A-9-322774 to construct pPK4_CspBss-AET-TPO1-PAS and pPK4_CspBss-AET-TPO2-PAS8, which are secretion expression plasmids of each TPO-PAS dimer peptide using the CspB signal sequence, respectively. As a result of nucleotide sequencing of the inserted fragment, it was confirmed that the expression cassettes of the TPO-PAS dimer peptides as designed were constructed. Nucleotide sequencing was performed using the BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the 3500xL Genetic Analyzer (Applied Biosystems).

[0158] (23-6) Secretory expression of each TPO-PAS dimer peptide in C. glutamicum Using pPK4_CspAss-TPO2-PAS8, pPK4_CspBss-AET-TPO1-PAS8, and pPK4_CspBss-AET-TPO2-PAS8 constructed above, the C. glutamicum YDK0107 strain described in WO2016 / 171224 was transformed to obtain the YDK0107 / pPK4_CspAss-TPO2-PAS8 strain, the YDK0107 / pPK4_CspBss-AET-TPO1-PAS8 strain, and the YDK0107 / pPK4_CspBss-AET-TPO2-PAS8 strain. Each of the obtained transformants was cultured at 30 °C for 72 hours in MMTG liquid medium (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, ferrous sulfate heptahydrate 0.03 g, manganese sulfate pentahydrate 0.03 g, thiamine hydrochloride 0.45 mg, biotin 0.45 mg, DL-methionine 0.15 g, soybean hydrochloride hydrolysate (total nitrogen content 0.2 g), calcium carbonate 50 g, adjusted to pH 7.0 with water to 1 L) containing 25 mg / L of kanamycin. After the cultivation was completed, 6.5 μL of the culture supernatant obtained by centrifuging each culture solution was subjected to reducing SDS-PAGE using NuPAGE® 12% Bis-Tirs Gel (Thermo Fisher Scientific) and then stained with Quick-CBB (Wako). As a result, a protein band presumed to be AET-TPO1-PAS8 (Figure 37, lanes 2-5) was detected in the culture supernatant of the YDK0107 / pPK4_CspBss-AET-TPO1-PAS8 strain, a protein band presumed to be TPO2-PAS8 (Figure 37, lanes 6-9) was detected in the culture supernatant of the YDK0107 / pPK4_CspAss-TPO2-PAS8 strain, and a protein band presumed to be AET-TPO2-PAS8 (Figure 37, lanes 10-13) was detected in the culture supernatant of the YDK0107 / pPK4_CspBss-AET-TPO2-PAS8 strain.

[0159] Example 24: Molecular Weight Analysis of TPO-PAS Dimer Peptide Regarding the bacterial cell filtrate of the TPO-PAS dimer peptide after culturing in Example 23, the molecular weight was measured using LC-MS (Waters, ACQUITY UPLC / SQD2). The analysis conditions are as described in Table 2. As shown in Figures 38 to 40, values almost identical to the theoretical values of the molecular weights of the oxidized dimer peptides each forming two disulfide bonds within the molecule were obtained. From this, it was confirmed that peptides with the correct amino acid sequence containing two disulfide bonds could be expressed.

[0160] Example 25: Activity Evaluation of TPO-PAS Dimer Peptide The thrombopoietin-like activity of the TPO-PAS dimer peptide was evaluated by quantifying the tyrosine phosphorylation level of HEL cells, a human leukemia-derived blood cell line. 200,000 HEL cells were cultured in RPMI1640 medium (Thermo Fisher Scientific) without FBS for 24 hours to make them in a starved state. These cells were stimulated for 20 minutes with RPMI1640 medium supplemented with recombinant human TPO (rhTPO, Peprotech) at 10 - 100 ng / mL or culture supernatants containing AET-TPO1-PAS8, TPO2-PAS8, or AET-TPO2-PAS8 at a predetermined dilution ratio. As a negative control, PBS (Mock group) or a supernatant not expressing the peptide by using an empty vector (Empty group) was added and stimulated for 20 minutes. After removing the medium and washing the cells once with PBS, the cells were lysed by adding 100 μL of Lysis buffer 17 (R&D Systems) supplemented with Protease inhibitor cocktail (Nacalai Tesque). The lysate was centrifuged at 14,000 G for 5 minutes, and the supernatant was obtained as the lysate. A PVDF membrane (Immobilon-P membrane 0.45 μm, Merck) was treated with methanol for 1 minute and then with ultrapure water for 5 minutes for hydrophilic treatment. The PVDF membrane was dried, and each cell lysate was spotted with a 1 μL pipette and dried. The PVDF membrane was blocked with PVDF Blocking Reagent for Can Get Signal (NOF Corporation) at room temperature for 1 hour, and then Can Get Signal Solution 2 (NOF Corporation) containing anti-pY antibody-HGF conjugate (Cell Signaling Technology) added at a ratio of 1:5000 was added, and an antibody reaction was carried out at 4 °C for 24 hours. The membrane was washed three times with TBST buffer, Luminata Crescendo Western HRP Substrate (Merck) was added, and a chemiluminescence reaction was carried out. Chemiluminescence was detected using an Amersham Imager 600 (GE Healthcare). The luminescence intensity of the detected dots was quantified using ImageJ. As shown in Fig. 41, an increase in tyrosine phosphorylation level equal to or higher than that of rhTPO was observed in all of AET-TPO1-PAS8, AET-TPO2-PAS8, and TPO2-PAS8. From this result, it became clear that AET-TPO1-PAS8, AET-TPO2-PAS8, and TPO2-PAS8 activated HEL cells in the same manner as TPO. Fig. 42 shows the concentration-dependent HEL cell activation ability of TPO2-PAS8. In the range of 1 / 100 - 1 / 10000 dilution, since the TPO2-PAS8-stimulated group showed a luminescence intensity equal to or higher than that of the 100 ng / mL rhTPO-stimulated group, it became clear that it had an HEL cell activation ability equal to or higher than that of rhTPO in this concentration range.

[0161] Example 26: Evaluation of Signal Characteristics of TPO-PAS Dimer Peptide The signal characteristics of the TPO-PAS dimer peptide were evaluated using the Proteome Profiler Human Phospho-Kinase Array Kit (R&D Systems). 1,000,000 HEL cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific) without FBS for 24 hours to induce a starved state. These cells were then stimulated with 100 ng / mL of recombinant human TPO (rhTPO, Peprotech) or RPMI 1640 medium supplemented with culture supernatant containing TPO2-PAS8 at a dilution of 1 / 100 - 1 / 1000 for 20 minutes. As a negative control, PBS was added and the cells were stimulated for 20 minutes (Mock group). After removing the medium and washing the cells once with PBS, the cells were lysed by adding 200 μL of Lysis buffer 17 (R&D Systems) supplemented with Protease inhibitor cocktail (Nacalai Tesque). The lysate was centrifuged at 14,000 G for 5 minutes, and the supernatant was obtained as the lysate. The lysate was analyzed using the Proteome Profiler Human Phospho-Kinase Array Kit. An Amersham Imager 600 (GE Healthcare) was used for chemiluminescence detection. The chemiluminescence intensity of the dots was quantified using ImageJ. As shown in Figure 43, it was confirmed that in cells stimulated with rhTPO or TPO2-PAS8 compared to cells stimulated with PBS (Mock group), the phosphorylation levels of Erk, CREB, Stat3, Stat5a, and Stat5b increased, and the phosphorylation patterns of both were similar. The signal pathways stimulated by rhTPO are mainly the JAK2 / Stat3,5 pathways, and all the proteins with increased phosphorylation levels are included in the pathways. From these results, it was confirmed that TPO2-PAS8 activates signals similar to rhTPO.

[0162] Example 27: Design of VEGFR_D2-PAS Heterodimer Peptide and Its Secretion and Expression in C. glutamicum (2) Overview of Design The peptide heterodimer molecule in which the VEGFR-binding cyclic peptides P-3 and P-4 reported in A. Shrivastava, et al. Protein Eng. Des. Select. 18, 417, 2005 are dimerized by a glutaric acid linker cannot be expressed in C. glutamicum as a single polypeptide chain for the following reasons. Dimerization is achieved by binding the ε-amino groups of the lysine residues located at the C-terminals of the cyclic peptides P-3 and P-4 with glutaric acid, and it cannot be expressed in C. glutamicum. Therefore, the molecule was designed into an expressible form by applying the following conversions. Instead of using a glutaric acid linker, a PAS sequence was used, and the peptide sequence was designed to be "the first VEGFR-binding sequence (P-3)" - "PAS linker" - "the second VEGFR-binding sequence (P-4)" so that it can be expressed as a single polypeptide chain. That is, the C-terminal of P-3 and the N-terminal of the PAS linker, and the N-terminal of P-4 and the C-terminal of the PAS linker are each linked by an amide bond and can be expressed as a single polypeptide chain.

[0163] (27-2) Preparation of VEGFR_D2-PAS heterodimer peptide The prepared VEGFR_D2-PAS heterodimer peptide is as follows. (w)AET-VEGFR_D2-PAS16 AETAGPTWCEDDWYYCWLFGTAAPAAPAPAAPAAPAPVCWEDSWGGEVCWLFGT (SEQ ID NO: 123)

[0164] (27-3) Expression of VEGFR_D2-PAS heterodimer peptide The expression of the VEGFR_D2-PAS heterodimer peptide was examined using Corynex (registered trademark). Hereinafter, examples of the expression examination using Corynex (registered trademark) will be described.

[0165] Construction of AET-VEGFR_D2-PAS16 Secretory Expression Plasmid Using CspB Signal Sequence As the VEGFR_D2-PAS heterodimer peptide, the amino acid sequence of the above (w)AET-VEGFR_D2-PAS16 (hereinafter, may be referred to as "AET-VEGFR_D2-PAS16" or simply "VEGFR_D2-PAS16") was designed, and the nucleotide sequence encoding this protein was designed considering the codon usage frequency of C. glutamicum. Furthermore, the following expression cassette was designed to enable secretory expression by C. glutamicum. AET-VEGFR_D2-PAS16 was secreted and expressed as a fusion protein of the signal peptide of 30 amino acid residues of CspB derived from C. glutamicum ATCC13869 strain and AET-VEGFR_D2-PAS16 (hereinafter, referred to as "CspBss-VEGFR_D2-PAS16"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AET-VEGFR_D2-PAS16 are shown in SEQ ID NO: 124 and SEQ ID NO: 125, respectively.

[0166] Nucleotide sequence encoding CspBss-AET-VEGFR_D2-PAS16 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagaccgctggtcctacctggtgtgaagacgactggtactactgctggctgtttggcaccgctgctcccgctgcccctgctcctgccgctccggcagccccagctcctgtgtgttgggaagattcttggggcggcgaagtttgctggctgtttggcacctaa (SEQ ID NO: 124) Amino acid sequence of CspBss-AET-VEGFR_D2-PAS16 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETAGPTWCEDDWYYCWLFGTAAPAAPAPAAPAAPAPVCWEDSWGGEVCWLFGT (SEQ ID NO: 125)

[0167] The promoter of the cspB gene derived from the C. glutamicum ATCC13869 strain was linked upstream of the nucleotide sequence described in CspBss-AET-VEGFR_D2-PAS16, and a KpnI site was added to the 5'-side and a BamHI site was added to the 3'-side to design and fully synthesize an expression cassette for the VEGFR_D2-PAS heterodimer peptide. The fully synthesized DNA fragment (expression cassette for the VEGFR_D2-PAS heterodimer peptide) was inserted into the KpnI-BamHI site of pPK4 described in JP-A-9-322774 to construct pPK4_CspBss-AET-VEGFR_D2-PAS16, a secretion expression plasmid for the VEGFR_D2-PAS heterodimer peptide using the CspB signal sequence. As a result of nucleotide sequence determination of the inserted fragment, it was confirmed that an expression cassette for the VEGFR_D2-PAS heterodimer peptide as designed was constructed. Nucleotide sequence determination was performed using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the 3500xL Genetic Analyzer (Applied Biosystems).

[0168] (27-5) Secretion expression of the VEGFR_D2-PAS heterodimer peptide in C. glutamicum Using pPK4_CspBss-AET-VEGFR_D2-PAS16 constructed above, the C. glutamicum YDK0107 strain described in WO2016 / 171224 was transformed to obtain the YDK0107 / pPK4_CspBss-AET-VEGFR_D2-PAS16 strain. The obtained transformants were cultured at 30 °C for 72 hours in MMTG liquid medium (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, ferrous sulfate heptahydrate 0.03 g, manganese sulfate pentahydrate 0.03 g, thiamine hydrochloride 0.45 mg, biotin 0.45 mg, DL-methionine 0.15 g, soybean hydrochloride hydrolysate (total nitrogen content 0.2 g), calcium carbonate 50 g, adjusted to 1 L with water and pH 7.0) containing 25 mg / L kanamycin. After the culture was completed, 6.5 μL each of the culture supernatant obtained by centrifuging the culture broth, or the culture broth containing the cells and the culture supernatant without centrifugation, was subjected to reducing SDS-PAGE using NuPAGE (registered trademark) 12% Bis-Tirs Gel (Thermo Fisher Scientific), and then stained with Quick-CBB (Wako). As a result, a very small amount of a protein band presumed to be AET-VEGFR_D2-PAS16 was detected in the culture supernatant of the YDK0107 / pPK4_CspBss-AET-VEGFR_D2-PAS16 strain (Figure 45, lane 2). On the other hand, a prominent band of the protein presumed to be AET-VEGFR_D2-PAS16 was detected in the culture broth sample containing the cells (Figure 45, lane 3). From the above results, it was considered that after secretion, most of AET-VEGFR_D2-PAS16 aggregated and existed in the culture supernatant, or adsorbed on the cell surface of the cultured cells.

[0169] (27-6) Solubilization of VEGFR_D2-PAS heterodimer peptide by urea treatment To the precipitate fraction containing the cells obtained by centrifuging the culture broth of the YDK0107 / pPK4_CspBss-AET-VEGFR_D2-PAS16 strain obtained above, an equal volume of the removed culture supernatant and 0.5 M Urea (50 mM Tris-HCl, pH 8.0) or 1.0 M Urea (50 mM Tris-HCl, pH 8.0) were added and mixed. 6.5 μL each of the supernatant fraction obtained by centrifuging each Urea-treated solution, or the mixed solution containing the cells and the supernatant uniformly without centrifugation, was subjected to reducing SDS-PAGE using NuPAGE (registered trademark) 12% Bis-Tirs Gel (Thermo Fisher Scientific), and then stained with Quick-CBB (Wako). As a result, protein bands presumed to be AET-VEGFR_D2-PAS16 were extracted into the supernatant in the 0.5 M Urea and 1.0 M Urea-treated samples and obtained as a soluble fraction (Figure 45, lanes 6 and 8).

[0170] Example 28: Molecular weight analysis of VEGFR_D2-PAS heterodimer Regarding the solution obtained by washing the cells expressing the VEGFR_D2-PAS heterodimer after culturing in Example 27 with 8 M Urea, after filtering the cells, the molecular weight was measured using LC-MS (Waters, ACQUITY UPLC / SQD2). The analysis conditions are shown in Table 5.

[0171]

Table 5

[0172] As shown in Figure 46, a value almost identical to the theoretical value of the molecular weight of the oxidized dimer peptide forming two disulfide bonds in the molecule was obtained. From this, it was confirmed that a peptide with the correct amino acid sequence containing two disulfide bonds could be expressed.

[0173] Example 29: Activity evaluation of VEGFR_D2-PAS heterodimer The VEGF inhibitory ability of the VEGFR_D2-PAS heterodimer peptide was evaluated by a luciferase assay. VEGFR / NFAT Reporter HEK293 cells (BPS Bioscience) at 40,000 cells / well were seeded and incubated overnight in a 37 °C, 5% CO2 incubator. The cells were incubated overnight in Assay medium (MEM medium (Nacalai Tesque), 1x MEM Non-Essential Amino Acids Solution (Thermo Fisher Scientific), 1 mM Sodium Pyruvate (Nacalai Tesque), 1% Penicillin-Streptomycin (Sigma Aldrich), 0.3% FBS (Thermo Fisher Scientific)) to be in a starved state. 10 ng / mL of human recombinant VEGF (R&D Systems) was added to the Assay medium, and VEGFR_D2-PAS16 or the monomeric peptide P3 (AGPTWCEDDWYYCWLFGT (SEQ ID NO: 71), synthesized by GenScript Japan K.K.), Pm4 (VCWEDSWGGEVCWLFGT (SEQ ID NO: 72), synthesized by GenScript Japan K.K.) purified by HPLC at a predetermined dilution ratio was added, and 100 μL of the Assay medium was added to the cells. The cells were stimulated by culturing in a 37 °C, 5% CO2 incubator for 4 hours. The One-Glo Luciferase Assay System (Promega) was used for detecting the signal intensity. 50 μL of the culture supernatant was removed, 50 μL of the One-Glo reagent was added, and the cells were lysed at room temperature for 10 minutes. The activation of the VEGFR-NFAT pathway was quantified by detecting the luminescence emitted from the cell lysate with a Nivo plate reader (Perkin Elmer), and the relative signal activity value with respect to the sample without the evaluation compound was determined and used as the relative reporter activity. As shown in Fig. 47, since VEGFR_D2-PAS16 showed higher inhibitory activity than the monomeric peptides P3 and Pm4, it was revealed that high inhibitory activity can be obtained by heterodimerization of cyclic peptides.

[0174] Example 30: Design of EPO-PAS dimer peptide linker variants and their secretory expression in C. glutamicum (30-1) Outline of the design of linker variants The erythropoietin-mimicking peptides AET-EPO-PAS8 and AET-EPO-PAS22 have a structure in which a first erythropoietin receptor-binding cyclic peptide and a second erythropoietin receptor-binding cyclic peptide are linked by a PAS linker consisting of 8 or 22 amino acids. By changing the length of this PAS linker, it is expected that the erythropoietin receptor-binding activity will change. Therefore, linker variants were designed in which the PAS linker region was changed to a PAS repeat sequence of 49 or 100 amino acids. In addition to the PAS linker, a GS linker, which is a repeating sequence of glycine and serine, is also known as a flexible linker. Therefore, a linker variant in which an EPO cyclic peptide was bound to the N-terminus and C-terminus of a GS linker consisting of a repeating sequence of 4 glycine residues and 1 serine residue (GGGGS), that is, a peptide sequence was designed so as to be "first erythropoietin receptor-binding cyclic peptide" - "GS linker" - "second erythropoietin receptor-binding cyclic peptide".

[0175] (30-2) Preparation of EPO-PAS dimer peptide linker variants The prepared EPO-PAS dimer peptide linker variants are as follows. (x) AET-EPO-PAS49 AETGGLYACHMGPMTWVCQPLRGAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAGGLYACHMGPMTWVCQPLRG (SEQ ID NO: 126) (y) AET-EPO-PAS49-b AETGGLYACHMGPMTWVCQPLRGSAPSPSSAPASASAPASPASASASASPASSPASASSASPAASSASPASSGGLYACHMGPMTWVCQPLRG (Sequence number: 127) (z)AET-EPO-PAS100 AETGGLYACHMGPMTWVCQPLRGAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAGGLYACHMGPMTWVCQPLRG ((Sequence number: 128) (A)AET-EPO-PAS100-b AETGGLYACHMGPMTWVCQPLRGSAPSPSSAPASASAPASPASASASASPASSPASASSASPAASSASPASSSASPSAPAAPSASAAPSASPASSSPAPASAPSPAAAASSPSPAPSSASSAAGGLYACHMGPMTWVCQPLRG (Sequence number: 129) (B)AET-EPO-GS8 AETGGLYACHMGPMTWVCQPLRGGGGGSGGGGGLYACHMGPMTWVCQPLRG (Sequence number: 130) (C)AET-EPO-GS22 AETGGLYACHMGPMTWVCQPLRGGGGGSGGGGSGGGGSGGGGLYACHMGPMTWVCQPLRG (Sequence number: 131)

[0176] (30-3) Expression of EPO-PAS dimer peptide linker variants The expression of these EPO-PAS dimer peptide linker variants was examined using Corynex (registered trademark). Hereinafter, examples of the expression examination using Corynex (registered trademark) will be described.

[0177] Construction of Secretion Expression Plasmids of AET-EPO-PAS49, AET-EPO-PAS49-b, AET-EPO-PAS100, AET-EPO-PAS100-b, AET-EPO-GS8, and AET-EPO-GS22 Using the CspB Signal Sequence As EPO-PAS dimer peptide linker variants, the six amino acid sequences of (x) AET-EPO-PAS49 (hereinafter sometimes referred to as "AET-EPO-PAS49"), (y) AET-EPO-PAS49-b (hereinafter sometimes referred to as "AET-EPO-PAS49-b"), (z) AET-EPO-PAS100 (hereinafter sometimes referred to as "AET-EPO-PAS100"), (A) AET-EPO-PAS100-b (hereinafter sometimes referred to as "AET-EPO-PAS100-b"), (B) AET-EPO-GS8 (hereinafter sometimes referred to as "AET-EPO-GS8"), and (C) AET-EPO-GS22 (hereinafter sometimes referred to as "AET-EPO-GS22") were designed respectively, and the nucleotide sequences encoding these proteins were designed considering the codon usage frequency of C. glutamicum. Furthermore, the following expression cassettes were designed respectively to enable secretion expression by C. glutamicum. AET-EPO-PAS49 was secreted and expressed as a fusion protein of the 30 amino acid residues of the signal peptide of CspB derived from C. glutamicum ATCC13869 strain and AET-EPO-PAS49 (hereinafter referred to as "CspBss-AET-EPO-PAS49"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AET-EPO-PAS49 are shown in SEQ ID NOs: 132 and 133, respectively.

[0178] Nucleotide Sequence Encoding CspBss-AET-EPO-PAS49 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagaccggtggtctctacgcctgtcacatgggtcctatgacctgggtgtgccaacctctccgtggtgccgcccccgccgcccccgcccccgccgccccagctgcaccagctccagcagccccagccgcaccagccccagcagctccagctgccccagcaccagcagcaccagccgctccagcaccagctgctccagcagctccagccccagccggcggcctttacgcctgccacatgggtcctatgacctgggtttgccagcctcttcgcggttag(SEQ ID NO: 132) Amino acid sequence of CspBss-AET-EPO-PAS49 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETGGLYACHMGPMTWVCQPLRGAAPAAPAPAAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAGGLYACHMGPMTWVCQPLRG(SEQ ID NO: 133)

[0179] AET-EPO-PAS49-b was secreted and expressed as a fusion protein of 30 amino acid residues of the signal peptide of CspB derived from C. glutamicum ATCC13869 strain and AET-EPO-PAS49-b (hereinafter referred to as "CspBss-AET-EPO-PAS49-b"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AET-EPO-PAS49-b are shown in SEQ ID NO: 134 and SEQ ID NO: 135, respectively.

[0180] Nucleotide sequence encoding CspBss-AET-EPO-PAS49-b atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagaccggcggactgtacgcctgccatatggggccaatgacctgggtgtgccaaccgttgcgtggatcagcgccatcgccctcttccgctccagcctctgcgtctgctcccgcatccccagcgtcagcttccgcatccgcttcgcctgcaagctcccctgctagcgcgtcctctgcctcccctgcagcatcctcggcttccccggcaagctcaggtggcctctatgcctgccacatgggtccgatgacttgggtctgtcagccacttcgcggctaa(SEQ ID NO: 134) Amino acid sequence of CspBss-AET-EPO-PAS49-b MFNNRIRTAALAGAIAISTAASGVAIPAFAAETGGLYACHMGPMTWVCQPLRG(SEQ ID NO: 135)

[0181] AET-EPO-PAS100 was secreted and expressed as a fusion protein of 30 amino acid residues of the signal peptide of CspB derived from C. glutamicum ATCC13869 strain and AET-EPO-PAS100 (hereinafter referred to as "CspBss-AET-EPO-PAS100"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AET-EPO-PAS100 are shown in SEQ ID NO: 136 and SEQ ID NO: 137, respectively.

[0182] Nucleotide sequence encoding CspBss-AET-EPO-PAS100 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagaccggtggtttgtacgcttgccacatgggtcctatgacctgggtttgtcagcctcttcgtggtgccgcccccgcagcccccgctcccgctgcaccagccgcaccagcaccagcagctccagcagctccagcaccagctgcaccagccgccccagcaccagcagccccagccgctccagcaccagctgccccagctgctccagctccagcagcaccagctgccccagccccagcagcaccagccgctccagccccagctgccccagcagctccagctccagctgctccagcagccccagcaccagccgccccagcagccccagctccagctgcaccagctgcaccagccccagccgcaccagccggcggtttgtacgcttgccacatgggtccgatgacctgggtttgtcagccccttcgcggttag(SEQ ID NO: 136) Amino acid sequence of CspBss-AET-EPO-PAS100 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETGGLYACHMGPMTWVCQPLRGAAPAAPAPAAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAGGLYACHMGPMTWVCQPLRG(SEQ ID NO: 137)

[0183] AET-EPO-PAS100-b was secreted and expressed as a fusion protein of the 30 amino acid residues of the signal peptide of CspB derived from the C. glutamicum ATCC13869 strain and AET-EPO-PAS100-b (hereinafter referred to as "CspBss-AET-EPO-PAS100-b"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AET-EPO-PAS100-b are shown in SEQ ID NO: 138 and SEQ ID NO: 139, respectively.

[0184] Nucleotide sequence encoding CspBss-AET-EPO-PAS100-b atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagaccggcggcttgtacgcctgccatatggggcctatgacctgggtctgccaaccactgcgtggaagcgcaccctcaccatcctccgcaccagctagcgcctccgctcctgcaagcccagcgtccgcatccgcttcggcctcaccagcgtcctctccagcttccgcttcgagcgctagcccagcggcatcctctgcctccccagcctcctcgtctgcatccccgtccgctccagccgcaccttctgcatctgcggctccgtccgcctccccggcatcttcctcgcctgctcccgcgtcagcgccttccccggctgcagcggcctcctctccgtcacccgctccttcaagcgcgtcgtctgcagcaggaggcctctatgcctgccacatgggtccgatgacttgggtgtgtcagccccttcgcggttaa (SEQ ID NO: 138) Amino acid sequence of CspBss-AET-EPO-PAS100-b MFNNRIRTAALAGAIAISTAASGVAIPAFAAETGGLYACHMGPMTWVCQPLRGSAPSPSSAPASASAPASPASASASASPASSPASASSASPAASSASPASSSASPSAPAAPSASAAPSASPASSSPAPASAPSPAAAASSPSPAPSSASSAAGGLYACHMGPMTWVCQPLRG (SEQ ID NO: 139)

[0185] AET-EPO-GS8 was secreted and expressed as a fusion protein of the 30 amino acid residues of the signal peptide of CspB derived from C. glutamicum ATCC13869 strain and AET-EPO-GS8 (hereinafter referred to as "CspBss-AET-EPO-GS8"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AET-EPO-GS8 are shown in SEQ ID NO: 140 and SEQ ID NO: 141, respectively.

[0186] Nucleotide sequence encoding CspBss-AET-EPO-GS8 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagaccggcggcctctatgcatgccatatggggccaatgacctgggtctgccaaccgttgcgtggcggaggtggcggttccggtggaggtggtggcctgtacgcttgccacatgggacccatgacttgggtgtgtcagcctcttcgcggctaa (SEQ ID NO: 140) Amino acid sequence of CspBss-AET-EPO-GS8 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETGGLYACHMGPMTWVCQPLRGGGGGSGGGGGLYACHMGPMTWVCQPLRG (SEQ ID NO: 141)

[0187] AET-EPO-GS22 was secreted and expressed as a fusion protein of the 30 amino acid residues of the signal peptide of CspB derived from the C. glutamicum ATCC13869 strain and AET-EPO-GS22 (hereinafter referred to as "CspBss-AET-EPO-GS22"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AET-EPO-GS22 are shown in SEQ ID NO: 142 and SEQ ID NO: 143, respectively.

[0188] Nucleotide sequence encoding CspBss-AET-EPO-GS22 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagaccggtgggctgtatgcttgccatatggggcctatgacttgggtgtgccaacccttgcgtggtggaggcggaggctccggcggtggtgggagcggaggaggcggttccggtggcggtggctctggcggaggcggcctctacgcatgccacatgggtccgatgacctgggtctgtcagccacttcgcggctaa (SEQ ID NO: 142) Amino acid sequence of CspBss-AET-EPO-GS22 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETGGLYACHMGPMTWVCQPLRGGGGGSGGGGSGGGGSGGGGSGGGGLYACHMGPMTWVCQPLRG (SEQ ID NO: 143)

[0189] The promoter of the cspB gene derived from the C. glutamicum ATCC13869 strain was ligated upstream of the nucleotide sequences described in CspBss-AET-EPO-PAS49, CspBss-AET-EPO-PAS49-b, CspBss-AET-EPO-PAS100, CspBss-AET-EPO-PAS100-b, CspBss-AET-EPO-GS8, and CspBss-AET-EPO-GS22, and a KpnI site was added to the 5'-side and a BamHI site was added to the 3'-side. Expression cassettes of each EPO-PAS dimer peptide linker variant were designed and fully synthesized. The fully synthesized DNA fragment (expression cassette of the EPO-PAS dimer peptide linker variant) was inserted into the KpnI-BamHI site of pPK4 described in JP-A-9-322774 to construct pPK4_CspBss-AET-EPO-PAS49, pPK4_CspBss-AET-EPO-PAS49-b, pPK4_CspBss-AET-EPO-PAS100, pPK4_CspBss-AET-EPO-PAS100-b, pPK4_CspBss-AET-EPO-GS8, and pPK4_CspBss-AET-EPO-GS22, which are secretion expression plasmids of each EPO-PAS dimer peptide linker variant using the CspB signal sequence. As a result of nucleotide sequence determination of the inserted fragment, it was confirmed that the expression cassettes of the EPO-PAS dimer peptide linker variants were constructed as designed. Nucleotide sequence determination was performed using the BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the 3500xL Genetic Analyzer (Applied Biosystems).

[0190] (30-5) Secretion expression of each EPO-PAS dimer peptide in C. glutamicum Using pPK4_CspBss-AET-EPO-PAS49, pPK4_CspBss-AET-EPO-PAS49-b, pPK4_CspBss-AET-EPO-PAS100, pPK4_CspBss-AET-EPO-PAS100-b, pPK4_CspBss-AET-EPO-GS8, and pPK4_CspBss-AET-EPO-GS22 constructed above, the C. glutamicum YDK0107 strain described in WO2016 / 171224 was transformed to obtain the YDK0107 / pPK4_CspBss-AET-EPO-PAS49 strain, YDK0107 / pPK4_CspBss-AET-EPO-PAS49-b strain, YDK0107 / pPK4_CspBss-AET-EPO-PAS100 strain, YDK0107 / pPK4_CspBss-AET-EPO-PAS100-b strain, YDK0107 / pPK4_CspBss-AET-EPO-GS8 strain, and YDK0107 / pPK4_CspBss-AET-EPO-GS22 strain. Each of the obtained transformants was cultured in MMTG liquid medium (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, iron (III) sulfate heptahydrate 0.03 g, manganese (II) sulfate pentahydrate 0.03 g, thiamine hydrochloride 0.45 mg, biotin 0.45 mg, DL-methionine 0.15 g, soybean hydrochloride hydrolysate (total nitrogen content 0.2 g), calcium carbonate 50 g, adjusted to 1 L with water and pH 7.0) containing 25 mg / L of kanamycin at 30 °C for 72 hours. After completion of the culture, 6.5 μL of the culture supernatant obtained by centrifuging each culture solution was subjected to reducing SDS-PAGE using NuPAGE (registered trademark) 12% Bis-Tirs Gel (Thermo Fisher Scientific), and then stained with Quick-CBB (Wako). As a result, protein bands presumed to be AET-EPO-PAS49 were detected in the culture supernatant of the YDK0107 / pPK4_CspBss-AET-EPO-PAS49 strain (Figure 48, lanes 2 to 5), protein bands presumed to be AET-EPO-PAS49-b were detected in the culture supernatant of the YDK0107 / pPK4_CspBss-AET-EPO-PAS49-b strain (Figure 48, lanes 6 to 9), protein bands presumed to be AET-EPO-PAS100 were detected in the culture supernatant of the YDK0107 / pPK4_CspBss-AET-EPO-PAS100 strain (Figure 48, lanes 10 to 13), protein bands presumed to be AET-EPO-PAS100-b were detected in the culture supernatant of the YDK0107 / pPK4_CspBss-AET-EPO-PAS100-b strain (Figure 48, lanes 14 to 17), protein bands presumed to be AET-EPO-GS8 were detected in the culture supernatant of the YDK0107 / pPK4_CspBss-AET-EPO-GS8 strain (Figure 48, lanes 18 to 21), and protein bands presumed to be AET-EPO-GS22 were detected in the culture supernatant of the YDK0107 / pPK4_CspBss-AET-EPO-GS22 strain (Figure 48, lanes 22 to 25).

[0191] Example 31: Molecular weight analysis of EPO-PAS dimer peptide linker variants Regarding the cell filtrate of the EPO-PAS dimer peptide linker variant after culturing in Example 30, the molecular weight was measured using LC-MS (Waters, ACQUITY UPLC / SQD2). The analysis conditions are as described in Table 2. As shown in Figures 49 to 54, values almost identical to the theoretical values of the molecular weights of the oxidized dimer peptides each forming two disulfide bonds in the molecule were obtained. From this, it was confirmed that peptides with the correct amino acid sequence containing two disulfide bonds could be expressed.

[0192] Example 32: Evaluation of the Activity of EPO-PAS Dimer Peptide Linker Variants The EpoR activation ability of the EPO-PAS dimer peptide linker variants was evaluated using the PathHunter® eXpress EpoR-JAK2 Functional Assay Kit (DiscoverX). Cells for EPO activity evaluation included in the PathHunter® eXpress EpoR-JAK2 Functional Assay Kit were seeded in a 96-well plate and cultured at 37 °C under 5% CO2 for 24 hours. Culture supernatants containing AET-EPO-PAS8, AET-EPO-GS8, AET-EPO-PAS22, or AET-EPO-GS22 diluted to a predetermined concentration (culturing was as described in Example 20 or 30) were added to the cells. As a negative control, a culture supernatant obtained by culturing as described in Example 30 except using a vector not containing the gene for expressing the EPO-PAS dimer peptide (Figure 48, lane 26), diluted at the same magnification as the culture supernatants containing AET-EPO-PAS8 or AET-EPO-PAS22, was added. The prepared Substrate Reagent was added to the cells stimulated at room temperature for 3 hours and incubated for 60 minutes. The chemiluminescence intensity was quantified using a Nivo plate reader (Perkin Elmer). As shown in FIGS. 55 to 57, it was revealed that all dimer peptides activate EpoR in a concentration-dependent manner. The EC50 values of each construct are as shown in Table 6 below. It was revealed that AET-EPO-PAS8 shows higher activity than AET-EPO-GS8, and AET-EPO-PAS22 has higher activity than AET-EPO-GS22. The EC50 values of rhEPO, AET-EPO-PAS8, and AET-EPO-PAS22 are those in Example 22. Also, it was revealed that AET-EPO-PAS100 has lower activity than AET-EPO-PAS49, AET-EPO-PAS22, and AET-EPO-PAS8. From this, it became clear that by using a PAS linker, higher activity can be achieved compared to a GS linker, and by using a PAS linker of 49 amino acids or less, a dimer peptide having high agonist activity can be developed.

[0193]

Table 6

[0194] Example 33: Design of Stabilized EPO-PAS Dimer Peptide and Its Secretion Expression in C. glutamicum (33-1) Outline of Design The EPO-PAS peptide with enhanced stability was designed as follows. As a method for enhancing the stability of peptides and proteins, a method of adding a long-chain PAS sequence has been reported (Special Table 2013-531480 and Special Table 2010-531139). As the EPO-PAS peptide with enhanced stability, a peptide was designed in which a PAS sequence consisting of 200 amino acids (PAS200) or a PAS sequence consisting of 600 amino acids (PAS600) was bound to the C-terminus of AET-EPO-PAS22. That is, the peptide sequence was designed to be "first erythropoietin receptor-binding cyclic peptide" - "PAS linker" - "second erythropoietin receptor-binding cyclic peptide" - "long-chain PAS sequence (PAS200 or PAS600)".

[0195] (33-2) Preparation of Stabilized EPO-PAS Dimer Peptide The prepared stabilized EPO-PAS dimer peptide is as follows. (D) AET-EPO-PAS22-PAS200 AETGGLYACHMGPMTWVCQPLRGAAPAAPAPAAPAAPAPAAPAAPGGLYACHMGPMTWVCQPLRGAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAP (SEQ ID NO: 144) (E) AET-EPO-PAS22-PAS600 AETGGLYACHMGPMTWVCQPLRGAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPGGLYACHMGPMTWVCQPLRGAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAP(SEQ ID NO: 145)

[0196] (33 - 3)Expression of Stabilized EPO - PAS Dimer Peptide The expression of the stabilized EPO - PAS dimer peptide was examined using Corynex (registered trademark). Hereinafter, examples of the expression examination using Corynex (registered trademark) will be described.

[0197] (33 - 4)Construction of AET - EPO - PAS22 - PAS200 Secretion Expression Plasmid Utilizing the CspB Signal Sequence As stabilized EPO-PAS dimer peptides, the amino acid sequences of (D) AET-EPO-PAS22-PAS200 (hereinafter sometimes referred to as "AET-EPO-PAS22-PAS200") and (E) AET-EPO-PAS22-PAS600 (hereinafter sometimes referred to as "AET-EPO-PAS22-PAS600") were designed, and the nucleotide sequences encoding these proteins were designed in consideration of the codon usage frequency of C. glutamicum. Furthermore, the following expression cassettes were designed to enable secretion expression by C. glutamicum. AET-EPO-PAS22-PAS200 was secreted and expressed as a fusion protein of the 30 amino acid residues of the signal peptide of CspB derived from C. glutamicum ATCC13869 strain and AET-EPO-PAS22-PAS200 (hereinafter referred to as "CspBss-AET-EPO-PAS22-PAS200"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AET-EPO-PAS22-PAS200 are shown in SEQ ID NOs: 146 and 147, respectively.

[0198] Nucleotide sequence encoding CspBss-AET-EPO-PAS22-PAS200 atgttcaacaaccgtattcgtaccgctgccctggccggcgccattgccatctccaccgctgcgtccggcgtcgccattcctgcgttcgcagccgaaaccggcggcctgtacgcatgccacatgggtccaatgacctgggtgtgccagccactgcgcggcgctgcaccagctgctccagcaccagcagccccagctgcaccagcaccagctgctccagctgcaccaggcggcctgtacgcttgtcatatgggtccaatgacttgggtctgccagccactccgcggcgctgcaccagcagctcctgctccagcagcacctgcagcaccagcaccagcagctccagcagcacctgccccagcagctcctgcagctcctgccccagcagcacctgccgctcctgctcctgcagctccagcagcacccgccccagcagctcccgcagctcctgcgccagcagcacctgccgcccctgctcccgcagctccagcagcaccggcacccgcagctccagcagctcctgcacctgcagcacctgccgctcccgctccggcagctccagcagcaccagcccctgcagctcctgctgcacctgctccagcagcacccgcggctcctgctccggctgctccagcagcccctgctccagcagctcccgctgcacctgcaccagcagcacctgccgcgcctgctcctgctgcccctgctgctcctgctcctgcagcccctgctgcacccgctccagcagcacctgcggctcctgctccggccgcccctgctgctccagcaccagctgcccctgcagcccctgcccctgcagccccagctgcaccagcaccagcagcaccagcagcacccgcaccagcagccccagcagccccagccccttag (SEQ ID NO: 146) Amino acid sequence of CspBss-AET-EPO-PAS22-PAS200 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETGGLYACHMGPMTWVCQPLRGAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPGGLYACHMGPMTWVCQPLRGAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAP(Accession number: 147)

[0199] AET-EPO-PAS22-PAS600 was secreted and expressed as a fusion protein of the 30 amino acid residues of the signal peptide of CspB derived from C. glutamicum ATCC13869 strain and AET-EPO-PAS22-PAS600 (hereinafter referred to as "CspBss-AET-EPO-PAS22-PAS600"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AET-EPO-PAS22-PAS600 are shown in SEQ ID NO: 148 and SEQ ID NO: 149, respectively.

[0200] Nucleotide sequence encoding CspBss-AET-EPO-PAS22-PAS600 Amino acid sequence of CspBss-AET-EPO-PAS22-PAS600 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETGGLYACHMGPMTWVCQPLRGAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPGGLYACHMGPMTWVCQPLRGAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAP(SEQ ID NO: 149)

[0201] The promoter of the cspB gene derived from the C. glutamicum ATCC13869 strain was ligated upstream of the nucleotide sequences described in CspBss-AET-EPO-PAS22-PAS200 and CspBss-AET-EPO-PAS22-PAS600, and a KpnI site was added to the 5'-side and a BamHI site was added to the 3'-side. Expression cassettes for each stabilized EPO-PAS dimer peptide were designed and fully synthesized. The fully synthesized DNA fragment (expression cassette for the stabilized EPO-PAS dimer peptide) was inserted into the KpnI-BamHI site of pPK4 described in JP-A-9-322774 to construct pPK4_CspBss-AET-EPO-PAS22-PAS200 and pPK4_CspBss-AET-EPO-PAS22-PAS600, which are secretion expression plasmids for each stabilized EPO-PAS dimer peptide using the CspB signal sequence. As a result of nucleotide sequence determination of the inserted fragment, it was confirmed that the expression cassette for the stabilized EPO-PAS dimer peptide was constructed as designed. Nucleotide sequence determination was performed using the BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the 3500xL Genetic Analyzer (Applied Biosystems).

[0202] (33-5) Secretion Expression of Stabilized EPO-PAS Dimer Peptide in C. glutamicum Using pPK4_CspBss-AET-EPO-PAS22-PAS200 and pPK4_CspBss-AET-EPO-PAS22-PAS600 constructed above, the C. glutamicum YDK0107 strain described in WO2016 / 171224 was transformed to obtain the YDK0107 / pPK4_CspBss-AET-EPO-PAS22-PAS200 strain and the YDK0107 / pPK4_CspBss-AET-EPO-PAS22-PAS600 strain. Each of the obtained transformants was cultured in MMTG liquid medium (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, ferrous sulfate heptahydrate 0.03 g, manganese sulfate pentahydrate 0.03 g, thiamine hydrochloride 0.45 mg, biotin 0.45 mg, DL-methionine 0.15 g, soybean hydrochloric acid hydrolysate (total nitrogen content 0.2 g), calcium carbonate 50 g, adjusted to 1 L with water and pH 7.0) containing 25 mg / L of kanamycin at 30 °C for 72 hours. After completion of the culture, 6.5 μL of the culture supernatant obtained by centrifuging each culture solution was subjected to reducing SDS-PAGE using NuPAGE (registered trademark) 4-12% Bis-Tirs Gel (Thermo Fisher Scientific), and then stained with Quick-CBB (Wako). As a result, protein bands presumably corresponding to AET-EPO-PAS22-PAS200 (Fig. 58, lanes 2-5) were detected in the culture supernatant of the YDK0107 / pPK4_CspBss-AET-EPO-PAS22-PAS200 strain, and protein bands presumably corresponding to AET-EPO-PAS22-PAS600 (Fig. 58, lanes 6-9) were detected in the culture supernatant of the YDK0107 / pPK4_CspBss-AET-EPO-PAS22-PAS600 strain.

[0203] Example 34: Molecular weight analysis of stabilized EPO-PAS dimer peptides For the cell filtrates of each stabilized EPO-PAS dimer peptide after the culture in Example 33, the molecular weight was measured using LC-MS (Waters, ACQUITY UPLC / SQD2). The analysis conditions were as described in Table 2. As shown in Figs. 59 and 60, values almost identical to the theoretical values of the molecular weights of the oxidized dimer peptides forming two disulfide bonds within the molecule were obtained. From this, it was confirmed that peptides with the correct amino acid sequence containing two disulfide bonds could be expressed.

[0204] Example 35: Activity evaluation of stabilized EPO-PAS dimer peptides The EpoR activation ability of the stabilized EPO-PAS dimer peptide was evaluated using the PathHunter® eXpress EpoR-JAK2 Functional Assay Kit (DiscoverX). Cells for EPO activity evaluation included in the PathHunter® eXpress EpoR-JAK2 Functional Assay Kit were seeded in a 96-well plate and cultured at 37 °C for 24 hours under 5% CO2. Culture supernatants containing 14.2 μg / L to 220 pg / L of AET-EPO-PAS22, AET-EPO-PAS22-PAS200, or AET-EPO-PAS22-PAS600 (cultivation was as described in Example 20 or 33) were added to the cells. As a negative control, a culture supernatant obtained by culturing as described in Example 33 except using a vector not containing the gene for expressing the EPO-PAS dimer peptide (Figure 58, lane 20) diluted at the same magnification as the culture supernatants containing AET-EPO-PAS22, AET-EPO-PAS22-PAS200, or AET-EPO-PAS22-PAS600 was added. The prepared Substrate Reagent was added to the cells stimulated at room temperature for 3 hours and incubated for 60 minutes. The chemiluminescence intensity was quantified using a Nivo plate reader (Perkin Elmer). As shown in Figure 61, it was revealed that AET-EPO-PAS22, AET-EPO-PAS22-PAS200, and AET-EPO-PAS22-PAS600 activate EpoR in a concentration-dependent manner. Also, the EC50 values were calculated as shown in Table 7 below, and it was clarified that the agonist ability of the EPO-PAS dimer peptide does not decrease even with the stabilization modification at the C-terminus. Note that the EC50 value of AET-EPO-PAS22 is that in Example 22.

[0205]

Table 7

[0206] Example 36: Cell-free expression of dimer peptide (36-1) Construction of Gene Construct of Dimer Peptide for Cell-Free Expression As dimer peptides for cell-free expression, the following were designed. (F) MAET-aMD4dY-PAS22 MAETCRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPAPAAPAAPGCRQFNRRTHEVWNLDC (SEQ ID NO: 150) (G) MAET-aMD4dY-PAS8 MAETCRQFNRRTHEVWNLDCGAAPAAPAPGCRQFNRRTHEVWNLDC (SEQ ID NO: 151) (H) MAET-aMD4-PAS22 MAETCYRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPAPAAPAAPGCRQFNRRTHEVWNLDC (SEQ ID NO: 152) (I) MAET-EPO-PAS8 MAETGGLYACHMGPMTWVCQPLRGAAPAAPAPGGLYACHMGPMTWVCQPLRG (SEQ ID NO: 153) (J) MAET-EPO-PAS49-b MAETGGLYACHMGPMTWVCQPLRGSAPSPSSAPASASAPASPASASASASPASSPASASSASPAASSASPASSGGLYACHMGPMTWVCQPLRG (SEQ ID NO: 154) (K) MAET-EPO-PAS100-b MAETGGLYACHMGPMTWVCQPLRGSAPSPSSAPASASAPASPASASASASPASSPASASSASPAASSASPASSSASPSAPAAPSASAAPSASPASSSPAPASAPSPAAAASSPSPAPSSASSAAGGLYACHMGPMTWVCQPLRG (SEQ ID NO: 155) (L) MAET-VEGFR_D2-PAS16 MAETAGPTWCEDDWYYCWLFGTAAPAAPAPAAPAAPAPVCWEDSWGGEVCWLFGT (SEQ ID NO: 156) (M)MAET-VEGFR_D2-PAS49-b MAETAGPTWCEDDWYYCWLFGTSAPSPSSAPASASAPASPASASASASPASSPASASSASPAASSASPASSVCWEDSWGGEVCWLFGT (SEQ ID NO: 157) (N)MAET-VEGFR_D2-PAS100-b MAETAGPTWCEDDWYYCWLFGTSAPSPSSAPASASAPASPASASASASPASSPASASSASPAASSASPASSSASPSAPAAPSASAAPSASPASSSPAPASAPSPAAAASSPSPAPSSASSAAVCWEDSWGGEVCWLFGT (SEQ ID NO: 158) (O)MAET-VEGFR_D2-GS16 MAETAGPTWCEDDWYYCWLFGTGGGGSGGGGSGGGGSGVCWEDSWGGEVCWLFGT (SEQ ID NO: 159)

[0207] The nucleotide sequences encoding the amino acid sequences of the above dimeric peptides were designed. Furthermore, a T7 promoter sequence and a ribosome binding sequence necessary for cell-free expression were added to the 5'-side of these, and a stop codon and an untranslated region were added to the 3'-side. The corresponding nucleotide sequences for cell-free expression are shown below. These DNA sequences were fully synthesized and cloned into the pEX-K4J2 vector at Eurofins Genomics Co., Ltd. Nucleotide sequence encoding MAET-aMD4dY-PAS22 gaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatataccaatgcagagacctgtcgtcagtttaaccgtcgtacgcacgaagtttggaatctggactgtggcgcagcccccgcagcaccagctccagcagcacccgcagctccagcaccagcagcccctgcagccccaggctgccgccaattcaatcgccgcacccatgaagtgtggaacttagattgctagtgaataactaatcc(SEQ ID NO: 160) Base sequence encoding MAET-aMD4dY-PAS8 gaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatataccaatggcagagacctgtcgtcagtttaaccgtcgtacgcacgaagtttggaatctggactgtggcgcagcacccgcagctccagcaccaggctgccgccaattcaatcgccgcacccatgaagtgtggaacttagattgctagtgaataactaatcc(SEQ ID NO: 161) Base sequence encoding MAET-aMD4-PAS22 gaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatataccaatggcagagacctgttatcgtcagtttaaccgtcgtacgcacgaagtttggaatctggactgtggcgcagcccccgcagcaccagctccagcagcacccgcagctccagcaccagcagcccctgcagccccaggctgctatcgccaattcaatcgccgcacccatgaagtgtggaacttagattgctagtgaataactaatcc(SEQ ID NO: 162) Base sequence encoding MAET-EPO-PAS8 gaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatataccaatggcagaaactggtggtttatacgcctgtcacatgggcccgatgacttgggtttgccagccattgcgtggcgcagcacccgcagctccagcaccaggtggcctgtatgcgtgccatatggggccaatgacctgggtgtgtcagccgttacgcggctagtgaataactaatcc(SEQ ID NO: 163) Base sequence encoding MAET-EPO-PAS49-b gaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatataccaatggcagaaactggtggtttatacgcctgtcacatgggcccgatgacttgggtttgccagccattgcgtggctctgctccatcaccttcttccgcaccagcatcagcttctgcaccagcgtcgcctgcttctgcatctgcgtctgcttcaccagcatcctcacctgcatcagcatcgtctgcttctccagcagcttcctcagcttcacctgcttcgtctggtggcctgtatgcgtgccatatggggccaatgacctgggtgtgtcagccgttacgcggctagtgaataactaatcc(SEQ ID NO: 164) Base sequence encoding MAET-EPO-PAS100-b gaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatataccaatggcagaaactggtggtttatacgcctgtcacatgggcccgatgacttgggtttgccagccattgcgtggctctgctccatcaccttcttccgcaccagcatcagcttctgcaccagcgtcgcctgcttctgcatctgcgtctgcttcaccagcatcctcacctgcatcagcatcgtctgcttctccagcagcttcctcagcttcacctgcttcgtcttccgcatccccatctgctccagcagcaccttcagcttctgcagctccatctgcttcacctgcctcatcgtctccagcacctgcgtcagctccatcccctgcagcagcggcttcatcgccatctccagcaccttcttcagcgtcatccgctgcaggtggcctgtatgcgtgccatatggggccaatgacctgggtgtgtcagccgttacgcggctagtgaataactaatcc(SEQ ID NO: 165) Base sequence encoding MAET-VEGFR_D2-PAS16 gaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatataccaatggcggaaaccgcgggtcccacgtggtgtgaggatgactggtactattgctggttattcgggactgcggcccctgctgccccggctccggcagcaccggccgcaccagcgccggtttgctgggaagatagctggggtggcgaagtgtgttggctgtttggcacctagtgaataactaatcc(SEQ ID NO: 166) Base sequence encoding MAET-VEGFR_D2-PAS49-b gaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatataccaatggcggaaaccgcgggtcccacgtggtgtgaggatgactggtactattgctggttattcgggacttctgctccatcaccttcttccgcaccagcatcagcttctgcaccagcgtcgcctgcttctgcatctgcgtctgcttcaccagcatcctcacctgcatcagcatcgtctgcttctccagcagcttcctcagcttcacctgcttcgtctgtttgctgggaagatagctggggtggcgaagtgtgttggctgtttggcacctagtgaataactaatcc(SEQ ID NO: 167) Base sequence encoding MAET-VEGFR_D2-PAS100-b gaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatataccaatggcagaaactggtggtttatacgcctgtcacatgggcccgatgacttgggtttgccagccattgcgtggctctgctccatcaccttcttccgcaccagcatcagcttctgcaccagcgtcgcctgcttctgcatctgcgtctgcttcaccagcatcctcacctgcatcagcatcgtctgcttctccagcagcttcctcagcttcacctgcttcgtcttccgcatccccatctgctccagcagcaccttcagcttctgcagctccatctgcttcacctgcctcatcgtctccagcacctgcgtcagctccatcccctgcagcagcggcttcatcgccatctccagcaccttcttcagcgtcatccgctgcaggtggcctgtatgcgtgccatatggggccaatgacctgggtgtgtcagccgttacgcggctagtgaataactaatcc(SEQ ID NO: 168) Base sequence encoding MAET-VEGFR_D2-GS16 gaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggagatataccaatggcggaaaccgcgggtcccacgtggtgtgaggatgactggtactattgctggttattcgggactggtagcggtagcggcagcggcagcggtagtggtagtggcagcggcagcgtttgctgggaagatagctggggtggcgaagtgtgttggctgtttggcacctagtgaataactaatcc(SEQ ID NO: 169)

[0208] (36-2) Cell-free expression of dimeric peptide The gene of the dimeric peptide constructed above was amplified by PCR. PCR was performed using primers of the sequences shown below and KOD-Plus-Ver.2 (Toyobo) as the PCR enzyme. Cell-free primer Fwd: gaaattaatacgactcactataggg(SEQ ID NO: 170) Cell-free primer Rev: cactgactggattagttattcac(SEQ ID NO: 171)

[0209] Next, based on the gene fragment amplified by PCR, a peptide dimer was expressed using a cell-free expression system. PUREfrex2.0 (Gene Frontier) was used for cell-free expression. When performing cell-free expression with PUREfrex2.0, 10 μL of Solution I, 1 μL of Solution II, 2 μL of Solution III, and 7 μL of water were added to 2 nM of the gene fragment amplified by PCR, and the translation reaction was carried out by incubating at 37 °C for 6 hours.

[0210] (36-3) Molecular weight analysis of cell-free expressed dimeric peptide Solutions expressing HGF-PAS dimer peptides (MAET-aMD4-PAS22, MAET-aMD4dY-PAS8, MAET-aMD4dY-PAS22), EPO-PAS dimer peptides (MAET-EPO-PAS8, MAET-EPO-PAS49-b, MAET-EPO-PAS100-b), and VEGFR_D2-PAS dimer peptides (MAET-VEGFR_D2-PAS16) in a cell-free expression system were mixed with α-Cyano-4-hydroxycinnamic Acid (CHCA), and the molecular weights were measured by MALDI-TOF-MS (Shimadzu, AXIMA TOF2). As shown in FIGS. 62 to 68, for the HGF-PAS dimer peptide, a value close to the molecular weight with two disulfide bonds formed and the N-terminus formylated was obtained. For the EPO-PAS dimer peptide and the VEGFR_D2-PAS dimer peptide, both the molecular weight with two disulfide bonds formed and the N-terminus formylated and the molecular weight with the N-terminus not de-formylated were obtained, confirming that dimer peptides containing two disulfide bonds were successfully expressed respectively.

[0211] Example 37: Activity Evaluation of Cell-Free Expressed HGF-PAS Dimer Peptide The Met activation ability of the cell-free expressed HGF-PAS dimer peptide was evaluated by a luciferase assay. To 25 μL of Opti-MEM medium (Thermo Fisher Scientific), 0.6 μL of Attractene Transfection Reagent (QIAGEN) was added and incubated at room temperature for 5 minutes. To the above mixed solution, a mixed solution of 25 μL of Opti-MEM and 1 μL of SRE reporter vector (QIAGEN) was added and incubated at room temperature for 20 minutes. This mixed solution was added to a 96-well plate, and 40,000 cells / well of HEK293E cells were seeded on it and incubated overnight in a 37 °C, 5% CO2 incubator. After transfection, all the culture supernatant was removed, and 100 μL of Opti-MEM medium (evaluation basal medium) containing 0.5% FBS (Thermo Fisher Scientific), 1% non-essential amino acid solution (Thermo Fisher Scientific), and penicillin-streptomycin (Nacalai Tesque) was added and cultured at 37 °C for 4 hours to make the cells in a starved state. To the evaluation basal medium, 0 - 100 ng / mL of HGF was added, or a cell-free translation solution that translated AET-aMD4dY-PAS22 was added to be diluted at a predetermined ratio with respect to the medium, or a cell-free translation solution that did not translate the peptide was added to be diluted at the same ratio, and 100 μL of the evaluation basal medium was added to the cells. Stimulation of the cells was performed by culturing overnight in a 37 °C, 5% CO2 incubator. The Dual-Luciferase Reporter Assay System (Promega) was used to detect signal intensity. 100 μL of the culture supernatant was removed, 50 μL of Glo reagent was added, and the cells were lysed at room temperature for 10 minutes. The activation of the HGF-Erk-SRE pathway was quantified by detecting the luminescence of Firefly luciferase emitted from the cell lysate using a plate reader. Subsequently, 50 μL of the Glo & Stop reagent solution was added, and after 10 minutes, the luminescence of Renilla luciferase, which is an internal standard, was detected to quantify the number of cells. The signal activity of each sample was quantified as SRE activity = (luminescence intensity of Firefly luciferase) / (luminescence intensity of Renilla luciferase). The relative signal activity value with respect to the sample without the evaluation compound was determined and defined as the relative reporter activity. As shown in Fig. 69, it was revealed that cell-free translated MAET-aMD4dY-PAS22, MAET-aMD4dY-PAS8, and MAET-aMD4-PAS22 have Met activation ability.

[0212] Example 38: Activity evaluation of cell-free expressed EPO-PAS dimer peptide The EpoR activation ability of the EPO-PAS dimer peptide linker variant was evaluated using the PathHunter® eXpress EpoR-JAK2 Functional Assay Kit (DiscoverX). Cells for evaluating EPO activity included in the PathHunter® eXpress EpoR-JAK2 Functional Assay Kit were seeded in a 96-well plate and cultured at 37 °C under 5% CO2 for 24 hours. Cell-free translation solutions translated from AET-EPO-PAS8, AET-EPO-PAS49-b, or AET-EPO-PAS100-b diluted to a predetermined concentration were added to the cells. As a negative control, a cell-free translation solution that did not translate the peptide, diluted at the same magnification as the cell-free translation solutions that translated AET-EPO-PAS8, AET-EPO-PAS49, or AET-EPO-PAS100, was added. The prepared Substrate Reagent was added to the cells stimulated at room temperature for 3 hours and incubated for 60 minutes. The chemiluminescence intensity was quantified using a Nivo plate reader (Perkin Elmer). As shown in Figure 70, it was revealed that all dimer peptides activated EpoR in a concentration-dependent manner. Also, MAET-EPO-PAS8 and MAET-EPO-PAS49-b showed higher activity than MAET-EPO-PAS100-b. From this, it became clear that dimer peptides with high activity can be constructed by using short-chain PAS linkers of 49 amino acids or less.

[0213] Example 39: Activity evaluation of cell-free expressed VEGFR_D2-PAS heterodimer The VEGF inhibitory ability of the cell-free expressed VEGFR_D2-PAS heterodimer peptide was evaluated by a luciferase assay. VEGFR / NFAT Reporter HEK293 cells (BPS Bioscience) at 40,000 cells / well were seeded and incubated overnight in a 37 °C, 5% CO2 incubator. The cells were incubated overnight in Assay medium (MEM medium (Nacalai Tesque), 1x MEM Non-Essential Amino Acids Solution (Thermo Fisher Scientific), 1 mM Sodium Pyruvate (Nacalai Tesque), 1% Penicillin-Streptomycin (Sigma Aldrich), 0.3% FBS (Thermo Fisher Scientific)) to be starved. 100 μL of 10 ng / mL human recombinant VEGF (R&D Systems) was added to the Assay medium, and further, a cell-free expression solution containing MAET-VEGFR_D2-PAS16, MAET-VEGFR_D2-PAS49-b, MAET-VEGFR_D2-PAS100-b, or MAET-VEGFR_D2-GS16, or a cell-free expression solution not expressing the peptide, was added at a predetermined dilution ratio, and 100 μL of the Assay medium was added to the cells. The cells were stimulated by culturing in a 37 °C, 5% CO2 incubator for 4 hours. The One-Glo Luciferase Assay System (Promega) was used for detecting the signal intensity. 50 μL of the culture supernatant was removed, 50 μL of the One-Glo reagent was added, and the cells were lysed at room temperature for 10 minutes. The activation of the VEGFR-NFAT pathway was quantified by detecting the luminescence emitted from the cell lysate with a Nivo plate reader (Perkin Elmer), and the relative signal activity value against the sample without the evaluation compound was determined and used as the relative reporter activity. As shown in FIGS. 71 and 72, since MAET-VEGFR_D2-PAS16 and MAET-VEGFR_D2-PAS49-b showed higher inhibitory activities compared to MAET-VEGFR_D2-PAS100-b, it was revealed that a dimer peptide having high activity can be constructed by using a short-chain PAS linker of 49 amino acids or less. In addition, since MAET-VEGFR_D2-PAS16 showed high inhibitory activity against MAET-VEGFR_D2-GS16, it was revealed that a dimer peptide having higher activity than the GS linker can be constructed by using the PAS linker.

[0214] Example 40: Design of HGF-PAS trimer peptide and its secretory expression in C. glutamicum (40-1) Outline of design The HGF-PAS trimer peptide was designed as follows. A peptide was designed in which an aMD4dY sequence was further linked to the C-terminus of the AET-aMD4dY-PAS22 via a PAS linker. That is, the peptide sequence was designed to be "first aMD4dY cyclic peptide" - "PAS linker" - "second aMD4dY cyclic peptide" - "PAS linker" - "third aMD4dY cyclic peptide".

[0215] (40-2) Preparation of HGF-PAS trimer peptide The prepared HGF-PAS trimer peptide is as follows. (P)AET-aMD4dY-PAS22-Trimer AETCRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPAPAAPAAPGCRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPAPAAPAAPGCRQFNRRTHEVWNLDC (SEQ ID NO: 172)

[0216] (40-3) Expression of HGF-PAS trimer peptide The expression of stabilized HGF-PAS trimer peptide was examined using Corynex (registered trademark). Hereinafter, examples of the expression examination using Corynex (registered trademark) will be described.

[0217] (40-4) Construction of AET-aMD4dY-PAS22-Trimer secretory expression plasmid using the CspB signal sequence As the stabilized HGF-PAS dimer peptide, the amino acid sequence of the above (P)AET-aMD4dY-PAS22-Trimer was designed, and the nucleotide sequence encoding this protein was designed in consideration of the codon usage frequency of C. glutamicum. Furthermore, the following expression cassette was designed to enable secretory expression by C. glutamicum. AET-aMD4dY-PAS22-Trimer was secreted and expressed as a fusion protein of 30 amino acid residues of the signal peptide of CspB derived from C. glutamicum ATCC13869 strain and AET-aMD4dY-PAS22-Trimer (hereinafter referred to as "CspBss-AET-aMD4dY-PAS22-Trimer"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-AET-aMD4dY-PAS22-Trimer are shown in SEQ ID NOs: 173 and 174, respectively.

[0218] Nucleotide sequence encoding CspBss-AET-aMD4dY-PAS22-Trimer atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagacctgccgccagttcaatcgccggacccacgaagtctggaatctggattgcggcgcagctcctgccgccccagcaccggctgcgccagctgctccagcgccggctgcccctgcggcaccgggttgccgtcagtttaaccgccgtactcacgaggtttggaaccttgactgtggagcggctcccgcagcaccagccccagccgcccctgctgcacccgcaccagcggcacccgccgctcctggctgtcgccaattcaaccgacgcacccatgaagtgtggaacctcgattgctaa(SEQ ID NO: 173) Amino acid sequence of CspBss-AET-aMD4dY-PAS22-Trimer MFNNRIRTAALAGAIAISTAASGVAIPAFAAETCRQFNRRTHEVWNLDCGAAPAAPAPAAPAAPAPAAPAAPGCRQFNRRTHEVWNLDC(SEQ ID NO: 174)

[0219] The promoter of the cspB gene derived from the C. glutamicum ATCC13869 strain was ligated upstream of the nucleotide sequence described in CspBss-AET-aMD4dY-PAS22-Trimer, and a KpnI site was added to the 5'-side and a BamHI site was added to the 3'-side. An expression cassette for the HGF-PAS trimer peptide was designed and fully synthesized. The fully synthesized DNA fragment (expression cassette for the HGF-PAS trimer peptide) was inserted into the KpnI-BamHI site of pPK4 described in JP-A-9-322774 to construct pPK4_CspBss-AET-aMD4dY-PAS22-Trimer, a secretion expression plasmid for the HGF-PAS trimer peptide utilizing the CspB signal sequence. As a result of nucleotide sequencing of the inserted fragment, it was confirmed that the expression cassette for the HGF-PAS trimer peptide was constructed as designed. Nucleotide sequencing was performed using the BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the 3500xL Genetic Analyzer (Applied Biosystems).

[0220] (40-5) Secretion expression of HGF-PAS trimer peptide in C. glutamicum Using the pPK4_CspBss-AET-aMD4dY-PAS22-Trimer constructed above, the C. glutamicum YDK010::phoS(W302C) strain described in WO2016 / 171224 was transformed to obtain the YDK010::phoS(W302C) / pPK4_CspBss-AET-aMD4dY-PAS22-Trimer strain. The obtained transformants were cultured at 30°C for 72 hours each in MMTG liquid medium (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, ferrous sulfate heptahydrate 0.03 g, manganese sulfate pentahydrate 0.03 g, thiamine hydrochloride 0.45 mg, biotin 0.45 mg, DL-methionine 0.15 g, soybean hydrochloric acid hydrolysate (total nitrogen content 0.2 g), calcium carbonate 50 g, adjusted to pH 7.0 with water to make 1 L) containing 25 mg / L of kanamycin. After completion of the culture, 6.5 μL of the culture supernatant obtained by centrifuging each culture solution was subjected to reducing SDS-PAGE using NuPAGE (registered trademark) 12% Bis-Tirs Gel (Thermo Fisher Scientific), and then stained with Quick-CBB (Wako). As a result, a protein band presumed to be AET-aMD4dY-PAS22-Trimer was detected in the culture supernatant of the YDK010::phoS(W302C) / pPK4_CspBss-AET-aMD4dY-PAS22-Trimer strain (Figure 73, lanes 2 to 5). In addition, the YDK010::phoS(W302C) / pPK4_CspBss-AET-aMD4dY-PAS22 strain obtained by the same method as above was cultured in the same manner as above, and a protein band presumed to be AET-aMD4dY-PAS22 (Figure 73, lane 6) was detected in its culture supernatant.

[0221] Example 41: Molecular weight analysis of HGF-PAS trimer peptide Regarding the cell filtrate of AET-aMD4dY-PAS22-Trimer after the culture in Example 40, the molecular weight was measured using LC-MS (Waters, ACQUITY UPLC / SQD2). The analysis conditions are as described in Table 2. As shown in Figure 74, a value almost identical to the theoretical value of the molecular weight of the oxidized dimer peptide forming three disulfide bonds in the molecule was obtained. From this, it was confirmed that a peptide with the correct amino acid sequence containing three disulfide bonds could be expressed.

[0222] Example 42: Activity Evaluation of HGF-PAS Trimer Peptide The Met activation ability of the HGF-PAS trimer peptide was evaluated by a luciferase assay. To 25 μL of Opti-MEM medium (Thermo Fisher Scientific), 0.6 μL of Attractene Transfection Reagent (QIAGEN) was added and incubated at room temperature for 5 minutes. To the above mixed solution, a mixed solution of 25 μL of Opti-MEM and 1 μL of SRE reporter vector (QIAGEN) was added and incubated at room temperature for 20 minutes. This mixed solution was added to a 96-well plate, and 40,000 cells / well of HEK293E cells were seeded on it and incubated overnight in a 37 °C, 5% CO2 incubator. After transfection, all the culture supernatant was removed, and 100 μL of Opti-MEM medium (evaluation basal medium) containing 0.5% FBS (Thermo Fisher Scientific), 1% non-essential amino acid solution (Thermo Fisher Scientific), and penicillin-streptomycin (Nacalai Tesque) was added and cultured at 37 °C for 4 hours to make the cells in a starved state. To the evaluation basal medium, 0 - 100 ng / mL of HGF was added, or the culture supernatant containing AET-aMD4dY-PAS22 or AET-aMD4dY-PAS22-Trimer was added so as to be diluted at a predetermined magnification with respect to the medium, and 100 μL of the evaluation basal medium was added to the cells. Stimulation of the cells was performed by culturing overnight in a 37 °C, 5% CO2 incubator. The Dual-Luciferase Reporter Assay System (Promega) was used for detecting signal intensity. 100 μL of the culture supernatant was removed, 50 μL of Glo reagent was added, and the cells were lysed at room temperature for 10 minutes. The activation of the HGF-Erk-SRE pathway was quantified by detecting the luminescence of Firefly luciferase emitted from the cell lysate using a plate reader. Subsequently, 50 μL of the Glo & Stop reagent solution was added, and after 10 minutes, the luminescence of Renilla luciferase, which is an internal standard, was detected to quantify the cell number. The signal activity of each sample was quantified as SRE activity = (luminescence intensity of Firefly luciferase) / (luminescence intensity of Renilla luciferase). The relative signal activity value with respect to the sample without the evaluation compound was determined and used as the relative reporter activity. As shown in Fig. 75, it was revealed that the HGF-PAS trimer peptide (AET-aMD4dY-PAS22-Trimer) has the ability to activate signals similar to that of the dimer peptide (AET-aMD4dY-PAS22).

[0223] Example 43: Activity comparison between HGF-PAS dimer peptide and monomer peptide The Met activation abilities of the HGF-PAS dimer peptide and the monomer peptide were compared by a luciferase assay. To 25 μL of Opti-MEM medium (Thermo Fisher Scientific), 0.6 μL of Attractene Transfection Reagent (QIAGEN) was added and incubated at room temperature for 5 minutes. To the above mixed solution, a mixed solution of 25 μL of Opti-MEM and 1 μL of SRE reporter vector (QIAGEN) was added and incubated at room temperature for 20 minutes. This mixed solution was added to a 96-well plate, and 40,000 cells / well of HEK293E cells were seeded on it and incubated overnight in a 37 °C, 5% CO2 incubator. After transfection, all the culture supernatants were removed, and 100 μL of Opti-MEM medium (evaluation basal medium) containing 0.5% FBS (Thermo Fisher Scientific), 1% non-essential amino acid solution (Thermo Fisher Scientific), and penicillin-streptomycin (Nacalai Tesque) was added and cultured at 37 °C for 4 hours to make the cells in a starvation state. 0 - 100 ng / mL of HGF was added to the evaluation basal medium, or the AET-aMD4dY-PAS22 or aMD4dY monomer peptide (CRQFNRRTHEVWNLDC (SEQ ID NO: 2), synthesized by GenScript Japan Inc.) was added at a predetermined concentration, and 100 μL was added to the cells. The cells were stimulated by incubating overnight in a 37 °C, 5% CO2 incubator. The Dual-Luciferase Reporter Assay System (Promega) was used to detect signal intensity. 100 μL of the culture supernatant was removed, 50 μL of Glo reagent was added, and the cells were lysed at room temperature for 10 minutes. The activation of the HGF-Erk-SRE pathway was quantified by detecting the luminescence of Firefly luciferase emitted from the cell lysate using a plate reader. Subsequently, 50 μL of the Glo & Stop reagent solution was added, and after 10 minutes, the luminescence of Renilla luciferase, which is an internal standard, was detected to quantify the number of cells. The signal activity of each sample was quantified as SRE activity = (luminescence intensity of Firefly luciferase) / (luminescence intensity of Renilla luciferase). The relative signal activity value with respect to the sample without the test compound was determined and defined as the Relative reporter activity. As shown in Fig. 76, HGF and the dimer peptide (AET-aMD4dY-PAS22) showed signal activation ability, while the monomer peptide (aMD4dY) did not show signal activation ability. From this result, it became clear that the peptide acquired signal activation ability by dimerization.

[0224] Example 44: Design of VEGFR_D2-PAS heterodimer peptide linker variants and their secretory expression in C. glutamicum (44-1) Outline of linker variant design AET-VEGFR_D2-PAS16 has a structure in which the first VEGFR binding sequence (P-3) and the second VEGFR binding sequence (P-4) are linked by a PAS linker consisting of 16 amino acids. By changing the length of this PAS linker, it is expected that the VEGFR binding activity will change. Therefore, linker variants were designed in which the PAS linker site was changed to a PAS repeat sequence of 49 amino acids or 100 amino acids. In addition to the PAS linker, a GS linker, which is a repeating sequence of glycine and serine, is also known as a flexible linker. Therefore, a linker variant was designed by binding a VEGFR-binding cyclic peptide to the N-terminus and C-terminus of a GS linker consisting of a repeating sequence of 4 glycine residues and 1 serine residue (GGGGS), that is, a peptide sequence was designed to be "the first VEGFR-binding sequence (P-3)" - "GS linker" - "the second VEGFR-binding sequence (P-4)". That is, the C-terminus of P-3 and the N-terminus of the PAS linker, and the N-terminus of P-4 and the C-terminus of the PAS linker are each bound by an amide bond and can be expressed as a single polypeptide chain.

[0225] (44-2) Preparation of VEGFR_D2-PAS heterodimer peptide linker variant The prepared VEGFR_D2-PAS heterodimer peptide linker variant is as follows. (Q) AET-VEGFR_D2-PAS49 AETAGPTWCEDDWYYCWLFGTAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAVCWEDSWGGEVCWLFGT (SEQ ID NO: 175) (R) AET-VEGFR_D2-PAS49-b AETAGPTWCEDDWYYCWLFGTSAPSPSSAPASASAPASPASASASASPASSPASASSASPAASSASPASSVCWEDSWGGEVCWLFGT (SEQ ID NO: 176) (S) AET-VEGFR_D2-PAS100 AETAGPTWCEDDWYYCWLFGTAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAVCWEDSWGGEVCWLFGT (SEQ ID NO: 177) (T) AET-VEGFR_D2-PAS100-b AETAGPTWCEDDWYYCWLFGTSAPSPSSAPASASAPASPASASASASPASSPASASSASPAASSASPASSSASPSAPAAPSASAAPSASPASSSPAPASAPSPAAAASSPSPAPSSASSAAVCWEDSWGGEVCWLFGT (SEQ ID NO: 178) (U)AET-VEGFR_D2-GS16 AETAGPTWCEDDWYYCWLFGTGGGGSGGGGSGGGGSGVCWEDSWGGEVCWLFGT (SEQ ID NO: 179)

[0226] (44-3) Expression of VEGFR_D2-PAS heterodimer peptide linker variants The expression of VEGFR_D2-PAS heterodimer peptide linker variants was examined using Corynex (registered trademark). Hereinafter, examples of the expression examination using Corynex (registered trademark) will be described.

[0227] (44-4) Construction of secretion expression plasmids for AET-VEGFR_D2-PAS49, AET-VEGFR_D2-PAS49-b, AET-VEGFR_D2-PAS100, AET-VEGFR_D2-PAS100-b, and AET-VEGFR_D2-GS16 using the CspB signal sequence As VEGFR_D2-PAS heterodimer peptide linker variants, the following five amino acid sequences: (Q) AET-VEGFR_D2-PAS49 (hereinafter sometimes referred to as "AET-VEGFR_D2-PAS49" and sometimes simply referred to as "VEGFR_D2-PAS49"), (R) AET-VEGFR_D2-PAS49-b (hereinafter sometimes referred to as "AET-VEGFR_D2-PAS49-b" and sometimes simply referred to as "VEGFR_D2-PAS49-b"), (S) AET-VEGFR_D2-PAS100 (hereinafter sometimes referred to as "AET-VEGFR_D2-PAS100" and sometimes simply referred to as "VEGFR_D2-PAS100"), (T) AET-VEGFR_D2-PAS100-b (hereinafter sometimes referred to as "AET-VEGFR_D2-PAS100-b" and sometimes simply referred to as "VEGFR_D2-PAS100-b"), and (U) AET-VEGFR_D2-GS16 (hereinafter sometimes referred to as "AET-VEGFR_D2-GS16" and sometimes simply referred to as "VEGFR_D2-GS16") were each designed, and the nucleotide sequences encoding these proteins were designed in consideration of the codon usage frequency of C. glutamicum. Furthermore, the following expression cassettes were designed respectively to enable secretion expression by C. glutamicum. VEGFR_D2-PAS49 was secreted and expressed as a fusion protein of the 30 amino acid residues of the signal peptide of CspB derived from C. glutamicum ATCC13869 strain and VEGFR_D2-PAS49 (hereinafter referred to as "CspBss-VEGFR_D2-PAS49"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-VEGFR_D2-PAS49 are shown in SEQ ID NOs: 180 and 181, respectively.

[0228] The nucleotide sequence encoding CspBss-VEGFR_D2-PAS49 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagaccgctggccctacctggtgtgaggacgattggtactactgttggctctttggcaccgccgcccctgctgctcccgcccccgccgcccctgccgcaccagctccagcagccccagctgcaccagcaccagccgctccagcagctccagcaccagctgctccagcagcaccagccccagcagctccagccgcaccagctcctgcggtttgctgggaggattcttggggcggcgaagtttgctggctctttggcacctaa(SEQ ID NO: 180) Amino acid sequence of CspBss-VEGFR_D2-PAS49 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETAGPTWCEDDWYYCWLFGTAAPAAPAPAAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAVCWEDSWGGEVCWLFGT(SEQ ID NO: 181)

[0229] VEGFR_D2-PAS49-b was secreted and expressed as a fusion protein of 30 amino acid residues of the signal peptide of CspB derived from C. glutamicum ATCC13869 strain and VEGFR_D2-PAS49-b (hereinafter referred to as "CspBss-VEGFR_D2-PAS49-b"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-VEGFR_D2-PAS49-b are shown in SEQ ID NO: 182 and SEQ ID NO: 183, respectively.

[0230] Nucleotide sequence encoding CspBss-VEGFR_D2-PAS49-b atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagaccgcaggacccacttggtgcgaagatgactggtactattgctggctctttggcacctcagcacccagcccatcctccgctccagcgtctgcgagcgctcctgcatccccagcctctgcctccgcatcggcatcaccggcttccagcccagcgtcggcatcgtcagcctctccggctgcctcctccgcttctcctgcgtcctccgtgtgttgggaggattcctggggtggtgaagtctgctggctgttcggcacctaa(SEQ ID NO: 182) Amino acid sequence of CspBss-VEGFR_D2-PAS49-b MFNNRIRTAALAGAIAISTAASGVAIPAFAAETAGPTWCEDDWYYCWLFGTSAPSPSSAPASASAPASPASASASASPASSPASASSASPAASSASPASSVCWEDSWGGEVCWLFGT(SEQ ID NO: 183)

[0231] VEGFR_D2-PAS100 was secreted and expressed as a fusion protein of 30 amino acid residues of the signal peptide of CspB derived from C. glutamicum ATCC13869 strain and VEGFR_D2-PAS100 (hereinafter referred to as "CspBss-VEGFR_D2-PAS100"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-VEGFR_D2-PAS100 are shown in SEQ ID NO: 184 and SEQ ID NO: 185, respectively.

[0232] Nucleotide sequence encoding CspBss-VEGFR_D2-PAS100 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagaccgccggtccaacctggtgtgaggatgactggtactactgctggctctttggcaccgctgctcccgctgctcccgcccctgctgctccagctgccccagccccagctgccccagctgctccagcaccagccgctccagctgccccagcaccagctgcaccagcagcaccagcaccagcagctccagccgctccagccccagccgctccagcagcaccagccccagcagctccagctgcaccagctccagccgccccagcagcaccagctccagcagccccagcagctccagctccagccgcaccagctgctccagccccagccgcaccagcagccccagctccagctgctcctgctgtctgttgggaggactcttggggcggcgaagtttgttggttgtttggcacctaa(SEQ ID NO: 184) Amino acid sequence of CspBss-VEGFR_D2-PAS100 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETAGPTWCEDDWYYCWLFGTAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAVCWEDSWGGEVCWLFGT(SEQ ID NO: 185)

[0233] VEGFR_D2-PAS100-b was secreted and expressed as a fusion protein of 30 amino acid residues of the signal peptide of CspB derived from C. glutamicum ATCC13869 strain and VEGFR_D2-PAS100-b (hereinafter referred to as "CspBss-VEGFR_D2-PAS100-b"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-VEGFR_D2-PAS100-b are shown in SEQ ID NO: 186 and 187, respectively.

[0234] The nucleotide sequence encoding CspBss-VEGFR_D2-PAS100-b atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagaccgccggacctacctggtgtgaggatgactggtactattgctggctcttcggcacttccgccccttccccttccagcgcaccagcgagcgcttcggctccggcctccccagcgtccgcatctgcgtctgcatcccctgcgtcgtcacccgcttcagcctcctcagcctcccctgcggcttcatccgcctcgcccgcatccagctccgcatcaccctctgcgccagcagctccatccgcttctgctgccccatcggcctctccggcttccagctctcccgcaccggcatccgctccgagcccagcagcagctgcatcctccccatctccggcaccatcctcggcctctagcgcggctgtgtgctgggaagattcctggggtggtgaagtctgctggctgtttggcacctaa(SEQ ID NO: 186) The amino acid sequence of CspBss-VEGFR_D2-PAS100-b MFNNRIRTAALAGAIAISTAASGVAIPAFAAETAGPTWCEDDWYYCWLFGTSAPSPSSAPASASAPASPASASASASPASSPASASSASPAASSASPASSSASPSAPAAPSASAAPSASPASSSPAPASAPSPAAAASSPSPAPSSASSAAVCWEDSWGGEVCWLFGT(SEQ ID NO: 187)

[0235] VEGFR_D2-GS16 was secreted and expressed as a fusion protein of the 30 amino acid residues of the signal peptide of CspB derived from the C. glutamicum ATCC13869 strain and VEGFR_D2-GS16 (hereinafter referred to as "CspBss-VEGFR_D2-GS16"). The nucleotide sequence and amino acid sequence encoding the designed CspBss-VEGFR_D2-GS16 are shown in SEQ ID NO: 188 and SEQ ID NO: 189, respectively.

[0236] Nucleotide sequence encoding CspBss-VEGFR_D2-GS16 atgtttaacaaccgtatccgcactgcagctctcgctggtgcaatcgcaatctccaccgcagcttccggcgtagctatcccagcattcgctgcagagaccgcgggtccgacctggtgtgaggatgactggtactactgttggttgttcggcaccggcggcggcggctccggcggcggcggctctggcggcggcggctctggcgtttgttgggaggactcttggggcggcgaagtttgctggctgtttggcacctaa (SEQ ID NO: 188) Amino acid sequence of CspBss-VEGFR_D2-GS16 MFNNRIRTAALAGAIAISTAASGVAIPAFAAETAGPTWCEDDWYYCWLFGTGGGGSGGGGSGGGGSGVCWEDSWGGEVCWLFGT (SEQ ID NO: 189)

[0237] The promoter of the cspB gene derived from the C. glutamicum ATCC13869 strain was ligated upstream of the nucleotide sequences described in CspBss-VEGFR_D2-PAS49, CspBss-VEGFR_D2-PAS49-b, CspBss-VEGFR_D2-PAS100, CspBss-VEGFR_D2-PAS100-b, and CspBss-VEGFR_D2-GS16. Furthermore, a KpnI site was added to the 5'-side and a BamHI site was added to the 3'-side, and the expression cassettes of each VEGFR_D2-PAS heterodimer peptide linker variant were designed and fully synthesized. The fully synthesized DNA fragment (the expression cassette of the VEGFR_D2-PAS heterodimer peptide linker variant) was inserted into the KpnI-BamHI site of pPK4 described in JP-A-9-322774 to construct pPK4_CspBss-VEGFR_D2-PAS49, pPK4_CspBss-VEGFR_D2-PAS49-b, pPK4_CspBss-VEGFR_D2-PAS100, pPK4_CspBss-VEGFR_D2-PAS100-b, and pPK4_CspBss-VEGFR_D2-GS16, which are secretion expression plasmids of each VEGFR_D2-PAS heterodimer peptide linker variant using the CspB signal sequence. As a result of nucleotide sequencing of the inserted fragment, it was confirmed that the expression cassettes of the VEGFR_D2-PAS heterodimer peptide linker variants were constructed as designed. Nucleotide sequencing was performed using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and the 3500xL Genetic Analyzer (Applied Biosystems).

[0238] (44-5) Secretion expression of each VEGFR_D2-PAS heterodimer peptide linker variant in C. glutamicum Using pPK4_CspBss-VEGFR_D2-PAS49, pPK4_CspBss-VEGFR_D2-PAS49-b, pPK4_CspBss-VEGFR_D2-PAS100, pPK4_CspBss-VEGFR_D2-PAS100-b, and pPK4_CspBss-VEGFR_D2-GS16 constructed above, the C. glutamicum YDK0107 strain described in WO2016 / 171224 was transformed to obtain the YDK0107 / pPK4_CspBss-VEGFR_D2-PAS49 strain, YDK0107 / pPK4_CspBss-VEGFR_D2-PAS49-b strain, YDK0107 / pPK4_CspBss-VEGFR_D2-PAS100 strain, YDK0107 / pPK4_CspBss-VEGFR_D2-PAS100-b strain, and YDK0107 / pPK4_CspBss-VEGFR_D2-GS16 strain. Each of the obtained transformants was cultured in MMTG liquid medium (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, iron sulfate heptahydrate 0.03 g, manganese sulfate pentahydrate 0.03 g, thiamine hydrochloride 0.45 mg, biotin 0.45 mg, DL-methionine 0.15 g, soybean hydrochloride hydrolyzate (total nitrogen content 0.2 g), calcium carbonate 50 g, adjusted to 1 L with water and pH 7.0) containing 25 mg / L of kanamycin at 30°C for 72 hours. After the completion of cultivation, 6.5 μL of the culture supernatant obtained by centrifuging each culture solution was subjected to reducing SDS-PAGE using NuPAGE (registered trademark) 12% Bis-Tirs Gel (Thermo Fisher Scientific), and then stained with Quick-CBB (Wako). As a result, protein bands presumed to be VEGFR_D2-PAS49 were detected in the culture supernatant of the YDK0107 / pPK4_CspBss-VEGFR_D2-PAS49 strain (Figure 77, lanes 2 to 5), protein bands presumed to be VEGFR_D2-PAS49-b were detected in the culture supernatant of the YDK0107 / pPK4_CspBss-VEGFR_D2-PAS49-b strain (Figure 77, lanes 6 to 9), protein bands presumed to be VEGFR_D2-PAS100 were detected in the culture supernatant of the YDK0107 / pPK4_CspBss-VEGFR_D2-PAS100 strain (Figure 77, lanes 10 to 13), protein bands presumed to be VEGFR_D2-PAS100-b were detected in the culture supernatant of the YDK0107 / pPK4_CspBss-VEGFR_D2-PAS100-b strain (Figure 77, lanes 14 to 17), and protein bands presumed to be VEGFR_D2-GS16 were detected in the culture supernatant of the YDK0107 / pPK4_CspBss-VEGFR_D2-GS16 strain (Figure 77, lanes 18 to 21).

[0239] Example 45: Molecular Weight Analysis of VEGFR_D2-PAS Heterodimer Peptide Linker Variants Regarding the cell filtrate expressing the VEGFR_D2-PAS heterodimer variant after cultivation in Example 44, after cell filtration, the molecular weight was measured using LC-MS (Waters, ACQUITY UPLC / SQD2). The analysis conditions were as described in Table 5. As shown in Figures 78 to 81, values almost identical to the theoretical values of the molecular weights of the oxidized dimer peptides each forming two disulfide bonds within the molecule were obtained. From this, it was confirmed that peptides with the correct amino acid sequence containing two disulfide bonds could be expressed.

[0240] Example 46: Activity Evaluation of VEGFR_D2-PAS Heterodimer Peptide Linker Variants The VEGF inhibitory ability of the VEGFR_D2-PAS heterodimer peptide linker variants was evaluated by a luciferase assay. VEGFR / NFAT Reporter HEK293 cells (BPS Bioscience) at 40,000 cells / well were seeded and incubated overnight in a 37 °C, 5% CO2 incubator. The cells were incubated overnight in Assay medium (MEM medium (Nacalai Tesque), 1x MEM Non-Essential Amino Acids Solution (Thermo Fisher Scientific), 1 mM Sodium Pyruvate (Nacalai Tesque), 1% Penicillin-Streptomycin (Sigma Aldrich), 0.3% FBS (Thermo Fisher Scientific)) to make them starved. Human recombinant VEGF (R&D Systems) at 10 ng / mL was added to the Assay medium, and further culture supernatants containing VEGFR_D2-PAS16, VEGFR_D2-PAS49, and VEGFR_D2-PAS100 at a predetermined concentration were added. 100 μL of the Assay medium was added to the cells. The cells were stimulated by culturing in a 37 °C, 5% CO2 incubator for 4 hours. VEGFR_D2-PAS16 was obtained according to the method described in Example 27. The One-Glo Luciferase Assay System (Promega) was used to detect the signal intensity. 50 μL of the culture supernatant was removed, 50 μL of the One-Glo reagent was added, and the cells were lysed at room temperature for 10 minutes. The activation of the VEGFR-NFAT pathway was quantified by detecting the luminescence emitted from the cell lysate with a Nivo plate reader (Perkin Elmer), and the relative signal activity value with respect to the sample without the evaluation compound was determined and used as the relative reporter activity. The evaluation results are shown in FIGS. 82 and 83, and the results of calculating the IC50 values of each peptide dimer are shown in Table 8 below. VEGFR_D2-PAS16 showed high inhibitory activity against VEGFR_D2-GS16. In addition, since VEGFR_D2-PAS16 and VEGFR_D2-PAS49 showed high inhibitory activity against VEGFR_D2-PAS100, it was revealed that by using the PAS linker, a dimer peptide having higher inhibitory activity than the GS linker can be developed, and a dimer peptide having high inhibitory activity can be developed by using a PAS linker of 49 amino acids or less.

[0241]

Table 8

[0242] Example 47: Design of TPO-PAS Dimer Peptide Linker Variants and Their Secretion and Expression in C. glutamicum (47-1) Outline of the Design of Linker Variants TPO2-PAS8 having thrombopoietin-like activity has a structure in which the first AF12285 peptide and the second first AF12285 peptide are linked by a PAS linker consisting of 8 amino acids. By changing the length of this PAS linker, it is expected that the thrombopoietin-like activity will change. Therefore, linker variants were designed in which the PAS linker region was changed to a PAS repeat sequence of 49 amino acids or 100 amino acids. In addition, as a flexible linker, in addition to the PAS linker, a GS linker, which is a repeating sequence of glycine and serine, is also known. Therefore, a linker variant in which an EPO cyclic peptide was bound to the N-terminus and C-terminus of a GS linker consisting of a repeating sequence of 4 glycine residues and 1 serine residue (GGGGS), that is, a peptide sequence was designed so as to be "first AF12285 peptide" - "GS linker" - "first AF12285 peptide".

[0243] (47-2) Preparation of TPO-PAS Dimer Peptide Linker Variants The prepared TPO-PAS dimer peptide linker variants are as follows. (V)TPO2-PAS49 GGCADGPTLREWISFCGGAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAGGCADGPTLREWISFCGG (SEQ ID NO: 190) (W)TPO2-PAS49-b GGCADGPTLREWISFCGGSAPSPSSAPASASAPASPASASASASPASSPASASSASPAASSASPASSGGCADGPTLREWISFCGG (SEQ ID NO: 191) (X)TPO2-PAS100 GGCADGPTLREWISFCGGAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAGGCADGPTLREWISFCGG (SEQ ID NO: 192) (Y)TPO2-PAS100-b GGCADGPTLREWISFCGGSAPSPSSAPASASAPASPASASASASPASSPASASSASPAASSASPASSSASPSAPAAPSASAAPSASPASSSPAPASAPSPAAAASSPSPAPSSASSAAGGCADGPTLREWISFCGG (SEQ ID NO: 193) (Z)TPO2-GS8 GGCADGPTLREWISFCGGGGGGSGGGGGCADGPTLREWISFCGG (SEQ ID NO: 194)

[0244] (47-3) Expression of the TPO-PAS dimer peptide linker variants The expression of these TPO-PAS dimer peptide linker variants was examined using Corynex®. Hereinafter, examples of the expression examination using Corynex® will be described.

[0245] Construction of Secretory Expression Plasmids of TPO2-PAS49, TPO2-PAS49-b, TPO2-PAS100, TPO2-PAS100-b, and TPO2-GS8 Using the CspA Signal Sequence As TPO-PAS dimer peptide linker variants, the following five amino acid sequences of (V) TPO2-PAS49 (hereinafter sometimes referred to as "TPO2-PAS49"), (W) TPO2-PAS49-b (hereinafter sometimes referred to as "TPO2-PAS49-b"), (X) TPO2-PAS100 (hereinafter sometimes referred to as "TPO2-PAS100"), (Y) TPO2-PAS100-b (hereinafter sometimes referred to as "TPO2-PAS100-b"), and (Z) TPO2-GS8 (hereinafter sometimes referred to as "TPO2-GS8") were each designed, and the nucleotide sequences encoding these proteins were designed in consideration of the codon usage frequency of C. glutamicum. Furthermore, the following expression cassettes were designed respectively to enable secretory expression by C. glutamicum. TPO2-PAS49 was secreted and expressed as a fusion protein of the 25 amino acid residues of the signal peptide of CspA derived from the C. ammoniagenes ATCC6872 strain and TPO2-PAS49 (hereinafter referred to as "CspAss-TPO2-PAS49"). The nucleotide sequence and amino acid sequence encoding the designed CspAss-TPO2-PAS49 are shown in SEQ ID NOs: 195 and 196, respectively.

[0246] Nucleotide Sequence Encoding CspAss-TPO2-PAS49 atgaaacgcatgaaatcgctggctgcggcgctcaccgtcgctggggccatgctggccgcacctgtggcaacggcaggtggctgtgctgacggtcctacccttcgtgagtggatttctttttgcggcggtgctgctcctgctgcccctgctcctgctgctcctgcagcaccagccccagcagctccagccgccccagctccagccgcaccagctgccccagcaccagccgctccagcagcaccagctccagcagccccagctgcaccagccccagcgggcggttgtgctgacggtcctacccttcgtgagtggatttccttctgtggtggctaa(SEQ ID NO: 195) Amino acid sequence of CspAss-TPO2-PAS49 MKRMKSLAAALTVAGAMLAAPVATAGGCADGPTLREWISFCGGAAPAAPAPAAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAGGCADGPTLREWISFCGG(SEQ ID NO: 196)

[0247] TPO2-PAS49-b was secreted and expressed as a fusion protein of 25 amino acid residues of the signal peptide of CspA derived from the C. ammoniagenes ATCC6872 strain and TPO2-PAS49-b (hereinafter referred to as "CspAss-TPO2-PAS49-b"). The nucleotide sequence and amino acid sequence encoding the designed CspAss-TPO2-PAS49-b are shown in SEQ ID NOs: 197 and 198, respectively.

[0248] Nucleotide sequence encoding CspAss-TPO2-PAS49-b atgaaacgcatgaaatcgctggctgcggcgctcaccgtcgctggggccatgctggccgcacctgtggcaacggcaggcgggtgtgcagacggacccactctgcgcgaatggatctccttttgcggcggttccgctccgtcgccatcctctgctccggcatccgcgtctgcgccagcatccccagccagcgcttcagcctcagcgtcccctgcctcctctccagcatccgccagcagcgcatctcctgcggctagctcggcatcacccgcttcgtccggaggttgcgctgatggccctaccctccgtgagtggatttccttctgcggtggctaa(SEQ ID NO: 197) Amino acid sequence of CspAss-TPO2-PAS49-b MKRMKSLAAALTVAGAMLAAPVATAGGCADGPTLREWISFCGGSAPSPSSAPASASAPASPASASASASPASSPASASSASPAASSASPASSGGCADGPTLREWISFCGG(SEQ ID NO: 198)

[0249] TPO2-PAS100 was secreted and expressed as a fusion protein of the 25 amino acid residues of the signal peptide of CspA derived from C. ammoniagenes ATCC6872 strain and TPO2-PAS100 (hereinafter referred to as "CspAss-TPO2-PAS100"). The nucleotide sequence and amino acid sequence encoding the designed CspAss-TPO2-PAS100 are shown in SEQ ID NOs: 199 and 200, respectively.

[0250] Nucleotide sequence encoding CspAss-TPO2-PAS100 atgaaacgcatgaaatcgctggctgcggcgctcaccgtcgctggggccatgctggccgcacctgtggcaacggcaggcggttgtgctgacggtcctaccttgcgtgaatggatttctttctgtggtggtgcggctccggctgctccggctcctgcggctccggctgcaccagcaccagccgcaccagctgcaccagccccagctgcaccagccgctccagcaccagctgccccagctgcaccagctccagccgcaccagcagccccagctccagcagccccagcagctccagcaccagccgctccagctgccccagcaccagctgctccagcagctccagcaccagcagctccagctgctccagccccagccgccccagccgctccagccccagcagcaccagccgccccagctccagccgccccagcaggtggctgtgctgatggtcctacccttcgtgaatggatttccttttgtggcggttag(SEQ ID NO:199) Amino acid sequence of CspAss-TPO2-PAS100 MKRMKSLAAALTVAGAMLAAPVATAGGCADGPTLREWISFCGGAAPAAPAPAAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAAPAPAAPAGGCADGPTLREWISFCGG(SEQ ID NO:200)

[0251] TPO2-PAS100-b was secreted and expressed as a fusion protein of 25 amino acid residues of the signal peptide of CspA derived from C. ammoniagenes ATCC6872 strain and TPO2-PAS100 (hereinafter referred to as "CspAss-TPO2-PAS100-b"). The nucleotide sequence and amino acid sequence encoding the designed CspAss-TPO2-PAS100-b are shown in SEQ ID NO:201 and SE...

Claims

1. (I)a first bioactive peptide site, (II)a linker site consisting of 49 or fewer amino acid residues, (III)a second bioactive peptide site located on the side opposite to the first bioactive peptide site via the linker site A peptide molecule comprising: wherein the first bioactive peptide site and the second bioactive peptide site may be the same as or different from each other, at least 90% of the amino acid sequence of the linker site consists of amino acid residues selected from alanine (A), proline (P), and serine (S), said peptide molecule.

2. The peptide molecule according to claim 1, wherein the first bioactive peptide site forms a cyclic peptide and / or the second bioactive peptide site forms a cyclic peptide.

3. The peptide molecule according to claim 1 or 2, wherein the first bioactive peptide site forms a cyclic peptide with a cyclic structure formed by a disulfide bond and / or the second bioactive peptide site forms a cyclic peptide with a cyclic structure formed by a disulfide bond.

4. The peptide molecule according to any one of claims 1 to 3, wherein the first bioactive peptide site and the second bioactive peptide site each form a cyclic peptide that binds to the c-Met protein, and may be the same as or different from each other.

5. The peptide molecule according to claim 4, wherein the cyclic peptide is a cyclic peptide having a cyclic structure formed by a disulfide bond and comprising an amino acid sequence selected from the following (a) to (i). (a)CYRQFNRRTHEVWNLDC (SEQ ID NO: 1); (b)CRQFNRRTHEVWNLDC (SEQ ID NO: 2); (c)CYWYYAWDQTYKAFPC (SEQ ID NO: 3); (d)CWYYAWDQTYKAFPC (SEQ ID NO: 4); (e)CYISWNEFNSPNWRFITC (SEQ ID NO: 5); (f)CISWNEFNSPNWRFITC (SEQ ID NO: 6); (g)a peptide in which one or two amino acid residues other than cysteine residues are substituted, deleted, or added in any of the amino acid sequences of (a) to (f) and which binds to the c-Met protein; (h)A peptide consisting of an amino acid sequence having 90% or more sequence identity with any one of the amino acid sequences of (a) to (f), provided that it has cysteine residues at both ends and binds to the c-Met protein; and (i)A peptide in which at least one amino acid other than the cysteine residues at both ends is modified in the amino acid sequence of any one of (a) to (h) (the modification of the amino acid is phosphorylation, methylation, acetylation, adenylation, ADP ribosylation or glycosylation).

6. The peptide molecule according to any one of claims 1 to 3, wherein the first bioactive peptide site and the second bioactive peptide site each constitute a cyclic peptide that binds to the erythropoietin receptor, and they may be the same or different from each other.

7. The peptide molecule according to claim 6, wherein the cyclic peptide is a cyclic peptide that forms a cyclic structure by a disulfide bond and contains an amino acid sequence selected from the following (j) to (n). (j)GGLYACHMGPMTWVCQPLRG (SEQ ID NO: 65); (k)CISWNEFNSPNWRFITC (SEQ ID NO: 66); (l)A peptide in which one or two amino acid residues other than the cysteine residues are substituted, deleted or added in the amino acid sequence of (j) or (k) and binds to the erythropoietin receptor; (m)A peptide consisting of an amino acid sequence having 90% or more sequence identity with any one of the amino acid sequences of (j) or (k), provided that it has cysteine residues at the same positions as in (j) or (k) and binds to the erythropoietin receptor; and (n)A peptide in which at least one amino acid other than the cysteine residues is modified in the amino acid sequence of any one of (j) to (m) (the modification of the amino acid is phosphorylation, methylation, acetylation, adenylation, ADP ribosylation or glycosylation).

8. The peptide molecule according to any one of claims 1 to 3, wherein the first bioactive peptide site and the second bioactive peptide site each consist of a bioactive peptide that binds to the thrombopoietin receptor, and they may be the same or different from each other.

9. The peptide molecule according to claim 8, wherein the bioactive peptide contains an amino acid sequence selected from the following (o) to (u). (o)IEGPTLRQWLAARA (SEQ ID NO: 67); (p) GGCADGPTLREWISFCGG (SEQ ID NO: 68); (q) GGCTLREWLHGGFCGG (SEQ ID NO: 69); (r) LAIEGPTLRQWLHGNGRDT (SEQ ID NO: 70); (s) A peptide in which one or two amino acid residues other than cysteine residues are substituted, deleted or added in any of the amino acid sequences of (o) to (r) and which binds to the thrombopoietin receptor; (t) A peptide having 90% or more sequence identity with any of the amino acid sequences of (o) to (r), provided that when (p) or (q) is used as a reference, it has cysteine residues at the same positions as in (p) or (q) and which binds to the thrombopoietin receptor; and (u) A peptide in which at least one amino acid other than cysteine residues is modified in any of the amino acid sequences of (o) to (t) (the modification of the amino acid is phosphorylation, methylation, acetylation, adenylation, ADP ribosylation or glycosylation).

10. The peptide molecule according to any one of claims 1 to 9, further comprising a functional modification site at a position not adjacent to the linker site (II).

11. The linker site (II) is a first linker site, The second bioactive peptide site further comprises a second linker site on the side opposite to the first linker site, The second linker site further comprises a third bioactive peptide site located on the side opposite to the second bioactive peptide site via the second linker site, The third bioactive peptide site may be the same as or different from the first bioactive peptide site and / or the second bioactive peptide site, The second linker site consists of 49 or fewer amino acid residues, and at least 90% of the amino acid sequence consists of amino acid residues selected from alanine (A), proline (P) and serine (S), and may be the same as or different from the first linker site. The peptide molecule according to any one of claims 1 to 10.

12. The linker region (II) contains two or more repetitions of an amino acid sequence selected from the group consisting of AP, AAP, AS, ASP, and ASS, and when a second linker region is present, the second linker region contains two or more repetitions of an amino acid sequence selected from the group consisting of AP, AAP, AS, ASP, and ASS. The peptide molecule according to any one of claims 1 to 11.

13. A method for producing a peptide molecule, generating the peptide molecule by expressing the polynucleotide in a host cell containing the polynucleotide encoding the peptide molecule, or by expressing the polynucleotide encoding the peptide molecule in a cell-free expression system. comprising the peptide molecule includes (I) a first bioactive peptide site, (II) a linker site consisting of amino acid residues, and (III) a second bioactive peptide site and / or an additional amino acid sequence consisting of at least one amino acid residue located on the opposite side of the first bioactive peptide site via the linker site. the first bioactive peptide site and the second bioactive peptide site each have a molecular weight of 10,000 or less and consist of 50 or fewer amino acid residues, and may be the same or different from each other. the additional amino acid sequence has a molecular weight of 10,000 or less and consists of 50 or fewer amino acid residues. The manufacturing method.

14. The manufacturing method according to claim 13, wherein the first bioactive peptide site constitutes a cyclic peptide and / or the second bioactive peptide site constitutes a cyclic peptide.

15. A method for producing a peptide molecule, generating the peptide molecule by expressing the polynucleotide in a host cell containing the polynucleotide encoding the peptide molecule, or by expressing the polynucleotide encoding the peptide molecule in a cell-free expression system. comprising the peptide molecule includes (I) a first bioactive peptide site that constitutes a cyclic peptide, (II) a linker site consisting of amino acid residues, and (III) a second bioactive peptide site and / or an additional amino acid sequence consisting of at least one amino acid residue located on the opposite side of the first bioactive peptide site via the linker site. The first bioactive peptide site and the second bioactive peptide site may be the same as or different from each other. The production method, wherein the additional amino acid sequence has a molecular weight of 10,000 or less and consists of 50 or fewer amino acid residues. **Claim 16** The production method according to claim 15, wherein the peptide molecule contains the second bioactive peptide site, and the second bioactive peptide site constitutes a cyclic peptide. **Claim 17** The production method according to any one of claims 13 to 16, wherein the linker site has a molecular weight of 10,000 or less and consists of 50 or fewer amino acid residues. **Claim 18** The production method according to any one of claims 13 to 16, wherein the linker site has a molecular weight of 10,000 or less and is a linker site consisting of 49 or fewer amino acid residues, and at least 90% of the amino acid sequence thereof consists of amino acid residues selected from alanine (A), proline (P), and serine (S). **Claim 19** The production method according to any one of claims 13 to 18, wherein the linker site contains two or more repetitions of an amino acid sequence selected from the group consisting of AP, AAP, AS, ASP, and ASS.

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