Antibodies binding to fibrin, fibrinolytic proteins containing the antibodies and pharmaceutical preparations containing the proteins

Abstract

The present disclosure provides antibodies that bind to fibrin, fibrinolytic proteins containing the antibodies, and pharmaceutical formulations containing the proteins.

Description

Technical Field

[0001] This invention relates to antibodies that bind to fibrin, fibrinolytic proteins containing the antibodies, and pharmaceutical formulations containing the proteins. Particularly, this invention relates to fibrinolytic proteins suitable for large-scale purification and pharmaceutical formulations containing the proteins. Further, this invention relates to methods for stabilizing fibrinolytic proteins and methods for manufacturing stabilized fibrinolytic proteins. Background Technology

[0002] Antibodies that bind to fibrin with a stronger affinity than fibrinogen have been developed (Patent Documents 1 and 2). These patent documents disclose the use of these antibodies in cancer treatment. Antibodies that bind strongly to fibrin can also be used for in vivo imaging (Patent Documents 1 and 2).

[0003] In addition, a fusion protein of an antibody that binds to fibrin and a urokinase mutant was developed and disclosed to have thrombolytic activity (Patent Document 3).

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: WO2014 / 133093

[0007] Patent Document 2: WO2018 / 203517

[0008] Patent Document 3: WO2021 / 200922 Summary of the Invention

[0009] This invention provides an antibody that binds to fibrin, a fibrinolytic protein containing the antibody, and a pharmaceutical formulation containing the protein. Specifically, this invention addresses the problems in mass production described in WO2021 / 200922 (Patent Document 3) (e.g., the problem of increased production limits in protein-producing cells, the problem of producing protein breakdown products, the problem of needing serum during culture, the problem of needing specific additives during purification, and the problem of accidental activation, etc.). Furthermore, the fusion protein disclosed in Patent Document 3 does not bind to mouse urokinase receptors but shows reactivity to human urokinase receptors. In one aspect of this invention, reactivity to human urokinase receptors can be reduced or eliminated.

[0010] According to the present invention, the following invention is provided.

[0011] [1] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin and a catalytic domain of urokinase having an amino acid sequence corresponding to the amino acid sequence from position 156 to position 411 of urokinase as described in sequence number 6 or 7, wherein the antibody or antigen-binding fragment thereof is directly or via a linker connected to the catalytic domain of urokinase, wherein in the fusion protein, The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. The phenylalanine in the catalytic domain corresponding to the phenylalanine at position 157 of prourokinase as recorded in sequence number 6 or 7 is replaced with other amino acids.

[0012] [2] According to the fusion protein described in [1] above, wherein the phenylalanine is replaced with isoleucine, glutamic acid, aspartic acid or cysteine.

[0013] [3] According to the fusion protein described in [1] or [2] above, wherein the phenylalanine is replaced with glutamic acid or aspartic acid.

[0014] [4] The fusion protein according to any one of [1] to [3] above, wherein, compared with the fusion protein having the amino acid sequence described in Serial No. 1, the binding ability to one or more urokinase receptors selected from the group consisting of human urokinase receptor and mouse urokinase receptor is reduced.

[0015] [5] The fusion protein according to any one of [1] to [4] above, wherein it does not show significant binding to one or more urokinase receptors selected from the group consisting of human urokinase receptor and mouse urokinase receptor.

[0016] [6] The fusion protein according to any one of [1] to [3] above, wherein, between the antibody or its antigen-binding fragment portion and the catalytic domain portion, there is an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence of the 7th to 43rd positions of the urokinase described in sequence number 6 or 7, wherein the amino acids of the above EGF-like domain corresponding to the asparagine at position 22, the asparagine at position 27, the histidine at position 29, the tryptophan at position 30 and the glutamine at position 40 of the urokinase described in sequence number 6 or 7 are replaced with other amino acids, thereby reducing or losing the ability to bind to the human urokinase receptor.

[0017] [7] The fusion protein according to any one of [1] to [4] above, wherein, between the antibody or its antigen-binding fragment portion and the catalytic domain portion, there is an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence of the 7th to 43rd positions of the urokinase described in sequence number 6 or 7, wherein the amino acids of the EGF-like domain corresponding to the asparagine at position 22, the asparagine at position 27, the histidine at position 29, the tryptophan at position 30 and the glutamine at position 40 of the urokinase described in sequence number 6 or 7 are respectively replaced with tyrosine, serine, arginine, arginine and glutamic acid, thereby reducing or losing the ability to bind to the human urokinase receptor.

[0018] [8] The fusion protein according to any one of [1] to [4] above, wherein, between the antibody or its antigen-binding fragment portion and the catalytic domain portion, there is an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence of the 7th to 43rd position of the urokinase described in sequence number 6 or 7, and an amino acid deletion of the above EGF-like domain corresponding to the amino acid from asparagine at position 22 to isoleucine at position 28 of the urokinase described in sequence number 6 or 7 {the deletion site may have an insertion of 1 to 7 amino acids (a peptide that does not have binding affinity to the human urokinase receptor, such as a 2-amino acid peptide, such as GG)}.

[0019] [9] A pharmaceutical preparation containing any one of the above [1] to [8] fusion proteins.

[0020]

[10] A method for manufacturing the fusion protein described in any one of [1] to [8] above, comprising the steps of: culturing a protein-producing cell having a nucleic acid encoding the fusion protein in an expressible manner, thereby expressing the fusion protein; and purifying the fusion protein from the resulting culture.

[0021]

[11] According to the method described in

[10] above, wherein the culture is carried out under serum-free conditions or in the presence of less than 5% serum.

[0022]

[12] The method described in

[10] or

[11] above, wherein the purification is carried out in the absence of L-arginine.

[0023]

[13] A method for manufacturing (preferably with good stability) a fusion protein modifier or a pharmaceutical composition containing the modifier, wherein, The fusion protein is a fusion protein containing an antibody or antigen-binding fragment thereof that binds to insoluble fibrin and a catalytic domain of urokinase having an amino acid sequence corresponding to the amino acid sequence from position 156 to position 411 of urokinase as described in sequence number 6 or 7, wherein the antibody or antigen-binding fragment thereof is directly or indirectly linked to the catalytic domain of urokinase. The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. The method includes the following steps: introducing an F157E or F157D mutation into the fusion protein, thereby conferring better stability than the unmodified fusion protein.

[0024]

[21] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin and a catalytic domain of urokinase having an amino acid sequence corresponding to the amino acid sequence from position 156 to position 411 of urokinase as described in sequence number 6 or 7, wherein the antibody or antigen-binding fragment thereof is directly or via a linker connected to the catalytic domain of urokinase, wherein in the fusion protein, The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. Between the antibody or its antigen-binding fragment portion and the catalytic domain portion, there is an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from position 7 to position 43 of the urokinase described in sequence number 6 or 7. The amino acids of the above-mentioned EGF-like domain corresponding to asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30, and glutamine at position 40 of the urokinase described in sequence number 6 or 7 are replaced with other amino acids, thereby reducing or eliminating the binding ability to the human urokinase receptor.

[0025]

[22] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin and a catalytic domain of urokinase having an amino acid sequence corresponding to the amino acid sequence from position 156 to position 411 of urokinase as described in sequence number 6 or 7, wherein the antibody or antigen-binding fragment thereof is directly or via a linker connected to the catalytic domain of urokinase, wherein in the fusion protein, The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. Between the antibody or its antigen-binding fragment portion and the catalytic domain portion, there is an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from position 7 to position 43 of the urokinase described in sequence number 6 or 7. The amino acids in the above-mentioned EGF-like domain corresponding to asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30, and glutamine at position 40 of the urokinase described in sequence number 6 or 7 are replaced with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing or eliminating the binding ability to human urokinase receptors.

[0026]

[23] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of urokinase having an amino acid sequence corresponding to amino acids 7 to 43 of urokinase as described in sequence number 6 or 7, a kringle domain of urokinase corresponding to amino acids 49 to 131 of urokinase, and a catalytic domain of urokinase having an amino acid sequence corresponding to amino acids 156 to 411 of urokinase, wherein the antibody or antigen-binding fragment thereof is directly or via a linker connected to the catalytic domain of urokinase, wherein in the fusion protein, The connector is a non-disintegrating connector. The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. Between the antibody or its antigen-binding fragment portion and the catalytic domain portion, there is an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from position 7 to position 43 of the urokinase described in sequence number 6 or 7. The amino acids of the above-mentioned EGF-like domain corresponding to asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30, and glutamine at position 40 of the urokinase described in sequence number 6 or 7 are replaced with other amino acids, thereby reducing or eliminating the binding ability to the human urokinase receptor.

[0027]

[24] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of urokinase having an amino acid sequence corresponding to amino acids 7 to 43 of urokinase as described in sequence number 6 or 7, a kringle domain of urokinase corresponding to amino acids 49 to 131 of urokinase, and a catalytic domain of urokinase having an amino acid sequence corresponding to amino acids 156 to 411 of urokinase, wherein the antibody or antigen-binding fragment thereof is directly or via a linker connected to the catalytic domain of urokinase, wherein the fusion protein, The connector is a non-disintegrating connector. The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. Between the antibody or its antigen-binding fragment portion and the catalytic domain portion, there is an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from position 7 to position 43 of the urokinase described in sequence number 6 or 7. The amino acids in the above-mentioned EGF-like domain corresponding to asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30, and glutamine at position 40 of the urokinase described in sequence number 6 or 7 are replaced with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing or eliminating the binding ability to human urokinase receptors.

[0028]

[25] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of urokinase having an amino acid sequence corresponding to amino acids 7 to 43 of urokinase as described in sequence number 6 or 7, a kringle domain of urokinase corresponding to amino acids 49 to 131, and a catalytic domain of urokinase having an amino acid sequence corresponding to amino acids 156 to 411, wherein the antibody or antigen-binding fragment thereof is directly or via a linker connected to the catalytic domain of urokinase, wherein the fusion protein, The connector is a non-disintegrating connector. The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. Between the antibody or its antigen-binding fragment portion and the catalytic domain portion, there is an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from position 7 to position 43 of the urokinase described in sequence number 6 or 7. The amino acid sequence of the above-mentioned EGF-like domain corresponding to the amino acid sequence from position 22 (asparagine) to position 28 (isoleucine) of the urokinase described in sequence number 6 or 7 is deleted {at the deletion site, there may be an insertion of 1 to 7 amino acids (a peptide that does not have binding affinity for the human urokinase receptor, such as a 2-amino acid peptide, such as GG)}, thereby reducing or preferably eliminating the binding ability to the human urokinase receptor.

[0029]

[26] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of urokinase having an amino acid sequence corresponding to amino acids 7 to 43 of urokinase as described in sequence number 6 or 7, a kringle domain of urokinase corresponding to amino acids 49 to 131 of urokinase, and a catalytic domain of urokinase having an amino acid sequence corresponding to amino acids 156 to 411 of urokinase, wherein the antibody or antigen-binding fragment thereof is directly or via a linker connected to the catalytic domain of urokinase, wherein the fusion protein, The connector is a non-disintegrating connector. The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. Between the antibody or its antigen-binding fragment portion and the catalytic domain portion, there is an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from position 7 to position 43 of the urokinase described in sequence number 6 or 7. The amino acid sequence of the above-mentioned EGF-like domain corresponding to the amino acid sequence from position 22 (asparagine) to position 28 (isoleucine) of the urokinase described in sequence number 6 or 7 is deleted. At the deletion site, there is an insertion of 1 to 7 amino acids (a peptide that does not have binding affinity for the human urokinase receptor, such as a 2-amino acid peptide, such as GG), which reduces or preferably eliminates the binding ability to the human urokinase receptor.

[0030] [31A] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin and a catalytic domain of urokinase having an amino acid sequence corresponding to the amino acid sequence from position 156 to position 411 of urokinase as described in sequence number 6 or 7, wherein the antibody or antigen-binding fragment thereof is directly or via a linker connected to the catalytic domain of urokinase, wherein in the fusion protein, The connector is a non-disintegrating connector. The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. The phenylalanine in the catalytic domain corresponding to the phenylalanine at position 157 of prourokinase as recorded in sequence number 6 or 7 is replaced with other amino acids.

[0031] [31B] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of urokinase having an amino acid sequence corresponding to amino acids 7 to 43 of urokinase as described in sequence number 6 or 7, and a catalytic domain of urokinase having an amino acid sequence corresponding to amino acids 156 to 411, wherein the antibody or antigen-binding fragment thereof is directly or via a linker to the catalytic domain of urokinase, wherein in the fusion protein, The connector is a non-disintegrating connector. The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. The phenylalanine in the catalytic domain corresponding to the phenylalanine at position 157 of prourokinase as recorded in sequence number 6 or 7 is replaced with other amino acids.

[0032] [31C] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, a kringle domain of urokinase corresponding to amino acids 49 to 131 of urokinase as described in sequence number 6 or 7, and a catalytic domain of urokinase having an amino acid sequence corresponding to amino acid sequences 156 to 411, wherein the antibody or antigen-binding fragment thereof is directly or via a linker to the catalytic domain of urokinase, wherein in the fusion protein, The connector is a non-disintegrating connector. The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. The phenylalanine in the catalytic domain corresponding to the phenylalanine at position 157 of prourokinase as recorded in sequence number 6 or 7 is replaced with other amino acids.

[0033] [31D] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of urokinase having an amino acid sequence corresponding to amino acids 7 to 43 of urokinase as described in sequence number 6 or 7, a kringle domain of urokinase corresponding to amino acids 49 to 131 of urokinase, and a catalytic domain of urokinase having an amino acid sequence corresponding to amino acids 156 to 411 of urokinase, wherein the antibody or antigen-binding fragment thereof is directly or via a linker connected to the catalytic domain of urokinase, wherein in the fusion protein, The connector is a non-disintegrating connector. The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. The phenylalanine in the catalytic domain corresponding to the phenylalanine at position 157 of prourokinase as recorded in sequence number 6 or 7 is replaced with other amino acids.

[0034]

[32] According to the fusion protein described in

[31] above (i.e., any one of [31A] to [31D] above), wherein the phenylalanine is replaced by isoleucine, glutamic acid, aspartic acid or cysteine.

[0035]

[33] According to the fusion protein described in

[31] or

[32] above, wherein the phenylalanine is replaced with glutamic acid or aspartic acid.

[0036]

[34] The fusion protein according to any one of

[31] to

[33] above, wherein, Between the antibody or its antigen-binding fragment portion and the catalytic domain portion, there is an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from position 7 to position 43 of the urokinase described in sequence number 6 or 7. The amino acids of the above-mentioned EGF-like domain corresponding to asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30, and glutamine at position 40 of the urokinase described in sequence number 6 or 7 are replaced with other amino acids, thereby reducing or eliminating the binding ability to the human urokinase receptor.

[0037]

[35] The fusion protein according to any one of

[31] to

[34] above, wherein, Between the antibody or its antigen-binding fragment portion and the catalytic domain portion, there is an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from position 7 to position 43 of the urokinase described in sequence number 6 or 7. The amino acids in the above-mentioned EGF-like domain corresponding to asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30, and glutamine at position 40 of the urokinase described in sequence number 6 or 7 are replaced with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing or eliminating the binding ability to human urokinase receptors.

[0038]

[36] The fusion protein according to any one of

[31] to

[34] above, wherein, Between the antibody or its antigen-binding fragment portion and the catalytic domain portion, there is also an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from position 7 to position 43 of the urokinase described in sequence number 6 or 7, and a deletion of the amino acid sequence of the EGF-like domain corresponding to the amino acid sequence from position 22 (asparagine) to position 28 (isoleucine) of the urokinase described in sequence number 6 or 7 (for example, an insertion of 1 to 7 amino acids (at least a peptide that does not bind to the human urokinase receptor, preferably a peptide that does not bind to both the human and mouse urokinase receptors, such as a 2-amino acid peptide, such as GG)) at the deletion site), resulting in reduced or preferably lost binding ability to the human urokinase receptor.

[0039]

[37] A pharmaceutical preparation containing any one of the fusion proteins described in any one of

[31] to

[36] above.

[0040]

[38] A method for manufacturing the fusion protein described in any one of

[31] to

[36] above, comprising the following steps: Protein-producing cells that have nucleic acids encoding the aforementioned fusion protein in an expressible manner are cultured to express the fusion protein, and the fusion protein is purified from the resulting culture.

[0041]

[39] According to the method described in

[38] above, the culture is carried out under serum-free conditions or in the presence of less than 5% serum.

[0042]

[40] The method described in

[38] or

[39] above, wherein the purification is carried out in the absence of L-arginine.

[0043]

[41] A fusion protein, wherein the fusion protein contains an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of urokinase having an amino acid sequence corresponding to amino acids 7 to 43 of urokinase as described in sequence number 6 or 7, a kringle domain of urokinase corresponding to amino acids 49 to 131, and a catalytic domain of urokinase having an amino acid sequence corresponding to amino acids 156 to 411, wherein the antibody or antigen-binding fragment thereof is directly or via a linker connected to the catalytic domain of urokinase, wherein the fusion protein, The connector is a non-disintegrating connector. The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. The phenylalanine in the catalytic domain corresponding to the phenylalanine at position 157 of prourokinase as described in sequence number 6 or 7 is replaced with other amino acids. The amino acids corresponding to the asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30, and glutamine at position 40 of the urokinase precursor described in sequence number 6 or 7 are replaced with other amino acids, resulting in a decrease or loss of the ability to bind to the human urokinase receptor.

[0044]

[42] According to the fusion protein described in

[41] above, wherein the phenylalanine in the catalytic domain corresponding to the phenylalanine at position 157 is replaced with an amino acid other than tyrosine.

[0045]

[43] According to the fusion protein described above

[41] , wherein the phenylalanine in the catalytic domain corresponding to the phenylalanine at position 157 is replaced with isoleucine, glutamic acid, aspartic acid or cysteine.

[0046]

[44] According to the fusion protein described in

[41] above, the phenylalanine in the catalytic domain corresponding to the phenylalanine at position 157 is glutamic acid or aspartic acid.

[0047]

[45] According to the fusion protein described above

[41] , the amino acids of the above-mentioned EGF-like domains corresponding to asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30 and glutamine at position 40 of the urokinase progenitor recorded in sequence number 6 or 7 are replaced with tyrosine, serine, arginine, arginine and glutamic acid, respectively, thereby reducing or eliminating the binding ability to human urokinase receptor.

[0048]

[46] According to the fusion protein described above

[42] , wherein the amino acids of the above-mentioned EGF-like domains corresponding to asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30 and glutamine at position 40 of the urokinase described in sequence number 6 or 7 are replaced with tyrosine, serine, arginine, arginine and glutamic acid respectively, thereby reducing or losing the ability to bind to human urokinase receptor.

[0049]

[47] According to the fusion protein described above

[43] , the amino acids of the above-mentioned EGF-like domains corresponding to asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30 and glutamine at position 40 of the urokinase progenitor recorded in sequence number 6 or 7 are replaced with tyrosine, serine, arginine, arginine and glutamic acid respectively, thereby reducing or losing the ability to bind to human urokinase receptor.

[0050]

[48] ​​According to the fusion protein described above

[44] , the amino acids of the above-mentioned EGF-like domains corresponding to asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30 and glutamine at position 40 of the urokinase progenitor recorded in sequence number 6 or 7 are replaced with tyrosine, serine, arginine, arginine and glutamic acid respectively, thereby reducing or losing the ability to bind to human urokinase receptor.

[0051]

[61] According to any of the preceding claims, the fusion protein wherein the antibody or its antigen-binding fragment specifically binds to insoluble fibrin (particularly, for one or more, preferably all, selected from the group consisting of fibrinogen, soluble fibrin and fibrin breakdown products, the binding is weak, particularly preferably insignificant, compared to the binding to insoluble fibrin).

[0052]

[62] The fusion protein according to any of the preceding claims, wherein the antibody or an antigen-binding fragment thereof is bound to a peptide consisting of the amino acid sequence described in Serial No. 8.

[0053]

[63] The fusion protein according to any of the preceding claims, wherein the antibody or its antigen-binding fragment has heavy chain CDR1-3 and light chain CDR1-3 respectively selected from the group consisting of antibody 102-10, antibody 34-105 and antibody Fib-0355 disclosed in WO2014 / 133093 as corresponding CDRs.

[0054]

[64] The fusion protein according to any of the preceding claims, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region having a heavy chain CDR1 having the amino acid sequence described in sequence number 10, a heavy chain CDR2 having the amino acid sequence described in sequence number 11, a heavy chain CDR3 having the amino acid sequence described in sequence number 12, and a light chain variable region having a light chain CDR1 having the amino acid sequence described in sequence number 13, a light chain CDR2 having the amino acid sequence described in sequence number 14, and a light chain CDR3 having the amino acid sequence described in sequence number 15.

[0055]

[65] The fusion protein according to any of the preceding claims, wherein the antibody or its antigen-binding fragment contains a heavy chain variable region having the amino acid sequence described in Serial No. 16 and a light chain variable region having the amino acid sequence described in Serial No. 17.

[0056]

[66] The fusion protein according to any one of the preceding claims comprises a heavy chain variable region having a heavy chain CDR1 having the amino acid sequence described in sequence number 18, a heavy chain CDR2 having the amino acid sequence described in sequence number 19, a heavy chain CDR3 having the amino acid sequence described in sequence number 20, and a light chain variable region having a light chain CDR1 having the amino acid sequence described in sequence number 21, a light chain CDR2 having the amino acid sequence described in sequence number 22, and a light chain CDR3 having the amino acid sequence described in sequence number 23.

[0057]

[67] The fusion protein according to any of the preceding claims, wherein the antibody or its antigen-binding fragment contains a heavy chain variable region of an amino acid sequence having the amino acid sequence described in Serial No. 24, and a light chain variable region of an amino acid sequence having the amino acid sequence described in Serial No. 25.

[0058]

[68] The fusion protein according to any of the preceding claims, wherein the antibody or its antigen-binding fragment competes with a reference antibody for binding to insoluble fibrin, the reference antibody containing a heavy chain variable region having an amino acid sequence having the amino acid sequence described in Serial No. 16 and a light chain variable region having an amino acid sequence having the amino acid sequence described in Serial No. 17.

[0059]

[69] The fusion protein according to any of the preceding claims, wherein the antibody or its antigen-binding fragment competes with a reference antibody for binding to insoluble fibrin, the reference antibody containing a heavy chain variable region having an amino acid sequence having the amino acid sequence described in sequence number 24, and a light chain variable region having an amino acid sequence having the amino acid sequence described in sequence number 25.

[0060]

[70] The fusion protein according to any of the preceding claims has an amino acid sequence of sequence number 107.

[0061]

[71] The fusion protein according to any one of the preceding claims, wherein the antibody or its antigen-binding fragment has 10 -6 M and below, 10 -7 M or below or 10 -8 The binding dissociation constant (KD) below M.

[0062]

[72] The fusion protein according to any of the preceding claims, wherein the antibody or its antigen-binding fragment is a Fab fragment or a Fab' fragment {preferably a fusion protein formed by linking the C-terminus of the heavy chain of the above fragment to the N-terminus of prourokinase or a portion thereof or a mutant thereof}.

[0063]

[81] The fusion protein according to any one of the preceding claims has the following characteristics selected from the group consisting of at least one, preferably at least two, more preferably at least three, and even more preferably all of the following: (i) binding specificity to insoluble fibrin; (ii) binding affinity to insoluble fibrin; (iii) resistance to inhibition caused by plasminogen activator inhibitor (PAI-1); and (iv) low inducibility of matrix metalloproteinase (MMP) activity in tissues surrounding thrombus or embolism.

[0064]

[82] The fusion protein according to any of the preceding claims is linked to an imaging agent.

[0065]

[91] A pharmaceutical preparation containing any of the fusion proteins described above.

[0066]

[92] The pharmaceutical preparation described above

[91] is used to treat thromboembolism or extravascular or intravascular diseases with accumulation of insoluble fibrin (e.g., extravascular diseases with accumulation of insoluble fibrin, preferably Alzheimer's disease).

[0067]

[93] The pharmaceutical preparation described above

[91] is a suitable treatment for the dissolution of fibrin clots or for the management of a disease.

[0068]

[94] The pharmaceutical preparation described above

[91] is used to treat cerebrovascular and cardiovascular disorders with thrombosis or embolism.

[0069]

[95] The pharmaceutical preparation described above

[91] is used to treat conditions or symptoms arising from cerebrovascular and cardiovascular disorders due to the formation of thrombi or embolisms.

[0070]

[96] The pharmaceutical preparation described above

[91] is used to treat the state or symptoms of cerebrovascular and cardiovascular disorders.

[0071]

[97] The pharmaceutical preparation described above

[91] is used to treat ischemic cerebrovascular disorders.

[0072]

[98] The pharmaceutical preparation described above

[91] is used to treat either cerebral infarction or transient ischemic attack.

[0073]

[99] The pharmaceutical preparation described above

[91] is used to treat ischemic cardiovascular disorders.

[0074]

[100] The pharmaceutical preparation described above

[91] is used to treat either angina pectoris or myocardial infarction.

[0075]

[101] The pharmaceutical preparation according to any one of

[91] to

[100] above is used to treat a disease in the acute phase.

[0076]

[102] The pharmaceutical preparation described above

[91] is used to dissolve fibrin clots in a subject having fibrin clots.

[0077]

[103] A pharmaceutical preparation for detecting thrombi or embolisms in the body, the pharmaceutical preparation containing the fusion protein described above

[82] .

[0078]

[104] The pharmaceutical preparation described in

[103] above is also used for the pharmaceutical uses specified in any one of

[92] to

[102] above.

[0079] According to the present invention, the following invention is provided.

[1001] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin and a catalytic domain of urokinase having an amino acid sequence corresponding to amino acid positions 156 to 411 of urokinase as described in sequence number 6 or 7, wherein the antibody or antigen-binding fragment thereof is directly or via a linker connected to the catalytic domain of urokinase, wherein in the fusion protein, The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. The arginine in the catalytic domain corresponding to the arginine at position 156 of the prourokinase described in sequence number 6 or 7 is replaced with other amino acids {here, the fusion protein preferably has significantly increased stability against degradation in human plasma compared to AMU1114, for example, it may have mutations of R156I, R156E or R156D}.

[1002] According to the fusion protein described above

[1001] , wherein the arginine is replaced with glutamic acid or aspartic acid.

[1003] According to the fusion protein described in

[1001] or

[1002] above, wherein, Compared to fusion proteins with the amino acid sequence described in Serial No. 1, the binding ability to more than one urokinase receptor selected from the group consisting of human urokinase receptor and mouse urokinase receptor is reduced.

[1004] The fusion protein according to any one of claims

[1001] to

[1003] above, wherein, Significant binding was not observed to one or more urokinase receptors selected from the group consisting of human and mouse urokinase receptors.

[1005] The fusion protein according to any one of

[1001] to

[1003] above, wherein, Between the antibody or its antigen-binding fragment portion and the catalytic domain portion, there is an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from position 7 to position 43 of the urokinase described in sequence number 6 or 7. The amino acids of the above-mentioned EGF-like domain corresponding to asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30, and glutamine at position 40 of the urokinase described in sequence number 6 or 7 are replaced with other amino acids, thereby reducing or eliminating the binding ability to the human urokinase receptor.

[1006] The fusion protein according to any one of

[1001] to

[1003] above, wherein, Between the antibody or its antigen-binding fragment portion and the catalytic domain portion, there is an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from position 7 to position 43 of the urokinase described in sequence number 6 or 7. The amino acids in the above-mentioned EGF-like domain corresponding to asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30, and glutamine at position 40 of the urokinase described in sequence number 6 or 7 are replaced with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing or eliminating the binding ability to human urokinase receptors.

[1007] The fusion protein according to any one of

[1001] to

[1003] above, wherein, Between the antibody or its antigen-binding fragment portion and the catalytic domain portion, there is also an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from position 7 to position 43 of the urokinase described in sequence number 6 or 7, and an amino acid deletion of the above-mentioned EGF-like domain corresponding to the amino acids from asparagine to isoleucine at position 22 of the urokinase described in sequence number 6 or 7 {the deletion site may have an insertion of 1 to 7 amino acids (a peptide that does not have binding affinity for the human urokinase receptor, such as a 2-amino acid peptide, such as GG)}.

[1008] The fusion protein according to any one of

[1001] to

[1007] above, wherein the amino acid of the catalytic domain corresponding to the phenylalanine at position 157 of the prourokinase described in sequence number 6 or 7 is phenylalanine.

[1009] A pharmaceutical preparation containing any one of the fusion proteins described in

[1001] to

[1008] above.

[1010] The pharmaceutical preparation described above

[1009] is used in a method for dissolving thrombi or embolisms in subjects with acute or chronic thrombotic or embolic diseases.

[1011] The pharmaceutical preparation described in

[1009] or

[1010] above is used in a method for dissolving thrombi or embolisms in a subject with acute infarction.

[1012] A method for manufacturing the fusion protein described in any one of

[1001] to

[1008] above, comprising the following steps: The fusion protein is expressed by culturing protein-producing cells that have nucleic acids encoding the fusion protein in an expressible manner; and the fusion protein is purified from the resulting culture.

[1013] According to the method described in

[1012] above, the culture is carried out under serum-free conditions or in the presence of less than 5% serum.

[1014] According to the method described in

[1012] or

[1013] above, the purification is carried out in the absence of L-arginine.

[1015] A method for modifying a fusion protein and a modified fusion protein obtained by the method, wherein... The fusion protein is a fusion protein containing an antibody or antigen-binding fragment thereof that binds to insoluble fibrin and a catalytic domain of urokinase having an amino acid sequence corresponding to the amino acid sequence from position 156 to position 411 of urokinase as described in sequence number 6 or 7, wherein the antibody or antigen-binding fragment thereof is directly or indirectly linked to the catalytic domain of urokinase. The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. The method includes the following steps: introducing a mutation (e.g., point mutations such as R156I, R156E, or R156D) into the amino acid corresponding to R156 of the above-mentioned fusion protein, thereby endowing it with superior stability and superior urokinase activity when bound to insoluble fibrin compared to the fusion protein before modification.

[1016] According to the method described above

[1015] and the modified fusion protein obtained by the method, the method includes the step of further introducing mutations (e.g., point mutations such as F157Y) into the amino acid corresponding to F157 of the fusion protein.

[1017] The method described in

[1015] or

[1016] above and the modified fusion protein obtained by the method include a step of further introducing mutations (e.g., point mutations such as P155G) into the amino acid corresponding to P155 of the fusion protein.

[1018] According to the fusion protein described above

[1007] , the antibody or its antigen-binding fragment that binds to insoluble fibrin is an antibody or its antigen-binding fragment that binds to insoluble fibrin, and the linker of the heavy chain variable region, EGF-like domain and catalytic domain is composed of the amino acid sequence of sequence number 64.

[1019] According to the fusion protein described above

[1007] , the fusion protein exhibits more rapid urokinase activity compared to the control protein under the conditions of no plasmin pretreatment and 5 μg / mL plasminogen, plasmin substrate (e.g., 15 μL / well TestzymS PLG, Sekisui Medical) and 37°C. The control protein is a protein containing a heavy chain variable region, an EGF-like domain and a catalytic domain, with the linker consisting of the amino acid sequence of sequence number 63 and the light chain consisting of the amino acid sequence of sequence number 17, and the linker and the light chain are complexed together.

[1021] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin and a catalytic domain of urokinase having an amino acid sequence corresponding to amino acid positions 156 to 411 of urokinase as described in sequence number 6 or 7, wherein the antibody or antigen-binding fragment thereof is directly or via a linker connected to the catalytic domain of urokinase, wherein in the fusion protein, The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. Between the antibody or its antigen-binding fragment portion and the catalytic domain portion, there is an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from position 7 to position 43 of the urokinase described in sequence number 6 or 7. The amino acids of the above-mentioned EGF-like domain corresponding to asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30, and glutamine at position 40 of the urokinase described in sequence number 6 or 7 are replaced with other amino acids, thereby reducing or eliminating the binding ability to the human urokinase receptor.

[1022] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin and a catalytic domain of urokinase having an amino acid sequence corresponding to amino acid positions 156 to 411 of urokinase as described in sequence number 6 or 7, wherein the antibody or antigen-binding fragment thereof is directly or via a linker connected to the catalytic domain of urokinase, wherein in the fusion protein, The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. Between the antibody or its antigen-binding fragment portion and the catalytic domain portion, there is an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from position 7 to position 43 of the urokinase described in sequence number 6 or 7. The amino acids in the above-mentioned EGF-like domain corresponding to asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30, and glutamine at position 40 of the urokinase described in sequence number 6 or 7 are replaced with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing or eliminating the binding ability to human urokinase receptors.

[1023] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of urokinase having an amino acid sequence corresponding to amino acids 7 to 43 of urokinase as described in sequence number 6 or 7, a kringle domain of urokinase corresponding to amino acids 49 to 131 of urokinase, and a catalytic domain of urokinase having an amino acid sequence corresponding to amino acids 156 to 411 of urokinase, wherein the antibody or antigen-binding fragment thereof is directly or via a linker connected to the catalytic domain of urokinase, wherein the fusion protein, The connector is a non-disintegrating connector. The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. Between the antibody or its antigen-binding fragment portion and the catalytic domain portion, there is an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from position 7 to position 43 of the urokinase described in sequence number 6 or 7. The amino acids of the above-mentioned EGF-like domain corresponding to asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30, and glutamine at position 40 of the urokinase described in sequence number 6 or 7 are replaced with other amino acids, thereby reducing or eliminating the binding ability to the human urokinase receptor.

[1024] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of urokinase having an amino acid sequence corresponding to amino acids 7 to 43 of urokinase as described in sequence number 6 or 7, a kringle domain of urokinase corresponding to amino acids 49 to 131 of urokinase, and a catalytic domain of urokinase having an amino acid sequence corresponding to amino acids 156 to 411 of urokinase, wherein the antibody or antigen-binding fragment thereof is directly or via a linker connected to the catalytic domain of urokinase, wherein the fusion protein, The connector is a non-disintegrating connector. The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. Between the antibody or its antigen-binding fragment portion and the catalytic domain portion, there is an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from position 7 to position 43 of the urokinase described in sequence number 6 or 7. The amino acids in the above-mentioned EGF-like domain corresponding to asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30, and glutamine at position 40 of the urokinase described in sequence number 6 or 7 are replaced with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing or eliminating the binding ability to human urokinase receptors.

[1025] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of urokinase having an amino acid sequence corresponding to amino acids 7 to 43 of urokinase as described in sequence number 6 or 7, a kringle domain of urokinase corresponding to amino acids 49 to 131 of urokinase, and a catalytic domain of urokinase having an amino acid sequence corresponding to amino acids 156 to 411 of urokinase, wherein the antibody or antigen-binding fragment thereof is directly or via a linker connected to the catalytic domain of urokinase, wherein the fusion protein, The connector is a non-disintegrating connector. The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. Between the antibody or its antigen-binding fragment portion and the catalytic domain portion, there is an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from position 7 to position 43 of the urokinase described in sequence number 6 or 7. The amino acid sequence of the above-mentioned EGF-like domain corresponding to the amino acid sequence from position 22 (asparagine) to position 28 (isoleucine) of the urokinase described in sequence number 6 or 7 is deleted {at the deletion site, there may be an insertion of 1 to 7 amino acids (a peptide that does not have binding affinity for the human urokinase receptor, such as a 2-amino acid peptide, such as GG)}, thereby reducing or preferably eliminating the binding ability to the human urokinase receptor.

[1026] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of urokinase having an amino acid sequence corresponding to amino acids 7 to 43 of urokinase as described in sequence number 6 or 7, a kringle domain of urokinase corresponding to amino acids 49 to 131 of urokinase, and a catalytic domain of urokinase having an amino acid sequence corresponding to amino acids 156 to 411 of urokinase, wherein the antibody or antigen-binding fragment thereof is directly or via a linker connected to the catalytic domain of urokinase, wherein the fusion protein, The connector is a non-disintegrating connector. The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. Between the antibody or its antigen-binding fragment portion and the catalytic domain portion, there is an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from position 7 to position 43 of the urokinase described in sequence number 6 or 7. The amino acid sequence of the above-mentioned EGF-like domain corresponding to the amino acid sequence from position 22 (asparagine) to position 28 (isoleucine) of the urokinase described in sequence number 6 or 7 is deleted. The deleted site has an insertion of 1 to 7 amino acids (a peptide that does not have binding affinity for the human urokinase receptor, such as a 2-amino acid peptide, such as GG), which reduces or preferably eliminates the binding ability to the human urokinase receptor. [1031A] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin and a catalytic domain of urokinase having an amino acid sequence corresponding to the amino acid sequence from position 156 to position 411 of urokinase as described in sequence number 6 or 7, wherein the antibody or antigen-binding fragment thereof is directly or via a linker connected to the catalytic domain of urokinase, wherein in the fusion protein, The connector is a non-disintegrating connector. The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. The arginine in the catalytic domain corresponding to the arginine at position 156 of prourokinase as recorded in sequence number 6 or 7 is replaced with other amino acids. [1031B] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of urokinase having an amino acid sequence corresponding to amino acids 7 to 43 of urokinase as described in sequence number 6 or 7, and a catalytic domain of urokinase having an amino acid sequence corresponding to amino acid sequences 156 to 411, wherein the antibody or antigen-binding fragment thereof is directly or via a linker to the catalytic domain of urokinase, wherein in the fusion protein, The connector is a non-disintegrating connector. The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. The phenylalanine in the catalytic domain corresponding to the arginine at position 156 of prourokinase as described in sequence number 6 or 7 is replaced with other amino acids. [1031C] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, a kringle domain of urokinase corresponding to amino acids 49 to 131 of urokinase as described in sequence number 6 or 7, and a catalytic domain of urokinase having an amino acid sequence corresponding to amino acid sequences 156 to 411, wherein the antibody or antigen-binding fragment thereof is directly or via a linker to the catalytic domain of urokinase, wherein in the fusion protein, The connector is a non-disintegrating connector. The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. The arginine in the catalytic domain corresponding to the arginine at position 156 of prourokinase as recorded in sequence number 6 or 7 is replaced with other amino acids. [1031D] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of urokinase having an amino acid sequence corresponding to amino acids 7 to 43 of urokinase as described in sequence number 6 or 7, a kringle domain of urokinase corresponding to amino acids 49 to 131 of urokinase, and a catalytic domain of urokinase having an amino acid sequence corresponding to amino acids 156 to 411 of urokinase, wherein the antibody or antigen-binding fragment thereof is directly or via a linker connected to the catalytic domain of urokinase, wherein in the fusion protein, The connector is a non-disintegrating connector. The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. The arginine in the catalytic domain corresponding to the arginine at position 156 of prourokinase as recorded in sequence number 6 or 7 is replaced with other amino acids.

[1032] The fusion protein according to any one of [1031A] to [1031D] {hereinafter collectively referred to as

[1031] } above, wherein the arginine is replaced with glutamic acid or aspartic acid.

[1033] According to the fusion protein described in

[1031] or

[1032] above, wherein, Compared to fusion proteins with the amino acid sequence described in Serial No. 1, the binding ability to more than one urokinase receptor selected from the group consisting of human urokinase receptor and mouse urokinase receptor is reduced.

[1034] The fusion protein according to any one of

[1031] to

[1033] above, wherein, Significant binding was not observed to one or more urokinase receptors selected from the group consisting of human and mouse urokinase receptors.

[1035] The fusion protein according to any one of

[1031] to

[1034] above, wherein, Between the antibody or its antigen-binding fragment portion and the catalytic domain portion, there is an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from position 7 to position 43 of the urokinase described in sequence number 6 or 7. The amino acids of the above-mentioned EGF-like domain corresponding to asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30, and glutamine at position 40 of the urokinase described in sequence number 6 or 7 are replaced with other amino acids, thereby reducing or eliminating the binding ability to the human urokinase receptor.

[1036] The fusion protein according to any one of

[1031] to

[1034] above, wherein, Between the antibody or its antigen-binding fragment portion and the catalytic domain portion, there is an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from position 7 to position 43 of the urokinase described in sequence number 6 or 7. The amino acids in the above-mentioned EGF-like domain corresponding to asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30, and glutamine at position 40 of the urokinase described in sequence number 6 or 7 are replaced with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing or eliminating the binding ability to human urokinase receptors.

[1037] The fusion protein according to any one of

[1031] to

[1034] above, wherein, Between the antibody or its antigen-binding fragment portion and the catalytic domain portion, there is also an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from position 7 to position 43 of the urokinase described in sequence number 6 or 7, and a deletion of the amino acid sequence of the EGF-like domain corresponding to the amino acid sequence from position 22 (asparagine) to position 28 (isoleucine) of the urokinase described in sequence number 6 or 7 (for example, an insertion of 1 to 7 amino acids (at least a peptide that does not bind to the human urokinase receptor, preferably a peptide that does not bind to both the human and mouse urokinase receptors, such as a peptide of 2 amino acids, such as GG)) at the deletion site), resulting in reduced or preferably lost binding ability to the human urokinase receptor.

[1038] The fusion protein according to any one of

[1031] to

[1037] above, wherein the amino acid of the catalytic domain corresponding to the phenylalanine at position 157 of the prourokinase described in sequence number 6 or 7 is phenylalanine.

[1039] A pharmaceutical preparation containing any one of the fusion proteins described in

[1031] to

[1036] above.

[1040] The pharmaceutical preparation described above

[1039] is used in a method for dissolving thrombi or embolisms in subjects with acute infarction, chronic thrombotic disease, or embolic disease.

[1041] The pharmaceutical preparation described in

[1039] or

[1040] above is used in a method for dissolving thrombi or emboli in a subject with acute infarction.

[1042] A method for manufacturing the fusion protein described in any one of

[1031] to

[1036] above, comprising the following steps: The fusion protein is expressed by culturing protein-producing cells that have nucleic acids encoding the fusion protein in an expressible manner; and the fusion protein is purified from the resulting culture.

[1043] According to the method described above

[1042] , the culture is carried out under serum-free conditions or in the presence of less than 5% serum.

[1044] According to the method described in

[1042] or

[1043] above, the purification is carried out in the absence of L-arginine.

[1051] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of urokinase having an amino acid sequence corresponding to amino acids 7 to 43 of urokinase as described in sequence number 6 or 7, a kringle domain of urokinase corresponding to amino acids 49 to 131 of urokinase, and a catalytic domain of urokinase having an amino acid sequence corresponding to amino acids 156 to 411 of urokinase, wherein the antibody or antigen-binding fragment thereof is directly or via a linker connected to the catalytic domain of urokinase, wherein the fusion protein, The connector is a non-disintegrating connector. The aforementioned antibody or antigen-binding fragment contains both heavy chain variable regions and light chain variable regions of the antibody that binds to insoluble fibrin. The arginine in the catalytic domain corresponding to the arginine at position 156 of prourokinase as described in sequence number 6 or 7 is replaced with other amino acids. The amino acids corresponding to the asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30, and glutamine at position 40 of the urokinase precursor described in sequence number 6 or 7 are replaced with other amino acids, resulting in a decrease or loss of the ability to bind to the human urokinase receptor.

[1052] According to the fusion protein described above

[1051] , the arginine in the catalytic domain corresponding to the arginine at position 156 is glutamic acid or aspartic acid.

[1053] According to the fusion protein of

[1051] above, the amino acids of the above-mentioned EGF-like domains corresponding to asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30 and glutamine at position 40 of the urokinase precursor described in sequence number 6 or 7 are replaced with tyrosine, serine, arginine, arginine and glutamic acid, respectively, thereby reducing or eliminating the binding ability to human urokinase receptor.

[1054] According to the fusion protein of

[1052] above, the amino acids of the above-mentioned EGF-like domains corresponding to asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30 and glutamine at position 40 of the urokinase described in sequence number 6 or 7 are replaced with tyrosine, serine, arginine, arginine and glutamic acid respectively, thereby reducing or losing the ability to bind to human urokinase receptor.

[1061] According to any of the foregoing fusion proteins, the antibody or its antigen-binding fragment specifically binds to insoluble fibrin (particularly, for one or more, preferably all, selected from the group consisting of fibrinogen, soluble fibrin and fibrin breakdown products, the binding is weak, particularly preferably insignificant, compared to the binding to insoluble fibrin).

[1062] According to any of the preceding claims, the fusion protein comprises an antibody or an antigen-binding fragment thereof bound to a peptide consisting of the amino acid sequence described in Serial No. 8.

[1063] According to any of the above-mentioned fusion proteins, wherein the antibody or its antigen-binding fragment has heavy chain CDR1-3 and light chain CDR1-3 selected from the group consisting of antibody 102-10, antibody 34-105 and antibody Fib-0355 disclosed in WO2014 / 133093 as corresponding CDRs.

[1064] According to any of the preceding claims, the fusion protein, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region having a heavy chain CDR1 having the amino acid sequence described in sequence number 10, a heavy chain CDR2 having the amino acid sequence described in sequence number 11, and a heavy chain CDR3 having the amino acid sequence described in sequence number 12, and a light chain variable region having a light chain CDR1 having the amino acid sequence described in sequence number 13, a light chain CDR2 having the amino acid sequence described in sequence number 14, and a light chain CDR3 having the amino acid sequence described in sequence number 15.

[1065] According to any of the preceding claims, the fusion protein, wherein the antibody or its antigen-binding fragment contains a heavy chain variable region having the amino acid sequence described in Serial No. 16, and a light chain variable region having the amino acid sequence described in Serial No. 17.

[1066] The fusion protein according to any one of the preceding claims comprises a heavy chain variable region having a heavy chain CDR1 having the amino acid sequence described in sequence number 18, a heavy chain CDR2 having the amino acid sequence described in sequence number 19, and a heavy chain CDR3 having the amino acid sequence described in sequence number 20, and a light chain variable region having a light chain CDR1 having the amino acid sequence described in sequence number 21, a light chain CDR2 having the amino acid sequence described in sequence number 22, and a light chain CDR3 having the amino acid sequence described in sequence number 23.

[1067] According to any of the preceding claims, the fusion protein, wherein the antibody or its antigen-binding fragment comprises a heavy chain variable region having the amino acid sequence described in Serial No. 24 and a light chain variable region having the amino acid sequence described in Serial No. 25.

[1068] According to any of the foregoing fusion proteins, wherein the antibody or its antigen-binding fragment competes with a reference antibody for binding to insoluble fibrin. The reference antibody comprises a heavy chain variable region having the amino acid sequence described in sequence number 16 and a light chain variable region having the amino acid sequence described in sequence number 17.

[1069] According to any of the foregoing fusion proteins, wherein the antibody or its antigen-binding fragment competes with a reference antibody for binding to insoluble fibrin. The reference antibody comprises a heavy chain variable region of an amino acid sequence having the amino acid sequence described in sequence number 24 and a light chain variable region of an amino acid sequence having the amino acid sequence described in sequence number 25.

[1070] The fusion protein according to any of the above has an amino acid sequence with sequence number 107.

[1071] According to any of the foregoing fusion proteins, wherein the antibody or its antigen-binding fragment has 10 - 6 M and below, 10 -7 M or below or 10 -8 The binding dissociation constant (KD) below M.

[1072] According to any of the above-mentioned fusion proteins, wherein the antibody or its antigen-binding fragment is a Fab fragment or a Fab' fragment {preferably, a fusion protein formed by linking the C-terminus of the heavy chain of the above-mentioned fragment to the N-terminus of prourokinase or a portion thereof or a mutant thereof}.

[1081] The fusion protein according to any one of the foregoing claims has the following properties selected from the group consisting of at least one, preferably at least two, more preferably at least three, and even more preferably all of the following: (i) binding specificity to insoluble fibrin; (ii) binding affinity to insoluble fibrin; (iii) resistance to inhibition caused by plasminogen activator inhibitor (PAI-1); and (iv) low inducibility of matrix metalloproteinase (MMP) activity in the surrounding tissues of thrombi or embolisms.

[1082] The fusion protein according to any of the above is linked to an imaging agent.

[1091] A pharmaceutical formulation or pharmaceutical composition comprising any of the fusion proteins described above.

[1092] The pharmaceutical preparation or composition described above

[1091] is used to treat thromboembolic diseases (e.g., thrombotic diseases, embolic diseases, thrombosis or embolism).

[1093] The pharmaceutical preparations or compositions described above

[1091] are suitable treatment methods for the dissolution of fibrin clots or for the management of a condition or disease.

[1094] The pharmaceutical preparations or compositions described above

[1091] are used to treat cerebrovascular and cardiovascular disorders with thrombosis or embolism.

[1095] The pharmaceutical preparations or pharmaceutical compositions described above

[1091] are used to treat conditions or symptoms arising from cerebrovascular and cardiovascular disorders due to the formation of thrombi or embolisms.

[1096] The pharmaceutical preparations or pharmaceutical compositions described above

[1091] are used to treat the state or symptoms of cerebrovascular and cardiovascular disorders.

[1097] The pharmaceutical preparation or pharmaceutical composition described above

[1091] is used to treat ischemic cerebrovascular disorders.

[1098] The pharmaceutical preparation or composition described above

[1091] is used to treat either cerebral infarction or transient ischemic attack.

[1099] The pharmaceutical preparation or pharmaceutical composition described above

[1091] is used to treat ischemic cardiovascular disorders.

[1100] The pharmaceutical preparation or composition described above

[1091] is used to treat either angina pectoris or myocardial infarction.

[1101] The pharmaceutical preparation or pharmaceutical composition according to any one of

[1091] to

[1100] above is used to treat a disease in the acute or chronic phase.

[1102] The pharmaceutical preparation or pharmaceutical composition according to any one of

[1091] to

[1100] above is used to treat a disease in the acute phase.

[1103] The pharmaceutical preparation or pharmaceutical composition according to any one of

[1091] to

[1100] above is used to treat a disease in the chronic phase.

[1104] The pharmaceutical preparation or composition described above

[1091] is used to dissolve fibrin clots in a subject having fibrin clots.

[1105] A pharmaceutical preparation or pharmaceutical composition containing the fusion protein described above

[1082] for the detection of thrombi or embolisms in vivo.

[1106] The pharmaceutical preparation or pharmaceutical composition described in

[1105] above is also used for the pharmaceutical uses specified in any one of

[1092] to

[1104] above.

[1201] A protein complex comprising a heavy chain having the amino acid sequence described in sequence number 64 and a light chain having the amino acid sequence described in sequence number 17.

[1202] A pharmaceutical preparation or pharmaceutical composition comprising the protein complex described above

[1201] .

[1203] The pharmaceutical preparation or composition described above

[1202] is used to treat thrombotic diseases, embolic diseases, thrombosis or embolism.

[1204] The pharmaceutical preparations or compositions described above

[1202] are suitable treatment methods for the dissolution of fibrin clots or for the management of a disease.

[1205] The pharmaceutical preparations or compositions described above

[1202] are used to treat cerebrovascular and cardiovascular disorders accompanied by thrombosis or embolism.

[1206] The pharmaceutical preparations or pharmaceutical compositions described above

[1202] are used to treat conditions or symptoms arising from cerebrovascular and cardiovascular disorders due to the formation of thrombi or embolisms.

[1207] The pharmaceutical preparations or pharmaceutical compositions described above

[1202] are used to treat the state or symptoms of cerebrovascular and cardiovascular disorders.

[1208] The pharmaceutical preparation or composition described above

[1202] is used to treat ischemic cerebrovascular disorders.

[1209] The pharmaceutical preparation or composition described above

[1202] is used to treat either cerebral infarction or transient ischemic attack.

[1210] The pharmaceutical preparation or composition described above

[1202] is used to treat ischemic cardiovascular disorders.

[1211] The pharmaceutical preparation or composition described above

[1202] is used to treat either angina pectoris or myocardial infarction.

[1212] The pharmaceutical preparation or pharmaceutical composition according to any one of

[1202] to

[1211] above is used to treat a disease in the acute or chronic phase.

[1213] The pharmaceutical preparation or pharmaceutical composition according to any one of

[1202] to

[1211] above is used to treat a disease in the acute phase.

[1214] The pharmaceutical preparation or pharmaceutical composition according to any one of

[1202] to

[1211] above is used to treat a disease in the chronic phase.

[1215] The pharmaceutical preparation or composition described above

[1202] is used to dissolve fibrin clots in a subject having fibrin clots.

[1216] A pharmaceutical formulation or composition containing the fusion protein described above

[1201] for detecting thrombi or embolisms in vivo, wherein the pharmaceutical formulation or composition is connected to an imaging agent.

[1217] The pharmaceutical preparation or pharmaceutical composition described in

[1216] above is also used for the pharmaceutical uses specified in any one of

[1203] to

[1215] above.

[1301] A method for treating thrombotic diseases, embolic diseases, infarct diseases, or thromboembolic diseases (preferably acute thrombotic diseases, embolic diseases, infarct diseases, or thromboembolic diseases) in a subject in need, comprising administering to the subject a therapeutically effective amount of an active ingredient, said active ingredient comprising a fusion protein containing a fibrin-binding domain (preferably an antigen-binding fragment of an antibody that binds to fibrin and is substantially not bound to fibrinogen, particularly a Fab fragment) and the EGF domain, kringle domain, and catalytic domain of prourokinase {here, the amino acid region corresponding to amino acid positions 22-28 in sequence number 1 is missing, and the missing site may contain an inactive amino acid sequence of several amino acids}, preferably comprising a protein containing the amino acid sequence described in sequence number 1 or 67.

[1302] According to the method described above

[1301] , the thrombotic disease, embolic disease, infarct disease or thromboembolism is an acute infarct disease or thromboembolism.

[1303] According to the method described in

[1301] or

[1302] above, the thrombotic disease, embolic disease, infarct disease or thromboembolism is selected from the group consisting of cerebral infarction, myocardial infarction, pulmonary embolism, peripheral arterial embolism and mesenteric infarction.

[1304] According to any one of

[1301] to

[1303] above, the method wherein a thrombus or embolism is opened in the subject (e.g., a human middle cerebral artery infarction or a rat model of middle cerebral artery infarction).

[1305] According to any one of

[1301] to

[1304] above, the method, by administering an equimolar amount, shows a thrombus opening rate or embolism opening rate higher than alteplase or preferably higher than tenecteplase.

[1306] According to any one of

[1301] to

[1305] above, the method, by administering an equimolar amount, shows a lower treatment-related bleeding frequency than alteplase and tenecteplase.

[1307] According to any one of

[1301] to

[1306] above, wherein, by administering an equimolar amount of the drug, a thrombus opening rate or embolism opening rate is shown to be higher than that of alteplase or preferably higher than that of tenecteplase, and a treatment-related bleeding frequency is shown to be lower than that of alteplase or preferably lower than that of tenecteplase.

[1308] A protein having the amino acid sequence described in Serial No. 1 or Serial No. 67, or a composition containing the protein, for use in the methods described in

[1301] to

[1307] above.

[1309] The method according to any of the preceding claims, wherein the protein further comprises a protein having the amino acid sequence described in sequence number 17 or 25.

[1310] A protein having the amino acid sequence described in Serial No. 1 or Serial No. 67, or a composition containing the protein, for use in the method described above

[1309] .

[1311] According to the composition described above

[1310] , the protein further includes a protein having the amino acid sequence described in sequence number 17 or 25.

[1321] A method for treating a disease with accumulation or deposition of insoluble fibrin (e.g., a disease with accumulation of insoluble fibrin outside the blood vessels, preferably Alzheimer's disease) in a subject in need, comprising administering to the subject a therapeutically effective amount of an active ingredient comprising a fusion protein containing a fibrin-binding domain (preferably an antigen-binding fragment of an antibody that binds to insoluble fibrin and substantially does not bind to fibrinogen, particularly a Fab fragment) and the EGF domain, kringle domain, and catalytic domain of prourokinase {here, the amino acid region corresponding to amino acid positions 22-28 in sequence number 1 is missing, and the missing site may contain an inactive amino acid sequence of several amino acids}, preferably comprising a protein containing the amino acid sequence described in sequence number 1 or 67.

[1322] According to the method described above

[1321] , the disease accompanied by the accumulation or deposition of insoluble fibrin is Alzheimer's dementia or vascular dementia.

[1323] According to the method described in

[1321] or

[1322] above, the subject is accompanied by the accumulation of β-amyloid protein.

[1324] According to any one of

[1321] to

[1323] above, the accumulation of β-amyloid protein is reduced.

[1325] A composition comprising a protein having an amino acid sequence described in Serial No. 1 or Serial No. 67, for use in any of the methods described in any of

[1321] to

[1324] above.

[1326] The composition described above

[1325] further contains a protein having the amino acid sequence described in sequence number 17 or 25. Attached Figure Description Figure 1 The reactivity of the AMU1114 modifier (particularly the F157 modifier of the urokinase moiety) to the substrate is shown. The vertical axis represents the absorbance at 405 nm. Figure 2 The reactivity of the plasmin-treated AMU1114 modifiers (particularly the F157 modifier of the urokinase moiety) to the substrate is shown. The vertical axis represents the absorbance at 405 nm. Figure 3A SDS-PAGE electrophoresis results of AMU1114 modifiers (particularly the F157 modifier of the urokinase moiety) after incubation in PBS at 37°C for 0 hours. Figure 3B SDS-PAGE electrophoresis results of AMU1114 modifiers (particularly the F157 modifier of the urokinase fraction) after incubation in PBS at 37°C for 24 hours. Figure 3C SDS-PAGE electrophoresis results of AMU1114 modifiers (particularly the F157 modifier of the urokinase fraction) after incubation in PBS at 37°C for 120 hours. Figure 4 The reactivity of the plasmin-treated AMU1114 modifier to the substrate is shown. The vertical axis represents the absorbance at 405 nm. Figure 5 The reactivity of the AMU1114 modifier targeting the human or mouse urokinase receptor to the substrate is shown. The vertical axis represents the absorbance at 405 nm. Figure 6AGel filtration chromatograms of urokinase expressed and purified in Chinese hamster ovary (CHO) cells in the presence of various concentrations of fetal bovine serum (FBS) are shown. Figure 6B Electrophoretic results of urokinase expressed and purified in Chinese hamster ovary (CHO) cells in the presence of various concentrations of fetal bovine serum (FBS) are shown. Figure 7A Electrophoresis results of AMU1114 expressed and purified in Chinese hamster ovary (CHO) cells with or without 20% FBS are shown. Figure 7B Electrophoresis results of the AMU1114 modifier expressed and purified in Chinese hamster ovary (CHO) cells in the absence of serum (in serum-free medium) are shown. Figure 8A The gel filtration chromatogram shows AMU1114 expressed and purified in Chinese hamster ovary (CHO) cells in the absence of serum (in serum-free medium). Figure 8B The gel filtration chromatogram of the AMU1114 modifier expressed and purified in Chinese hamster ovary (CHO) cells in the absence of serum (in serum-free medium) is shown. Figure 9 The comparison between AMU1114 and its modified counterpart is shown. Figure 10 The reactivity of the plasmin-treated AMU1114 modifier to the substrate is shown. The vertical axis represents the absorbance at 405 nm. Figure 11 The reactivity of the AMU1114 modifier targeting the human or mouse urokinase receptor to the substrate is shown. The vertical axis represents the absorbance at 405 nm. Figure 12A Images of lung tissue stained from mice with a thrombin thrombosis model who received PBS, AMU1114 (Δ22-28, F157E) (0.08 mg / kg body weight or 0.32 mg / kg body weight; denoted as Amu0.08 and Amu0.32, respectively), or tenepracetamol (0.05 mg / kg body weight or 0.2 mg / kg body weight; denoted as Tene0.05 and Tene0.2, respectively). Figure 12B It is a graph obtained by plotting the total area of ​​the signal corresponding to the thrombus. Figure 13This is the result of comparing the amino acid sequences of the heavy chains of AMU1114, AMU1114(Δ22-28, F157E), AMU1114(Δ22-28, R156E), AMU1114(Δ22-28, R156I, F157Y), and AMU1114(Δ22-28, P155G, R156I, F157Y) with those of AMU1114 and its modified counterparts. The modified counterparts respectively deleted the amino acid sequences from asparagine at position 22 to isoleucine at position 28 of urokinase, corresponding to the aforementioned EGF-like domain sequence described in sequence numbers 6 or 7, and replaced these deletions with the insertion of two glycine residues. Figure 14 The results show the stability of AMU1114 and its various modified versions. Figure 15 The resistance of AMU1114 and its modified counterpart to plasmin is shown. Figure 16A The urokinase activities of AMU1114 and its modified counterparts under conditions of pretreatment with plasmin are shown. Figure 16B The urokinase activities of AMU1114 and its modified counterparts under conditions without plasmin pretreatment are shown. Figure 17 The urokinase activities of AMU1114 and AMU1114(Δ22-28, F157E) are shown under conditions of pretreatment with and without plasmin. Figure 18 The urokinase activity of AMU1114 and various modified organisms is shown under conditions of pretreatment with and without plasmin. Figure 18 In the diagram, AMU1114(Δ22-28, R156E), AMU1114(Δ22-28, R156I, F157Y) and AMU1114(Δ22-28, P155G, R156I, F157Y) are denoted as modified bodies A, B and C, respectively. Figure 19 The decomposition stability of AMU1114 and AMU1114(Δ22-28, F157E) is shown. Figure 20 The decomposition stability of AMU1114 and various modified versions is shown. Figure 20 In the diagram, AMU1114(Δ22-28, R156E), AMU1114(Δ22-28, R156I, F157Y) and AMU1114(Δ22-28, P155G, R156I, F157Y) are denoted as modified bodies A, B and C, respectively. Figure 21 It is used in thrombosis experiments to create a rat model of cerebral infarction. Figure 22 This diagram illustrates the process of using a catheter to place a created thrombus in a branch of the left middle cerebral artery. Figure A is a frontal view, and Figure B is a side view. Figure 23 The images show the vascular images before and after thrombus placement. After thrombus placement, blood flow to the middle cerebral artery (MCA) stops, and no MCA image can be observed. Figure 24 These are images taken using an angiography device 1, 3, 6, and 24 hours after intravenous injection of the thrombolytic agent. The top image is the negative control, and the bottom image is the AMU1114(Δ22-28) administration group. The bottom image shows thrombolysis 1 hour after intravenous injection. Figure 25 The results of AMU1114 (Δ22-28) dosing experiments in a mouse model of Alzheimer's dementia are shown. Detailed Implementation In this specification, "object" refers to mammals, such as dogs, cats, cows, horses, pigs, primates (e.g., monkeys, gorillas, orangutans, bonobos, chimpanzees, and humans), such as humans. In this specification, "fibrin" refers to the insoluble coagulated substance formed by the cleavage of the C-terminus of the three polypeptide chains (Aα, Bβ, and γ chains) that constitute fibrinogen. In this specification, fibrin is sometimes referred to as insoluble fibrin. More specifically, the C-terminus of fibrinogen is cleaved to form a state called fibrin monomers, which polymerize under the influence of calcium to form insoluble fibrin polymers. These fibrin polymers cross-link between polymers under the influence of factor XIII, forming stable fibrin (insoluble fibrin or fibrin gel as defined in this specification). Insoluble fibrin can be broken down by plasmin. Plasmin is present in plasma in the form of plasminogen as a precursor. When the Arg-Val peptide of plasminogen is broken down by plasminogen activators (such as urokinase, tissue plasminogen activator, and streptokinase), plasminogen produces plasmin. Plasmin can be inhibited by proteins called plasmin inhibitors, thus limiting its activity. The Aα chain of fibrinogen can be the Aα chain of human fibrinogen. Examples of human fibrinogen Aα chains include those with the amino acid sequence registered in GenBank (accession number: AAI01936.1) and those with the corresponding amino acid sequence. The Bβ chain of fibrinogen can be the Bβ chain of human fibrinogen. Examples of human fibrinogen Bβ chains include the β chain of human fibrinogen with the amino acid sequence registered with NCBI reference number: NP_005132.2, and the β chain of human fibrinogen with the corresponding amino acid sequence. The γ chain of fibrinogen can be the γ chain of human fibrinogen. Examples of human fibrinogen γ chains include those with the amino acid sequence registered in GenBank (accession number: AAH07044.1) and those with the corresponding amino acid sequence. In this specification, "urokinase" refers to one of the serine proteases known as urokinase-type plasminogen activator (uPA) (e.g., an enzyme registered under EC 3.4.21.73). Urokinase is produced as a precursor, prourokinase, and the active form of urokinase is formed by cleaving the peptide bond between Lys158 and Lys159 (the cleaved chains are linked together by disulfide bonds). Urokinase has three domains: an EGF-like domain, a kringle domain, and a catalytic domain. Additionally, cleavage between Lys135 and Lys136 results in low molecular weight urokinase. It should be noted that the amino acid numbering of prourokinase is based on the amino acid sequence after cleavage of the signal peptide of prourokinase (e.g., the amino acid sequence described in Serial No. 6). Examples of prourokinase include, for example, human prourokinase. Examples of human prourokinase include, for example, prourokinase having the amino acid sequence registered under GenBank accession number: AAA61253.1 and prourokinase having the corresponding amino acid sequence. In the amino acid sequence accessed via GenBank accession number AAA61253.1, amino acid positions 1-20 correspond to the signal peptide. The EGF-like domain may contain the sequence of prourokinase corresponding to positions 7-43 of the amino acid sequence described in sequence number 6, the kringle domain may contain the sequence of prourokinase corresponding to positions 49-131 of the amino acid sequence described in sequence number 6, and the catalytic domain may contain the sequence of prourokinase corresponding to positions 136-403. Various prourokinases can be used as long as they possess the ability to be activated into a double-stranded prourokinase by plasmin and kallikrein, and to decompose plasminogen into plasmin. In this specification, the term "corresponding" refers to a sequence that corresponds to the first amino acid sequence when the second amino acid sequence has 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the first amino acid sequence and has the same function as a peptide having the first amino acid sequence (e.g., in the case of urokinase, the ability to break down plasminogen and convert it into plasmin). In this specification, "antibody" refers to an immunoglobulin. Antibodies can be of various isotypes, such as IgG. Antibodies are preferably monoclonal antibodies. Antibodies can be human chimeric antibodies, humanized antibodies, or human antibodies. Human chimeric antibodies are prepared by replacing the constant region of a non-human antibody with the constant region of a human antibody. Humanized antibodies are prepared by replacing the six CDRs of a human antibody with the six corresponding CDRs of a non-human antibody. Human antibodies can be prepared using animals (e.g., mice) whose variable regions of at least the heavy chain of an immunoglobulin are replaced with the corresponding regions of human loci. When the constant region is non-human, a human antibody can be obtained by replacing the constant region with the amino acid sequence of a human antibody. In this specification, antibodies are preferably humanized antibodies. In this specification, antibodies are preferably human antibodies. Antibodies possess a signal peptide when produced intracellularly, but this signal peptide is cleaved when secreted extracellularly. Therefore, when administered as a drug, the antibody does not require a signal peptide. In this specification, "CDR" refers to the complementarity-determining region (CDR) present in the heavy chain and light chain variable regions of the antibody. There are three CDRs in each of the heavy and light chain variable regions, designated CDR1, CDR2, and CDR3 from the N-terminus. CDRs can be identified, for example, based on the designations of Kabat et al. (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., 1991, Bethesda: US Dept. of Health and Human Services, PHS, NIH.). In this specification, "antigen-binding fragment of an antibody" refers to a fragment of an antibody that maintains its binding affinity to an antigen. Examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv, scFv (single-chain Fv), bispecific antibodies, and sc(Fv)2 (single-chain (Fv)2). For example, Fab can be obtained by digesting an antibody with papain. Alternatively, F(ab')2 can be obtained by digesting an antibody with pepsin, and Fab' can be obtained by further reducing it. Other antigen-binding fragments of antibodies can also be prepared by methods known to those skilled in the art. Such antigen-binding fragments of antibodies can be used in this invention. In this specification, a "fusion protein" refers to a protein composed of two or more peptides from different proteins linked by peptide bonds. In this specification, a fusion protein of a first peptide and a second peptide may contain a third peptide and further peptides without significantly reducing the function of the invention, or it may contain only the first and second peptides. In this specification, regarding a fusion protein of a first peptide and a second peptide, without significantly reducing the function of the invention, it may be either a fusion protein containing the first and second peptides sequentially or a fusion protein containing them in a different order, preferably a fusion protein containing the first and second peptides sequentially. In a fusion protein, two or more peptides from different proteins may be linked by a connector or without a connector without significantly reducing the function of the invention. If a connector is used, the connector may be a flexible connector. In this specification, "thrombotic disease" refers to a disease in which a blood clot (thrombus) blocks a blood vessel, impedes blood flow, and causes organ dysfunction or other disorders. The term "thrombotic disease" is used broadly in this specification to include embolic diseases in which the thrombus moves from its original site and follows the bloodstream to block a blood vessel. Therefore, "thrombotic disease" includes the meaning of thromboembolism. Thrombi and embolisms include conditions that partially (e.g., more than 50%, 60%, 70%, 80%, or 90%) obstruct blood flow and conditions that completely (e.g., about 100%) obstruct blood flow. In this specification, "infarction" refers to a condition where a blood vessel is blocked by a thrombus or embolism, resulting in tissue necrosis. <Modified form of the fusion protein of the present invention> (A) According to the present invention, a fusion protein is provided, comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin and a catalytic domain of urokinase having an amino acid sequence corresponding to the amino acid sequence of urokinase at positions 136 to 403 as described in Serial No. 6, wherein the antibody or antigen-binding fragment thereof is directly or via a linker connected to the catalytic domain of urokinase. Here, either or both (preferably both) of the amino acids at positions 135 and 136 (lysine at position 135) of the amino acid sequence corresponding to the amino acid sequence of urokinase at positions 136 to 403 as described in Serial No. 6 are replaced with other amino acids, thereby inhibiting cleavage from the catalytic domain of urokinase. In a preferred embodiment, both of the amino acids corresponding to the amino acids at positions 135 and 136 of urokinase as described in Serial No. 6 are replaced with glycine. Therefore, in a particularly preferred embodiment, the urokinase of the fusion protein or a portion thereof may have an amino acid sequence or a portion thereof corresponding to the amino acid sequence of a mutant of urokinase as described in Serial No. 7. Before binding to insoluble fibrin, the fusion protein of this invention has an inactive prourokinase moiety. Upon binding to insoluble fibrin, the prourokinase moiety is cleaved by plasminogen present nearby, generating double-stranded urokinase, which is then activated and cleaves nearby plasminogen to produce a large amount of plasmin. This effectively dissolves the insoluble fibrin. According to the present invention, the urokinase progenitor of the fusion protein may further have a kringle domain. The urokinase progenitor may also have an EGF-like domain and a kringle domain. Preferably, the fusion protein sequentially has an EGF-like domain, a kringle domain, and a catalytic domain. More preferably, the fusion protein has a urokinase progenitor region corresponding to positions 7 to 403 of the amino acid sequence described in sequence number 6 or 7. Most preferably, the fusion protein may contain a urokinase progenitor portion having an amino acid sequence corresponding to the amino acid sequence described in sequence number 6 or 7. According to the present invention, the fusion protein contains an antibody or an antigen-binding fragment thereof that binds to insoluble fibrin. The antibody or antigen-binding fragment thereof that binds to insoluble fibrin may contain a heavy chain variable region and a light chain variable region of the antibody or antigen-binding fragment thereof that binds to insoluble fibrin. According to the present invention, the antibody or antigen-binding fragment portion thereof that binds to insoluble fibrin in the fusion protein can be a Fab fragment or a Fab' fragment. In one embodiment, the heavy chain variable region of the Fab fragment or Fab' fragment is linked to prourokinase or a portion thereof in the manner described above. In another embodiment, the light chain variable region of the Fab fragment or Fab' fragment is linked to prourokinase or a portion thereof in the manner described above. It should be noted that the antigen-binding fragment may include a complex of a heavy chain variable region and a light chain variable region constituting another peptide (e.g., the case of a Fab fragment or Fab' fragment) or a case in which the heavy chain variable region and the light chain variable region are linked to form a fusion peptide (e.g., the case of an scFv fragment). In this specification, when used in the context of antigen-binding fragments, the terms "heavy chain" and "light chain" refer to either the "heavy chain" or the "light chain" derived from the source antibody, and are not terms indicating the molecular weight of the antigen-binding fragment. In a preferred embodiment of the present invention, the arginine in the catalytic domain corresponding to the arginine at position 156 of the urokinase precursor described in Serial No. 6 in the fusion protein is replaced with glutamic acid or aspartic acid (preferably glutamic acid). In a preferred embodiment of the present invention, the phenylalanine in the catalytic domain corresponding to the phenylalanine at position 157 of the urokinase precursor described in Serial No. 6 in the fusion protein is preferably phenylalanine, but it may also be replaced with other amino acids. (B) Therefore, according to the present invention, a fusion protein is provided, comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin and a catalytic domain of urokinase having an amino acid sequence corresponding to the amino acid sequence from position 136 to position 403 of urokinase as described in Serial No. 7, wherein the antibody or antigen-binding fragment thereof is directly or via a linker connected to the catalytic domain of urokinase, wherein the arginine in the catalytic domain corresponding to the arginine at position 156 of urokinase as described in Serial No. 7 is replaced with glutamic acid or aspartic acid (preferably glutamic acid). Preferably, the phenylalanine in the catalytic domain corresponding to the phenylalanine at position 157 of urokinase as described in Serial No. 6 is phenylalanine, but may also be replaced with other amino acids. (C) In a preferred embodiment, according to the present invention, a fusion protein is provided, comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin and a urokinase region having an amino acid sequence corresponding to the amino acid sequence from position 7 to position 403 of the urokinase described in Serial No. 7, wherein the antibody or antigen-binding fragment thereof is directly or via a linker connected to the catalytic domain of the urokinase, wherein in the fusion protein, the arginine in the catalytic domain corresponding to the arginine at position 156 of the urokinase described in Serial No. 7 is replaced with glutamic acid or aspartic acid (preferably glutamic acid). In a more preferred embodiment, the urokinase region may contain a urokinase region having an amino acid sequence corresponding to the amino acid sequence from position 1 to position 411 of the urokinase described in Serial No. 7. In this embodiment, the phenylalanine in the catalytic domain corresponding to the phenylalanine at position 157 of the urokinase described in Serial No. 6 is preferably phenylalanine, but may also be replaced with other amino acids. According to the present invention, the aforementioned phenylalanine or arginine is replaced with various amino acids (e.g., amino acids other than tyrosine). Therefore, the fusion protein of the present invention preferably exhibits enhanced resistance to cleavage by proteolytic enzymes (e.g., plasmin). In a preferred embodiment, the aforementioned phenylalanine or arginine is replaced with alanine, lysine, cysteine, serine, isoleucine, glycine, glutamic acid, or aspartic acid. In a more preferred embodiment, the aforementioned phenylalanine or arginine is replaced with isoleucine, glycine, glutamic acid, or aspartic acid. This inhibits fusion protein degradation and associated activation during fusion protein expression, purification, and / or storage. In a further preferred embodiment, the aforementioned phenylalanine or arginine is replaced with glutamic acid or aspartic acid. This, in addition to inhibiting fusion protein degradation and associated activation during fusion protein expression, purification, and / or storage, allows for plasminogen cleavage activity equivalent to that of the fusion protein without the aforementioned phenylalanine or arginine replacement. Furthermore, when fusion proteins without the aforementioned phenylalanine or arginine substitutions are generated from protein expression cells, fragmented fusion proteins are produced. However, by replacing the phenylalanine or arginine in the fusion protein with an amino acid other than tyrosine, preferably glutamic acid or aspartic acid, the amount of fragmented fusion proteins can be reduced or prevented from increasing, even when protein expression cells expressing the fusion protein are cultured in low-serum medium (e.g., containing less than 5% serum) or serum-free medium. Additionally, when fusion proteins without the aforementioned phenylalanine or arginine substitutions are generated from protein expression cells, although the amount of fusion protein produced continues to increase for 5–7 days, the amount of fusion protein produced no longer increases after this time, and the proportion of light chains in the produced protein increases. In contrast, by replacing the phenylalanine or arginine in the fusion protein with an amino acid other than tyrosine, preferably glutamic acid or aspartic acid, the proportion of light chains in the produced protein can be reduced. Furthermore, by replacing the phenylalanine or arginine in the aforementioned fusion protein with an amino acid other than tyrosine, preferably glutamic acid or aspartic acid, the production of the fusion protein continues to increase even after 7 days. Thus, the production of the fusion protein is significantly increased (e.g., 3-5 times) compared to fusion proteins without this replacement. This can reduce or avoid the use of serum, suppress fluctuations in protein production caused by serum quality variations, and lower culture costs, which may be beneficial. Therefore, replacing the arginine in the catalytic domain corresponding to the arginine at position 156 of the urokinase precursor described in Serial No. 7 with other amino acids, such as glutamic acid or aspartic acid (preferably glutamic acid), and especially replacing it with glutamic acid or aspartic acid, is of great significance from the perspective of increasing yield, increasing purity, increasing urokinase activity in the presence of fibrin, and / or saving costs in manufacturing. In this manner, the phenylalanine in the catalytic domain corresponding to the phenylalanine at position 157 of the urokinase precursor described in Serial No. 6 is preferably phenylalanine, but it can also be replaced with other amino acids. (D) In ​​a preferred embodiment of the present invention, a fusion protein is provided, comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin and a urokinase region having an amino acid sequence corresponding to the amino acid sequence from position 7 to position 403 of the urokinase described in Serial No. 7. The antibody or antigen-binding fragment is directly or via a linker connected to the catalytic domain of the urokinase. In the fusion protein, the arginine in the catalytic domain corresponding to the arginine at position 156 of the urokinase described in Serial No. 7 is replaced with other amino acids, specifically glutamic acid or aspartic acid (preferably glutamic acid). In a preferred embodiment, the urokinase region has an amino acid sequence corresponding to the amino acid sequence from position 1 to position 411 of the urokinase described in Serial No. 7. In this embodiment, the phenylalanine in the catalytic domain corresponding to the phenylalanine at position 157 of the urokinase described in Serial No. 6 is preferably phenylalanine, but may also be replaced with other amino acids. <Further modifications of the fusion protein of the present invention> According to the present invention, a modified version of the fusion protein is provided, which has a reduced affinity for the urokinase receptor compared to the fusion protein described above. Specifically, in a further modified version of the present invention, in the amino acid sequence of any of the above-mentioned fusion proteins (e.g., any of (A) to (D)), the amino acid of the EGF-like domain corresponding to the tryptophan at position 30 of prourokinase as described in sequence number 6 or 7 is replaced with other amino acids (especially arginine). As a result, the binding affinity of the further modified version of the present invention to the human urokinase receptor is reduced compared to the above-mentioned fusion protein. Furthermore, in a further modified version of the present invention, in the amino acid sequence of any of the above-mentioned fusion proteins (e.g., any of (A) to (D)), the amino acids corresponding to the asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30, and glutamine at position 40 of prourokinase as described in sequence number 6 or 7 are replaced with other amino acids. As a result, the binding affinity of the further modified version of the present invention to the human urokinase receptor is reduced compared to the above-mentioned fusion protein. In one embodiment, in a further modified version of the invention, in the amino acid sequence of any of the above-mentioned fusion proteins (e.g., any of (A) to (D)), the amino acids corresponding to the asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30, and glutamine at position 40 of prourokinase, as described in sequence number 6 or 7, are respectively replaced with tyrosine, serine, arginine, arginine, and glutamic acid. As a result, the binding affinity of the further modified version of the invention to the human urokinase receptor is reduced compared to the above-mentioned fusion protein. In one embodiment, in a further modified version of the present invention, the amino acids in the amino acid sequence of the fusion protein (A) corresponding to the asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30, and glutamine at position 40 of urokinase, as described in sequence number 6 or 7, are respectively replaced with tyrosine, serine, arginine, arginine, and glutamic acid. Consequently, the binding affinity of the further modified version of the present invention to the human urokinase receptor is reduced compared to the aforementioned fusion protein. In one embodiment, in a further modified version of the present invention, the amino acids in the amino acid sequence of the fusion protein (B) corresponding to the asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30, and glutamine at position 40 of urokinase, as described in sequence number 6 or 7, are respectively replaced with tyrosine, serine, arginine, arginine, and glutamic acid. Consequently, the further modified version of the present invention exhibits a lower binding affinity to the human urokinase receptor compared to the aforementioned fusion protein. In one embodiment, in a further modified version of the present invention, the amino acids in the amino acid sequence of the fusion protein (C) corresponding to the asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30, and glutamine at position 40 of urokinase, as described in sequence number 6 or 7, are respectively replaced with tyrosine, serine, arginine, arginine, and glutamic acid. Consequently, the further modified version of the present invention exhibits a lower binding affinity to the human urokinase receptor compared to the aforementioned fusion protein. In one embodiment, in a further modified version of the present invention, the amino acids in the amino acid sequence of the fusion protein (D) corresponding to the asparagine at position 22, asparagine at position 27, histidine at position 29, tryptophan at position 30, and glutamine at position 40 of urokinase, as described in sequence number 6 or 7, are respectively replaced with tyrosine, serine, arginine, arginine, and glutamic acid. As a result, the binding affinity of the further modified version of the present invention to the human urokinase receptor is reduced compared to the fusion protein described above. In a preferred embodiment, in a further modified version of the invention, the amino acid sequence of the EGF-like domain corresponding to the amino acid sequence of asparagine at position 22 to isoleucine at position 28 of prourokinase as described in Serial No. 6 or 7 is deleted in any of the above-mentioned fusion proteins (e.g., any of (A) to (D)). This is considered to cause the modified body to lose its binding ability to the urokinase receptor. In another embodiment, in a further modified version of the invention, the amino acid sequence of the EGF-like domain corresponding to the amino acid sequence of asparagine at position 22 to isoleucine at position 28 of prourokinase as described in Serial No. 6 or 7 is deleted in any of the above-mentioned fusion proteins (e.g., any of (A) to (D)). At the deleted site, there is an insertion of 1 to 7 amino acids (a peptide that has at least no binding affinity to the human urokinase receptor, preferably a peptide that has no binding affinity to both the human and mouse urokinase receptors, such as a peptide of 2 to 7 amino acids, such as a 2-amino acid peptide, such as GG). Amino acid sequences can be inserted to mitigate the shortening of protein length caused by deletions. The inserted amino acid sequence does not need to exert any particular activity and can consist of or contain neutral amino acids such as G and S. Therefore, further modifications of the present invention do not exhibit significant binding affinity to human urokinase receptors (particularly human and mouse urokinase receptors) or further reduce the binding affinity of the aforementioned fusion protein to these receptors; preferably, they do not exhibit significant binding affinity to human and mouse urokinase receptors. In a particularly preferred embodiment, a further modified body of the present invention comprises an antibody (anti-insoluble fibrin antibody) or an antigen-binding fragment thereof that binds to insoluble fibrin, and a urokinase region corresponding to positions 7 to 403 of the amino acid sequence described in Serial No. 6 or 7. The amino acid sequence corresponding to the amino acid sequence of asparagine at position 22 to isoleucine at position 28 of the urokinase described in Serial No. 6 or 7 is deleted. The arginine in the catalytic domain corresponding to arginine at position 156 of the urokinase described in Serial No. 7 is replaced with another amino acid, preferably glutamic acid or aspartic acid (more preferably glutamic acid). Furthermore, in a more preferred embodiment, the amino acid in the catalytic domain corresponding to phenylalanine at position 157 of the urokinase described in Serial No. 6 is phenylalanine. Additionally, the amino acids at positions 135 and 136 of the urokinase described in Serial No. 6 or 7 are preferably both replaced with glycine. In a further modified version of the present invention, one or more of the amino acids in the catalytic domain corresponding to the amino acids at positions 155 to 157 of the prourokinase described in Serial No. 7 (e.g., the amino acid at position 156, the amino acids at positions 156 and 157, and the amino acids at positions 155 to 157) are replaced with other amino acids (preferably, glycine at position 155, glutamic acid or aspartic acid (more preferably glutamic acid) at position 156, or tyrosine at position 157), preferably exhibiting superior degradation stability in human plasma compared to AMU1114. Representative examples of these modified versions include AMU1114(Δ22-28), AMU1114(Δ22-28, R156E), AMU1114(Δ22-28, R156I, F157Y), and AMU1114(Δ22-28, P155G, R156I, F157Y). Δ22-28 may have amino acid positions 22-28 missing, replaced by an inactive amino acid (e.g., GG) containing several bases at that position. Furthermore, these modified forms preferably exhibit urokinase activity earlier in human plasma than AMU1114 (Δ22-28, F157E) with sequence number 63 (i.e., early activation type), without pretreatment with plasmin. This early activation type of modified form is preferably administered to patients with chronic thrombotic diseases, embolic diseases, thromboembolism, or infarction, or preferably acute thrombotic diseases, embolic diseases, thromboembolism, or infarction, and more preferably to patients with acute infarction. In contrast, AMU1114 (Δ22-28, F157E) is more preferably administered to patients with chronic thrombotic diseases or infarction.Further modifications of the present invention may have further mutations (e.g., mutations, particularly point mutations) that can have more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% sequence identity with AMU1114 and exhibit superior degradation stability in human plasma compared to AMU1114. In a preferred embodiment, the antibody that binds to insoluble fibrin (anti-insoluble fibrin antibody) or its antigen-binding fragment may be humanized or be a human antibody or its antigen-binding fragment. In a preferred embodiment, the antibody (anti-insoluble fibrin antibody) binding to insoluble fibrin or its antigen-binding fragment comprises: a heavy chain variable region containing a heavy chain CDR1 having the amino acid sequence described in sequence number 10, a heavy chain CDR2 having the amino acid sequence described in sequence number 11, and a heavy chain CDR3 having the amino acid sequence described in sequence number 12, and a light chain variable region containing a light chain CDR1 having the amino acid sequence described in sequence number 13, a light chain CDR2 having the amino acid sequence described in sequence number 14, and a light chain CDR3 having the amino acid sequence described in sequence number 15. In a preferred embodiment, the antibody binding to insoluble fibrin (anti-insoluble fibrin antibody) or its antigen-binding fragment comprises: a heavy chain CDR1 having the amino acid sequence described in sequence number 18, a heavy chain variable region having a heavy chain CDR2 having the amino acid sequence described in sequence number 19 and a heavy chain CDR3 having the amino acid sequence described in sequence number 20, and a light chain variable region having a light chain CDR1 having the amino acid sequence described in sequence number 21, a light chain CDR2 having the amino acid sequence described in sequence number 22 and a light chain CDR3 having the amino acid sequence described in sequence number 23. In a preferred embodiment, the antibody binding to insoluble fibrin (anti-insoluble fibrin antibody) or its antigen-binding fragment comprises: The heavy chain variable region of the amino acid sequence having the amino acid sequence described in sequence number 16, and The light chain variable region of the amino acid sequence having the amino acid sequence described in sequence number 17. In a preferred embodiment, the antibody binding to insoluble fibrin (anti-insoluble fibrin antibody) or its antigen-binding fragment comprises: The heavy chain variable region of the amino acid sequence containing the amino acid sequence described in sequence number 24, and The light chain variable region of the amino acid sequence having the amino acid sequence described in sequence number 25. In a preferred embodiment, in a further modified version of the present invention, the heavy chain has the amino acid sequence described in serial number 64, 65, or 66. In a preferred embodiment, in a further modified version of the present invention, the light chain has the amino acid sequence described in serial number 17. In a preferred embodiment, in a further modified version of the present invention, the heavy chain has the amino acid sequence described in serial number 64, 65, or 66, and the light chain has the amino acid sequence described in serial number 17. In a preferred embodiment, in a further modified version of the invention, the heavy chain has the amino acid sequence described in Serial No. 64, and the light chain has the amino acid sequence described in Serial No. 17. This modified version can be used to treat thrombotic diseases, embolic diseases, thromboembolism, and preferably acute infarction. In a preferred embodiment, a further modified version of the invention does not have a mutation that confers resistance to plasmin cleavage. This modified version can be used to treat thrombotic diseases, embolic diseases, thromboembolic disorders, or infarcts, preferably chronic thrombotic diseases, and more preferably acute thrombotic diseases, embolic diseases, thromboembolic disorders, preferably acute infarcts, etc. In a preferred embodiment, in a further modified version of the invention, the heavy chain has the amino acid sequence described in Serial No. 63, and the light chain has the amino acid sequence described in Serial No. 17. This modified version can be used to treat thrombotic diseases, embolic diseases, thromboembolism, or infarction, preferably chronic thrombotic diseases, and more preferably acute thrombotic diseases, embolic diseases, thromboembolism, preferably acute infarction, etc. These modified bodies are preferably cleaved between the amino acids corresponding to lysine at position 158 and isoleucine at position 159 of the amino acid sequence described in sequence number 6 or 7, and the peptides before and after the cleavage site are linked by disulfide bonds (i.e., becoming active urokinase). The binding affinity to the urokinase receptor in humans can be less than 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 20, 1 / 30, 1 / 40, 1 / 50, 1 / 60, 1 / 70, 1 / 80, 1 / 90, or 1 / 100 compared to the fusion protein described in sequence number 1. Binding affinity can be determined by an ELISA assay measuring the amount of fusion protein bound to a plate immobilized with the urokinase receptor, after subtracting the background value based on a negative control (e.g., phosphate-buffered saline (PBS)). In the ELISA assay, the amount of fusion protein bound to the urokinase receptor can be detected using an enzyme-labeled antibody. As an enzyme-labeled antibody, antibodies that bind to fusion proteins (e.g., anti-human heavy or light chain antibodies) can be used; as an enzyme, horseradish peroxidase (HRP) can be used; and as a substrate, 3,3',5,5'-tetramethylbenzidine (TMB) can be used. Therefore, even if the fusion protein of the present invention is exposed around a thrombus or embolism, it will not bind to the urokinase receptor of the surrounding tissue, thereby inhibiting the production of MMP enzymes in the tissue and preventing tissue damage. Thus, it is believed that undesirable side effects caused by binding to the urokinase receptor can be prevented. In the modified fusion protein of the present invention, the antibody or its antigen-binding fragment partially binds to insoluble fibrin. In a preferred embodiment, in the modified fusion protein of the present invention, the antibody or its antigen-binding fragment partially specifically binds to insoluble fibrin. Here, "specifically binds to insoluble fibrin" means: binding to insoluble fibrin, but binding only weakly, and particularly preferably not significantly, to one or more of the group consisting of fibrinogen, soluble fibrin, and fibrin breakdown products, more preferably all of them, compared to insoluble fibrin. In this invention, the antibody that binds to insoluble fibrin is an antibody that binds to insoluble fibrin with a stronger affinity than that to fibrinogen (i.e., with a lower dissociation constant K than that to fibrinogen). DAntibodies binding to insoluble fibrin. Antibodies binding to insoluble fibrin with a stronger affinity than to fibrinogen can be, for example, antibodies binding to a peptide consisting of the amino acid sequence described in sequence number 8 or 9 (see WO2014 / 133093). The amino acid sequence described in sequence number 8 corresponds to the amino acid sequence described in sequence number 1 of WO2014 / 133093, and the amino acid sequence described in sequence number 9 corresponds to the amino acid sequence described in sequence number 2 of WO2014 / 133093. The peptide portion consisting of the amino acid sequence described in sequence number 8 or 9 is not exposed on the protein surface of fibrinogen, but is exposed only on the protein surface of insoluble fibrin in a manner accessible to the antibody. Therefore, antibodies binding to the peptide consisting of the amino acid sequence described in sequence number 8 or 9 can have binding selectivity for insoluble fibrin. <Antibody or its antigen-binding fragment> Examples of antibodies or antigen-binding fragments thereof that bind to insoluble fibrin with a stronger affinity for fibrinogen include insoluble fibrin-specific antibodies (e.g., antibodies that bind to insoluble fibrin but not substantially to fibrinogen), such as antibodies selected from WO2014 / 133093 (containing heavy chain variable regions having heavy chain CDR1-3 as described in serial numbers 27-29 and light chain variable regions having light chain CDR1-3 as described in serial numbers 30-32), and 34-105. The antibody (containing the heavy chain variable regions of heavy chain CDR1-3 as described in Serial Nos. 33-35 and the light chain variable regions of light chain CDR1-3 as described in Serial Nos. 36-38) and the Fib-0355 antibody (containing the heavy chain variable regions of heavy chain CDR1-3 as described in Serial Nos. 39-41 and the light chain variable regions of light chain CDR1-3 as described in Serial Nos. 42-44) in the group consist of antibodies whose heavy chain CDR1-3 and light chain CDR1-3 are respectively used as antibodies or antigen-binding fragments of the corresponding CDRs. Examples of antibodies or antigen-binding fragments thereof that bind to insoluble fibrin with a stronger affinity for fibrinogen include, for instance, the 99 antibody (containing heavy chain variable regions having heavy chain CDR1-3 as described in serial numbers 45-47 and light chain variable regions having light chain CDR1-3 as described in serial numbers 48-50) and the 1101 antibody (containing heavy chain CDR1-3 as described in serial numbers 51-53) disclosed in WO2018 / 203517. The heavy chain CDRs 1-3 and light chain CDRs 1-3 of the antibodies in the group consisting of the heavy chain variable regions of CDRs 1-3 as described in Serial Numbers 54-56, and the light chain variable regions of CDRs 1-3 as described in Serial Numbers 57-59, and the light chain variable regions of CDRs 1-3 as described in Serial Numbers 60-62, respectively, are antibodies or antigen-binding fragments thereof that are the corresponding CDRs. Antibodies against insoluble fibrin that bind to insoluble fibrin with a stronger affinity than that for fibrinogen can be listed as examples. Antibodies or antigen-binding fragments thereof that bind to insoluble fibrin with a stronger affinity than fibrinogen may include, for example, heavy chain variable regions comprising a heavy chain CDR1 having the amino acid sequence described in sequence number 10, a heavy chain CDR2 having the amino acid sequence described in sequence number 11, and a heavy chain CDR3 having the amino acid sequence described in sequence number 12, and light chain variable regions comprising a light chain CDR1 having the amino acid sequence described in sequence number 13, a light chain CDR2 having the amino acid sequence described in sequence number 14, and a light chain CDR3 having the amino acid sequence described in sequence number 15. The antibody may be a human chimeric antibody or a humanized antibody. According to the present invention, a humanized antibody having the aforementioned heavy chain variable region and light chain variable region is also provided. Examples of antibodies or antigen-binding fragments thereof that bind to insoluble fibrin with a stronger affinity than fibrinogen include, for instance, antibodies or antigen-binding fragments thereof containing a heavy chain variable region having the amino acid sequence described in Serial No. 16 and a light chain variable region having the amino acid sequence described in Serial No. 17. In addition to containing the heavy chain variable region, the antibody may also contain a heavy chain constant region or a portion thereof within the Fab fragment. The antibody may be a human chimeric antibody or a humanized antibody. According to the present invention, a humanized antibody having the aforementioned heavy chain variable region and light chain variable region is also provided. As an antibody or antigen-binding fragment thereof that binds to insoluble fibrin with a stronger affinity than fibrinogen, examples include antibodies or antigen-binding fragments thereof comprising a heavy chain variable region containing a heavy chain CDR1 having the amino acid sequence described in sequence number 18, a heavy chain CDR2 having the amino acid sequence described in sequence number 19, and a heavy chain CDR3 having the amino acid sequence described in sequence number 20, and a light chain variable region containing a light chain CDR1 having the amino acid sequence described in sequence number 21, a light chain CDR2 having the amino acid sequence described in sequence number 22, and a light chain CDR3 having the amino acid sequence described in sequence number 23. The antibody may be a human chimeric antibody or a humanized antibody. According to the present invention, a humanized antibody having the aforementioned heavy chain variable region and light chain variable region is also provided. Examples of antibodies or antigen-binding fragments thereof that bind to insoluble fibrin with a stronger affinity than fibrinogen include, for instance, antibodies or antigen-binding fragments thereof containing a heavy chain variable region having the amino acid sequence described in Serial No. 24 and a light chain variable region having the amino acid sequence described in Serial No. 25. In addition to the heavy chain variable region, the antibody may also contain a heavy chain constant region or a portion thereof within the Fab fragment. The antibody may be a human chimeric antibody or a humanized antibody. According to the present invention, a humanized antibody having the aforementioned heavy chain variable region and light chain variable region is also provided. According to the present invention, the following invention is provided. [1] An antibody or an antigen-binding fragment thereof that binds to insoluble fibrin, comprising: (1) A heavy chain variable region containing a heavy chain CDR1 having the amino acid sequence described in sequence number 74, a heavy chain CDR2 having the amino acid sequence described in sequence number 75, and a heavy chain CDR3 having the amino acid sequence described in sequence number 76, and A light chain variable region containing light chain CDR1 with the amino acid sequence described in sequence number 77, light chain CDR2 with the amino acid sequence described in sequence number 78, and light chain CDR3 with the amino acid sequence described in sequence number 79; or (2) A heavy chain variable region containing a heavy chain CDR1 having the amino acid sequence described in sequence number 82, a heavy chain CDR2 having the amino acid sequence described in sequence number 83, and a heavy chain CDR3 having the amino acid sequence described in sequence number 84, and The light chain variable region contains light chain CDR1 with the amino acid sequence described in sequence number 85, light chain CDR2 with the amino acid sequence described in sequence number 86, and light chain CDR3 with the amino acid sequence described in sequence number 87. [2] An insoluble fibrin antibody or its antigen-binding fragment, comprising: (3) A heavy chain variable region having the amino acid sequence described in sequence number 80 and a light chain variable region having the amino acid sequence described in sequence number 81; or (4) The heavy chain variable region having the amino acid sequence recorded in sequence number 88 and the light chain variable region having the amino acid sequence recorded in sequence number 89. According to the present invention, antibodies that compete with the antibodies described in (3) above for binding to insoluble fibrin can be provided, preferably competing antibodies. Here, the antibody is preferably a humanized antibody. According to the present invention, antibodies that compete with the antibodies described in (4) above for binding to insoluble fibrin can be provided, preferably competing antibodies. Here, the antibody is preferably a humanized antibody. According to the present invention, the antibody described in (1) above is a humanized antibody, which may be an insoluble fibrin-specific antibody. According to the present invention, the antibody described in (2) above is a humanized antibody, which may be an insoluble fibrin-specific antibody. According to the present invention, the antibody described in (3) above is a humanized antibody, which may be an insoluble fibrin-specific antibody. According to the present invention, the antibody described in (4) above is a humanized antibody, which may be an insoluble fibrin-specific antibody. According to the present invention, the antibody that competes with the antibody described in (3) above for binding to insoluble fibrin, preferably the competing antibody, is a humanized antibody, which may be an insoluble fibrin-specific antibody. According to the present invention, the antibody that competes with the antibody described in (4) above for binding to insoluble fibrin, preferably the competing antibody, is a humanized antibody, which may be an insoluble fibrin-specific antibody. The antibody of the present invention and its antigen-binding fragment portion can bind to a peptide consisting of the amino acid sequence described in Serial No. 8. Here, the antibody of the present invention can be a humanized antibody. In one embodiment, the modified fusion protein of the present invention has the amino acid sequence described in Serial Number 1. In one embodiment, the modified fusion protein of the present invention has the amino acid sequence described in Serial Number 2. In one embodiment, the modified fusion protein of the present invention has the amino acid sequence described in Serial Number 3. In one embodiment, the modified fusion protein of the present invention has the amino acid sequence described in Serial Number 26. In one embodiment, the modified fusion protein of the present invention has the amino acid sequence described in Serial Number 4. In one embodiment, the modified fusion protein of the present invention has the amino acid sequence described in Serial Number 5. In one embodiment, the modified fusion protein of the present invention has the amino acid sequence described in Serial Number 63. <Characteristics of the Modified Body> The modified fusion protein of the present invention may possess any one or more, preferably two or more, more preferably three or more, and further preferably all of the following properties selected from the group consisting of: (i) binding specificity to insoluble fibrin; (ii) binding affinity to insoluble fibrin; (iii) resistance to inhibition caused by plasminogen activator inhibitor (PAI-1); and (iv) low inducibility of matrix metalloproteinase (MMP) activity in perithrombus tissue. Therefore, a safer and more effective treatment can be provided than conventional tissue plasminogen activator (tPA) therapy. Furthermore, it provides a new technological innovation for thrombolytic therapy utilizing urokinase. In particular, it exhibits significant advantages in manufacturing and reduces side effects compared to the fusion protein disclosed in WO2021 / 200922A. <Method for manufacturing the modified fusion protein of the present invention> According to the present invention, a method for manufacturing a modified fusion protein of the present invention is provided. The modified fusion protein of the present invention can be produced by protein production cells. For example, a gene encoding the modified protein can be transiently or preferably stably introduced into protein production cells, and the protein production cells can be cultured under conditions suitable for producing the modified protein, thereby producing the modified protein from the protein production cells. Therefore, according to the present invention, a protein production cell having a gene encoding a modified fusion protein of the present invention in an expressible manner is provided. The protein production cell may be contained in a cell cryoprotectant or in a frozen state. Therefore, according to the present invention, a cell cryopreservation solution containing a protein production cell having a gene encoding a modified fusion protein of the present invention in an expressible manner is provided, as well as a working cell bank, a master cell bank, and a research cell bank. The modified fusion protein of the present invention is particularly operatively ligable to a control sequence. Thus, expression can be performed in the protein production cell. According to the present invention, the fusion protein of the present invention can be prepared by methods known to those skilled in the art. For example, the fusion protein of the present invention can be expressed in cells (insect cells, avian cells, Escherichia coli, yeast, and mammalian cells), preferably mammalian cells (e.g., cells suitable for protein expression, such as Chinese hamster ovary cells (CHO cells) and 293 cells and their derivatives, etc., human cells suitable for protein expression). CHO cells (e.g., ExpiCHO cells) can also be used as protein production cells. TM Cells, ExpiCHO-S TM Cells, Free Style TM(e.g., CHO cells). Expression can be performed, for example, using an expression vector containing nucleic acid encoding the fusion protein of the present invention, operatively linked to a promoter operatively responsive to the expression cell. If the fusion protein requires a light chain, the light chain can be co-expressed in the expression cell. The modified fusion protein of the present invention can solve the problem of low expression levels of fusion proteins in protein-producing cells. This is because, in the case of conventional fusion proteins, even increasing the culture time does not increase the production of the fusion protein, but rather the production of fragments increases. In contrast, the modified fusion protein of the present invention can increase the production of the modified protein by increasing the culture time. Moreover, the plasminogen lysate activity in the presence of plasmin is equivalent to that of conventional fusion proteins. In one approach, a method is provided for manufacturing a modified fusion protein with excellent stability or a pharmaceutical composition containing the modified protein, comprising the step of introducing an F157E or F157D mutation into the fusion protein of the present invention, thereby conferring better stability than the unmodified fusion protein. In another approach, a method is provided for improving the stability of a modified fusion protein, comprising the step of introducing an F157E or F157D mutation into the fusion protein of the present invention, thereby conferring better stability than the unmodified fusion protein. The modification can be performed on the DNA encoding the modified protein. Those skilled in the art can appropriately introduce the targeted modification into DNA and proteins by referring to codons in animals such as humans. The culture medium can be a serum-containing medium, a low-serum medium (e.g., serum concentration less than 5%), a serum-free medium, a xeno-free medium, or a medium with a defined chemical composition. From the viewpoint of impurity contamination, serum-free medium, xeno-free medium, or a medium with a defined chemical composition is preferred. Medium exchange can be performed appropriately during cultivation. Furthermore, from the viewpoint of reducing production costs, serum-free medium, xeno-free medium, or a medium with a defined chemical composition is preferred. The modified fusion protein of the present invention (including the modification of R156) can inhibit fragmentation in the absence of serum, thus offering manufacturing advantages compared to the unmodified form. Fusion proteins can be generated extracellularly by adding a signal peptide to the fusion protein. Therefore, in this case, the cell culture supernatant can be recovered and the fusion protein can be recovered from the supernatant. The fusion protein can be purified using various purification techniques known to those skilled in the art, such as gel filtration and affinity purification. The modified fusion protein of the present invention (containing the R156 modification) is resistant to fragmentation during purification, thus facilitating the management of conditions, including temperature and storage conditions during purification. For example, conventional fusion proteins require purification in the presence of L-arginine, but the above-described modified protein can be purified in the absence of L-arginine. Furthermore, due to its high storage stability, the composition of the formulation can be flexibly set. In particular, the formulation does not need to contain L-arginine. The modified fusion protein can be immobilized on the bottom of a plate, activated with plasmin, and the plasmin can be washed off to evaluate plasminogen lysate activity. For example, for a plate immobilized with an activated mutant, the plasminogen lysate can be reacted with substrates such as HD-valine-L-leucyl-L-lysyl-p-nitroaniline dihydrochloride in the presence of plasminogen. The plasminogen lysate activity of the modified protein can be determined by colorimetric quantification of p-nitroaniline, which develops color as the substrate decomposes. <Compositions and Pharmaceutical Preparations> According to the present invention, compositions or pharmaceutical formulations containing a modified fusion protein of the present invention can be provided. In addition to the modified fusion protein of the present invention, the compositions and pharmaceutical formulations may also contain pharmaceutically acceptable additives. Examples of additives include pH buffers, isotonic agents, salts, preservatives, and analgesics. The pharmaceutical formulations can be prepared for intravenous or intra-arterial administration. In one embodiment, the pharmaceutical formulation of the present invention can be a lyophilized pharmaceutical formulation. In another embodiment, the present invention provides a kit containing a lyophilized pharmaceutical formulation and water for injection (i.e., a kit prepared on demand). Pharmaceutical preparations can be used to dissolve blood clots formed in the body. Therefore, pharmaceutical preparations can be used to treat thrombosis. Pharmaceutical preparations can also be used to treat conditions or diseases caused by thrombosis. Pharmaceutical preparations can also be used to treat conditions or diseases for which the dissolution of fibrin clots is a suitable treatment method. Pharmaceutical preparations can also be used to treat cerebrovascular and cardiovascular disorders accompanied by thrombosis. Pharmaceutical preparations can, for example, treat conditions or symptoms arising from thrombosis in cerebrovascular and cardiovascular disorders, or thereby treat conditions or symptoms of cerebrovascular and cardiovascular disorders. Treatment includes therapeutic treatment and preventive treatment. Therapeutic treatment includes slowing the rate of deterioration of conditions or symptoms, weakening deterioration, stopping deterioration, or improving conditions or symptoms; preventive treatment includes preventing the occurrence of conditions or symptoms or reducing morbidity. Ischemic cerebrovascular disorders can be listed as examples of cerebrovascular disorders. Examples of ischemic cerebrovascular disorders include, for example, cerebral infarction and transient ischemic attack. Cerebral infarction can be caused by cerebral thrombosis or cerebral embolism. Cerebral thrombosis is a condition in which a thrombus blocks a blood vessel in the brain, while cerebral embolism is a condition in which a thrombus originating in a blood vessel outside the brain is transported to the brain and blocks a blood vessel in the brain. Cardiovascular disorders include ischemic heart disease, such as angina pectoris and myocardial infarction. The pharmaceutical preparation is preferably used during the acute phase of these diseases. This is because use during the acute phase can prevent tissue damage caused by thrombus or embolism. The acute phase can be within 1 hour, 2 hours, 3 hours, 4 hours, 4.5 hours, 5 hours, 6 hours, 12 hours, or 24 hours after onset. For example, for ischemic cerebrovascular disorders, it is recommended to administer a thrombolytic agent within 4.5 hours of onset; for ischemic cardiovascular disorders, it is recommended to administer a thrombolytic agent within 6 hours of onset. The pharmaceutical preparation of the present invention can also be used in the same manner. According to the present invention, the modified fusion protein can dissolve fibrin clots. In one embodiment, the fibrin clot is a thrombus. <Imaging Agent> According to the present invention, a modified version of the fusion protein can be linked (including covalently) to an imaging agent. The modified version and the imaging agent can be linked via a connector or directly without a connector. The modified version linked to the imaging agent enables imaging of fibrin clots (especially thrombi) in vivo, thereby allowing, for example, observation of thrombi in vivo. Examples of imaging agents include contrast agents. Contrast agents can be those used in magnetic resonance imaging (MRI), computed tomography (CT), and positron emission tomography (PET). Therefore, according to the present invention, a formulation or composition for in vivo imaging is provided, comprising a modified version of the fusion protein of the present invention linked to an imaging agent. According to the present invention, a method for treating thrombosis or dissolving fibrin clots in a subject in need is provided, comprising the step of administering a therapeutically effective amount of a modified fusion protein of the present invention to the subject. According to the present invention, a pharmaceutical preparation is provided for use in a method of treating thrombosis or dissolving fibrin clots in a subject in need, said pharmaceutical preparation containing a therapeutically effective amount of a modified fusion protein of the present invention. According to the present invention, a modified version of the fusion protein of the present invention is provided for use in methods for treating thrombosis or dissolving fibrin clots in subjects in need. According to the present invention, the use of a modified fusion protein of the present invention in a medicament for use in a method of treating thrombosis in a subject in need or in a method of dissolving fibrin clots is provided. According to the present invention, a method is provided for treating the state or symptoms of cerebrovascular and cardiovascular disorders in a subject in need, comprising administering to the subject a therapeutically effective amount of a modified fusion protein of the present invention. According to the present invention, a pharmaceutical formulation containing a modified fusion protein of the present invention is provided, which is used for treating states or symptoms of cerebrovascular and cardiovascular disorders in subjects in need. According to the present invention, a modified version of the fusion protein of the present invention is provided, which is used for a method of treating the state or symptoms of cerebrovascular and cardiovascular disorders in subjects in need. The invention provides the use of modified fusion proteins in the manufacture of medicaments for treating conditions or symptoms of cerebrovascular and cardiovascular disorders in subjects in need. Example Example 1: Preparation of fusion protein for dissolving fibrin clots In WO2021 / 200922A, a fusion protein (hereinafter referred to as "AMU1114") containing an antibody fragment that specifically binds to insoluble fibrin and a mutant of prourokinase was synthesized. AMU1114 is a complex of a fusion protein (heavy chain) (serial number 1) formed by linking the Fab region of the antibody and a mutant of prourokinase with the light chain of the antibody (serial number 17). AMU1114 was expressed in Chinese hamster ovary cells (CHO cells). The expression level peaked 5–7 days after the start of culture, and thereafter, even with continued culture, the expression level of AMU1114 did not increase. More specifically, such as... Figure 8A As shown, the amount of AMU1114 produced does not increase; only the amount of light chain (L chain) recycled increases. The cultivation conditions should be studied, such as Figure 8A and 8BAs shown, when AMU1114 was expressed in CHO cells in serum-free medium, the yield of light chains increased while the yield of AMU1114 decreased. However, when AMU1114 was expressed in CHO cells in the presence of 20% fetal bovine serum (FBS), the yield of AMU1114 increased while the yield of light chains decreased. Furthermore, purification of AMU1114 from CHO cells must be performed in the presence of L-arginine. The recovered amount of purified AMU1114 was 25–35 mg / L. Example 2: Preparation of modified fusion protein In this embodiment, a modified version of AMU1114 was created. Specifically, a site-specific amino acid substitution mutation was performed on the F396 (corresponding to F157 of urokinase) insertion site of the heavy chain of AMU1114. The amino acid substitution mutation was introduced by PCR using DNA encoding AMU1114 as a template and a mutation-introducing primer. PrimeStar HSDNA polymerase (Takara) was used as the PCR enzyme. AMU1114 integrated into pcDNA3.4 (ThermFisher) and all PCR products were reacted with XhoI (Takara) and NotI (Takara) for 2 hours according to the manufacturer's protocol, followed by 1% agarose gel electrophoresis and purification using the FastGeneGel / PCR Extraction Kit (Nippon Genetics). Transformation was performed using Competent High DH5α (toyobo) and cultured in LB medium (50 μg / mL) plates for 16 hours. Plasmids were extracted from the transformed E. coli using the FastGene Plasmid Mini-Prep Kit (Nippon Genetics) for sequence analysis. This confirmed that the target mutation was introduced. AMU1114 and various mutants were expressed in CHO cells. Specifically, AMU1114 and various mutants were transiently expressed using the ExpiCHO expression system (ThermoFisher). The culture supernatant was recovered after 7 days of culture. FBS was added to the culture medium to reach 20%. The culture supernatant from transient expression was added to CaptoL (Cytiva) equilibrated with PBS, washed with the same buffer, and eluted with 100 mM glycine buffer (pH 3.0) as the final product. Each mutant was adjusted to 10 μg / mL with PBS, and 100 μL / well was aliquoted into 96-well plates for immobilization at room temperature for 90 minutes. After immobilization, 300 μL / well of 1% BSA-T was aliquoted into TBS-T for blocking, and the plates were incubated at room temperature for 60 minutes. After blocking, the plates were washed with TBS-T, and 150 μL / well of a reaction mixture containing 5 μg / mL plasminogen and 15 μL / well of substrate (TestzymS PLG Sekisui Medical) adjusted with PBS was added. Absorbance at 405 nm and 505 nm was measured every 5 minutes for 2 hours. The results are as follows Figure 1 As shown. Figure 1 As shown, except for the F157Y mutant, all mutants exhibited reduced reactivity to the substrate. Some mutants (F157A, F157K, F157C, and F157S) showed significantly reduced reactivity, and it was further clarified that some mutants (F157I, F157G, F157D, and F157E) showed even more significant reductions in reactivity. This is attributed to the fact that these mutants possess resistance to plasmin. Each modified organism was adjusted to 10 μg / mL with PBS and aliquoted into 96-well plates at 100 μL / well, and allowed to stand at room temperature for 90 minutes to immobilize. After immobilization, the plates were blocked by aliquoting 300 μL / well of 1% BSA-T with TBS-T and allowing to stand at room temperature for 60 minutes. For the blocked plates, plasmin solution adjusted to 5 μg / mL was added at 100 μL / well, and the reaction was carried out at 37°C for 60 minutes. After washing with TBS-T, a reaction solution containing 5 μg / mL plasminogen and 15 μL / well of substrate (TestzymSPLG Sekisui Medical) adjusted with PBS was added at 150 μL / well. The absorbance at 405 nm and 505 nm was measured every 5 minutes for 2 hours. The results are as follows Figure 2 As shown. Figure 2 As shown, all mutants exhibited high reactivity to the substrate after plasmin treatment. In particular, the activities of the F157E and F157D mutants were equivalent to those of AMU1114. The purified F157 modifiers were adjusted to 1 mg / mL with PBS, incubated at 37°C, and compared with the results by SDS-PAGE electrophoresis after 0 hours. Figure 3A ), 24 hours later ( Figure 3B ), 120 hours later ( Figure 3C The samples were confirmed. Figures 3A-3CIn this study, various mutants are represented by single-letter symbols for the amino acids substituted at the F157 position. The F157D and F157E mutants exhibited particularly high stability. Furthermore, the yields of the F157E and F157D mutants were 3 to 5 times that of AMU1114. Example 3: Regarding the binding ability to urokinase receptors In this embodiment, the binding of AMU1114 to the human urokinase receptor was confirmed. Since the binding was confirmed, the F157D mutant and the F157E mutant were further introduced with mutations of N22Y, N27S, H29R, W30R and Q40E, respectively (see Miyake T, et al., J Biochem. 1988; 104(4): 643-647, Magdolen V, et al., Eur J Biochem. 1996; 237(3): 743-751, and Lin L, et al., J Biol Chem. 2010; 285(14): 10982-92). In addition, the AMU1114 mutant (N22G, K23G, Y24_I28del, F157E) was created using a site-specific mutation introduction method. Hereinafter, this mutant will be referred to as the AMU1114(Δ22-28, F157E) mutant. The AMU1114(Δ22-28, F157E) mutant has the amino acid sequence described in SEQ ID NO. 63. Multiple alignments of the amino acid sequence of the created mutant are shown below. Figure 9 The calculated yield is 100 mg / L. As urokinase receptors, mutants of human urokinase with the amino acid sequence described in SEQ ID NO: 6 and mouse urokinase receptors with the amino acid sequence described in SEQ ID NO: 7 were used. Affinity purification was performed by adding a 6×His tag to the C-terminus of each urokinase. Specifically, for the PCR products of pcDNA3.3 (ThermFisher) and the urokinase receptor, EcoRI (Takara) and XhoI (Takara) were reacted for 2 hours according to the manufacturer's protocol, followed by 1% agarose gel electrophoresis, and then purified using the FastGeneGel / PCR Extraction Kit (Nippon Genetics). The vector (pcDNA3.3) and insert were reacted with Ligation High (Takara) for 30 minutes. Transformation was performed using Competent High DH5α (Toyobo) and cultured in LB medium (50 μg / mL) for 16 hours. Plasmids were extracted from the transformed E. coli using the FastGene Plasmid Mini-Prep Kit (Nippon Genetics) for sequence analysis. The urokinase mutant was transiently expressed using the ExpiCHO expression system (Thermo Fisher). After 12 days of culture, the culture supernatant was recovered. The culture supernatant of transient expression was dialyzed with 50 mM Tris-HCl pH 8.5 and 300 mM NaCl, added to a Ni column (Cytiva) equilibrated with the same buffer, washed with the same buffer, and eluted with PBS containing 400 mM imidazole as the final product. Purification of the mutants was performed. AMU1114 (N22Y, N27S, H29R, W30R, Q40E, F157D) mutants, AMU1114 (N22Y, N27S, H29R, W30R, Q40E, F157E) mutants, and AMU1114 (Δ22-28, F157E) mutants were transiently expressed in serum-free medium using only the ExpiCHO expression system (Thermo Fisher). After 12 days of culture, the culture supernatant was recovered. The culture supernatant from transient expression was added to CaptoL (Cytiva) equilibrated with PBS, washed with the same buffer, and eluted with 100 mM glycine buffer at pH 3.0. The eluted sample was purified by gel filtration using Superdex 200 pg (Cytiva) as the final product. Each modified organism was adjusted to 10 μg / mL with PBS and aliquoted into 96-well plates at 100 μL / well, and allowed to stand at room temperature for 90 minutes to immobilize. For the immobilized plates, 300 μL / well of 1% BSA-T was aliquoted into TBS-T to block the plates, and the plates were allowed to stand at room temperature for 60 minutes. For the blocked plates, 100 μL / well of plasmin solution was added to the plates to a concentration of 5 μg / mL, and the reaction was carried out at 37°C for 60 minutes. After washing with TBS-T, 150 μL / well of a reaction solution prepared with 5 μg / mL plasminogen and 15 μL / well of substrate (TestzymSPLG Sekisui Medical) was added to the plates. The absorbance at 405 nm and 505 nm was measured every 5 minutes for 2 hours. The results are as follows Figure 4 and 10 As shown. Figure 4 and 10 As shown, all three mutants exhibited the same reactivity as AMU1114 in the presence of plasmin. Human and mouse urokinase receptors were adjusted to 1 μg / mL with PBS and aliquoted into 96-well plates at 50 μL / well, and incubated at room temperature for 90 min to immobilize. For the immobilized plates, 300 μL / well of TBS-T containing 1% BSA was aliquoted, and the plates were incubated at room temperature for 60 min to block. For the blocked plates, 50 μL / well of each sample (mutant) adjusted to 1000 ng / mL, 111.1 ng / mL, and 12.3 ng / mL were added, and the plates were reacted at room temperature for 60 min. After washing with TBS-T, 50 μL / well of HRP-conjugated anti-human Kappa chain antibody (Bethyl) diluted 1 / 50000 with TBS-T containing 1% BSA was aliquoted, and the plates were reacted at room temperature for 60 min. After washing, the plates were reacted with TMB (3,3',5,5'-tetramethylbenzidine) chromogenic reagent for 15 min. The reaction was stopped by adding 30 μL of 2N H2SO4 to each well, and the absorbance was measured at 450 nm. The results are as follows Figure 5 As shown. Figure 5 As shown, the binding affinity of AMU1114 to the human urokinase receptor was clearly demonstrated for the first time. Therefore, the binding affinity of the three mutants to the human urokinase receptor was tested. Figure 5 and 11 As shown, the three mutants lost their binding affinity to mutants of human urokinase. Additionally, as... Figure 11As can be understood by comparing it to PBS as a negative control, the AMU1114 (Δ22-28, F157E) mutant did not show significant binding to either the human or mouse urokinase receptor. Furthermore, when creating the AMU1114 mutant with F157E / D and W30R, this mutant also resulted in an extreme reduction in binding to the human urokinase receptor. Urokinase with a His-tagged C-terminus was expressed using ExpiCHO containing 5%, 10%, and 20% FBS, respectively. The culture supernatant after 12 days of culture was dialyzed with 50 mM Tris-HCl (pH 8.5) and 300 mM NaCl, then added to a Ni column (Cytiva) equilibrated with the same buffer. After washing with the same buffer, the sample was eluted with PBS containing 400 mM imidazole. The eluted sample was then gel-filtered using Superdex 75 pg (Cytiva) as the final sample. The results are as follows Figure 6A and 6B As shown. Figure 6A and 6B As shown, low molecular weight uPA was observed when cells were cultured in the presence of 5% FBS, and the amount of low molecular weight uPA decreased when cells were cultured in the presence of 20% FBS. Therefore, this indicates that cell culture in the presence of FBS is necessary for uPA expression. AMU1114 was expressed using ExpiCHO in the presence of 20% FBS or in serum-free conditions. AMU1114 was purified and electrophoresed as described above, and the presence of various fragments was confirmed in serum-free conditions (see [reference]). Figure 7A In the presence of 20% FBS, the amount of these fragments is significantly reduced (see reference). Figure 7A ). Then, the four AMU1114 mutants were expressed using ExpiCHO under serum-free conditions. When the mutants were purified and electrophoresed as described above, no fragmentation occurred when these mutants were cultured under serum-free conditions (see [reference]). Figure 7B ). AMU1114 and its modified form were affinity purified and analyzed by gel filtration. Results are as follows: Figure 8A and 8B As shown. Figure 8A and 8B As shown, in the case of AMU1114, the peak of the light chain is observed more strongly relative to the peak of AMU1114. The AMU1114 modified body suppresses the appearance of the light chain, and the peak of the AMU1114 modified body is relatively increased. Based on the above results, the F157 mutant of AMU1114 exhibits the same physiological activity as AMU1114 in the presence of plasmin, but with higher stability and less contamination of free light chains, resulting in higher purity and consequently, a production yield approximately 3 to 5 times higher than that of AMU1114. AMU1114 (Δ22-28, F157E) was administered to mice with a thrombin thrombosis model, and the degree of thrombolysis was compared with that of the tenecteplase group and the negative control group. Specifically, thrombin thrombosis model mice were established by intravenous injection of 600 U / kg thrombin into Balb / c female mice. Thirty minutes after intravenous injection, PBS (negative control), tenecteplase (positive control), and AMU1114 (Δ22-28, F157E) were administered intravenously, respectively. Then, 60 minutes later, the lungs were removed from the mice under deep anesthesia, and the thrombi were stained. Staining was performed using a direct method with a labeled anti-insoluble fibrin antibody (1101 antibody). The total area of ​​the signal (brown) corresponding to the thrombus was calculated. The higher the thrombus dissolution, the smaller the total area. The results are as follows: Figure 12A and Figure 12B As shown in Table 1, numerous thrombi were observed in the negative control group, while almost no thrombi were found in the positive control group and the AMU1114 (Δ22-28, F157E) administration group. Example 4: AMU1114(Δ22-28, R156E), AMU1114(Δ22-28, R156I, F157Y), AMU1114 Production and functional analysis of (Δ22-28, P155G, R156I, F157Y) AMU1114(Δ22-28, R156E), AMU1114(Δ22-28, R156I, F157Y), and AMU1114(Δ22-28, P155G, R156I, F157Y) were fabricated (refer to...). Figure 13 The heavy chains of AMU1114(Δ22-28, R156E), AMU1114(Δ22-28, R156I, F157Y), and AMU1114(Δ22-28, P155G, R156I, F157Y) were designed with amino acid sequences having sequence numbers 64, 65, and 66, respectively. The light chain has an amino acid sequence having sequence number 17. Hereinafter, AMU1114(Δ22-28, R156E), AMU1114(Δ22-28, R156I, F157Y), and AMU1114(Δ22-28, P155G, R156I, F157Y) are sometimes referred to as mutants A, B, and C, respectively. PCR reaction The gene sequence of AMU1114 (Δ22-28, Phe157Glu) inserted into pcDNA3.4 (ThermoFisher) was amplified by PCR using the following primers. PCR enzyme: PrimeStar HS DNA polymerase (Takara) AGGAGGCTCGAGTAGCAATGAACTTC (Serial Number 70) GTTTCAATGCGGCCAGAAAACATTGAGACCGGAATTTAAGATCATAGGCGGGGAGTTCACGAC (serial number 71) TCTCAATGTTTTCTGGCCGCATTGAAAC (Serial Number 72) CAAACTCATTACTAACCGGTAGGGATCG (Serial Number 73) restriction enzyme treatment For AMU1114 integrated into pcDNA3.4 (ThermFisher) and each PCR product, XhoI (Takara) and NotI (Takara) were reacted for 2 hours according to the manufacturer's instructions, followed by 1% agarose gel electrophoresis and purification using the FastGene Gel / PCR Extraction Kit (Nippon Genetics). Connection reaction Using Ligation High (Takara), allow the vector (pcDNA3.4) and insert to react for 30 minutes. Transformation Transformation was performed using Competent High DH5α (toyobo), and the cells were cultured in LB medium (50 μg / mL) plates for 16 hours. Gene confirmation Plasmids were extracted from transformed E. coli using the FastGene plasmid miniprep kit (Nippon Genetics) and sequenced to confirm the creation of AMU1114 (Δ22-28, R156E) with the amino acid sequence described in sequence number 64. Production and purification of AMU1114 modified organism using CHO Transient expression was performed using the ExpiCHO expression system (Thermo Fisher). After 12 days of culture, the culture supernatant was recovered. Culture was conducted in serum-free medium. The culture supernatant obtained from transient expression was added to CaptoL (Cytiva) equilibrated with PBS, washed with the same buffer, and eluted with 100 mM glycine buffer at pH 3.0. The eluted sample was gel-filtered using Superdex 200 pg 16 / 600 (Cytiva) equilibrated with PBS, 150 mM L-Arg, and pH 7.4, as the final product. The addition of L-arginine improved the solubility of the modified organism. The yields of AMU1114 (Δ22-28, R156E) were approximately 80 mg / L and approximately 85 mg / L, respectively, which were equivalent. The light chains of AMU1114(Δ22-28, R156E) and AMU1114(Δ22-28, F157E) have the amino acid sequence described in Serial No. 17. The heavy chain of AMU1114(Δ22-28, F157E) has the amino acid sequence described in Serial No. 63. Urokinase activity comparison experiment - pretreatment without plasmin utilization AMU1114, AMU1114(Δ22-28, Phe157Glu), and AMU1114(Δ22-28, Arg156Glu) were adjusted to 10 μg / mL using PBS, and 100 μL / well was aliquoted into a 96-well plate and allowed to stand at room temperature for 90 minutes to immobilize. For the immobilized plate, 300 μL / well of 1% BSA-T was aliquoted into TBS-T and allowed to stand at room temperature for 60 minutes to block the plate. After blocking, the plate was washed with TBS-T, and 150 μL / well of a reaction solution containing 5 μg / mL plasminogen and 15 μL / well of substrate (TestzymS PLG, Sekisui Medical) adjusted with PBS was added. The absorbance at 405 nm and 505 nm was measured every 5 minutes for 2 hours. Under physiological conditions, plasminogen is converted to plasmin by urokinase, and plasmin breaks down fibrin to generate fibrin degradation products. In this experiment, urokinase activity was estimated based on the amount of substrate reacted. Comparative experiment on urokinase activity - utilizing plasmin pretreatment AMU1114, AMU1114(Δ22-28, Phe157Glu), and AMU1114(Δ22-28, Arg156Glu) were adjusted to 10 μg / mL using PBS, and 100 μL / well was aliquoted into 96-well plates and incubated at room temperature for 90 minutes to immobilize the plate. For the immobilized plate, 300 μL / well of 1% BSA-T was aliquoted into TBS-T and the plate was incubated at room temperature for 60 minutes to block it. For the blocked plate, 100 μL / well of plasminogen lysate was added and the plate was reacted at 37°C for 60 minutes. After washing with TBS-T, 150 μL / well of a reaction mixture containing 5 μg / mL plasminogen lysate and 15 μL / well of substrate (TestzymS PLG Sekisui Medical) adjusted with PBS was added. The absorbance at 405 nm and 505 nm was measured every 5 minutes for 2 hours. Stability test The purified AMU1114, AMU1114(Δ22-28, Phe157Glu), and AMU1114(Δ22-28, Arg156Glu) were adjusted to 1 mg / mL with PBS, incubated at 37°C, and the samples were confirmed by SDS-PAGE electrophoresis after 48 hours. plasmin resistance test The purified AMU1114, AMU1114(Δ22-28, Phe157Glu), and AMU1114(Δ22-28, Arg156Glu) were adjusted to 1 mg / mL, and the plasmin was adjusted to 0.1 mg / mL (1 / 10 volume), 0.02 mg / mL (1 / 50 volume), and 0.004 mg / mL (1 / 250 volume), respectively. These were mixed at a 1:1 ratio and reacted at 37°C for 60 minutes. The resulting samples were confirmed by SDS-PAGE electrophoresis. result Stability test results of AMU1114 and its two modified versions are shown in Figure 14 AMU1114 decomposed upon incubation at 37°C for 48 hours, clearly confirming the presence of H chain fragments. In contrast, AMU1114(Δ22-28, F157E), AMU1114(Δ22-28, R156E), AMU1114(Δ22-28, R156I, F157Y), and AMU1114(Δ22-28, P155G, R156I, F157Y) were all stable, with no H chain fragments detected. Next, the resistance of AMU1114 and its two modified forms to plasmin was tested. For AMU1114 and its modified forms, 1 / 10, 1 / 50, or 1 / 250 of the amount of plasmin were reacted under the above conditions. The obtained samples were confirmed by electrophoresis. Figure 15 As shown, AMU1114 was observed to be almost completely decomposed by 1 / 10 of the amount of plasmin. AMU1114(Δ22-28, F157E) showed strong resistance to plasmin, while AMU1114(Δ22-28, R156E) showed moderate resistance to plasmin. Then, the urokinase activities of AMU1114 and its modified counterparts were confirmed under both pretreatment conditions with and without plasmin. Under pretreatment conditions with plasmin, such as... Figure 16A As shown, AMU1114 and its modified counterpart exhibit equivalent urokinase activity. In contrast, under conditions without pretreatment utilizing plasmin (under conditions closer to physiological conditions), such as... Figure 16B As shown, AMU1114 (Δ22-28, R156E) exhibited the highest urokinase activity, exceeding that of AMU1114 (Δ22-28, F157E) and being equivalent to that of AMU1114. The results above indicate that AMU1114 (Δ22-28, R156E) exhibits relatively high blood stability and moderate plasmin resistance, and is more easily activated than AMU1114 (Δ22-28, F157E) under fibrin-binding conditions. These characteristics of AMU1114 (Δ22-28, R156E) suggest its preference for thrombolytic therapy. Furthermore, it is believed that AMU1114 (Δ22-28, R156E), being Δ22-28, does not show significant binding to the human urokinase receptor. Furthermore, AMU1114 unexpectedly fragmented when expressed in cells under serum-free conditions, while AMU1114(Δ22-28, F157E) did not exhibit fragmentation even when expressed in cells under serum-free conditions. Similarly, AMU1114(Δ22-28, R156E) also did not exhibit fragmentation when expressed in cells under serum-free conditions and could be recovered in equivalent quantities to AMU1114(Δ22-28, F157E). Therefore, AMU1114(Δ22-28, R156E) also demonstrates manufacturing advantages. In addition, such as Figure 17 As shown, AMU1114 (Δ22-28, F157E) requires a certain activation time compared to AMU1114 (see reference). Figure 17(See right figure of I). When the reaction time is short, AMU1114 (Δ22-28, F157E) shows almost no thrombolytic effect (see reference). Figure 17 (II). On the other hand, AMU1114 (Δ22-28, F157E) also maintained stability after 48 hours of incubation in PBS at 37°C (see reference). Figure 19 Therefore, it can be expected that AMU1114 (Δ22-28, F157E) will exert a stable thrombolytic effect over the long term. For example, compared with acute infarction, AMU1114 (Δ22-28, F157E) can be preferred for chronic thrombotic diseases. In contrast, such as Figure 18 As shown, AMU1114(Δ22-28, R156E), AMU1114(Δ22-28, R156I, F157Y) and AMU1114(Δ22-28, P155G, R156I, F157Y) require a certain amount of time to activate compared to AMU1114 (see reference). Figure 18 (See the right figure of I), but it is activated immediately even with a short reaction time, showing activity comparable to AMU1114 (see Figure I). Figure 18 (II). Additionally, AMU1114 (Δ22-28, R156E), AMU1114 (Δ22-28, R156I, F157Y), and AMU1114 (Δ22-28, P155G, R156I, F157Y) maintained stability after 48 hours of incubation in PBS at 37°C (see reference). Figure 20 Therefore, it can be expected that AMU1114(Δ22-28, R156E), AMU1114(Δ22-28, R156I, F157Y), and AMU1114(Δ22-28, P155G, R156I, F157Y) will rapidly exert thrombolytic effects with long-lasting effects. For example, AMU1114(Δ22-28, R156E), AMU1114(Δ22-28, R156I, F157Y), and AMU1114(Δ22-28, P155G, R156I, F157Y) can be preferentially used for acute infarction and other similar conditions. Example 5: Therapeutic Experiment of Rat Model of Cerebral Infarction (1) Production of UK active human AMU1114 (Δ22-28) The ExpiCHO expression system (Thermo Fisher) was used to transiently express human AMU1114 (Δ22-28). After 7 days of incubation, the culture supernatant was recovered. The supernatant was added to CaptoL (Cytiva) equilibrated with PBS, washed with the same buffer, and then eluted with 100 mM glycine buffer (pH 3.0). The eluted target was recovered and purified by gel filtration using Superdex 200 pg (Cytiva) as the final product. (2) Establishment of a rat model of cerebral infarction A method for creating a cerebral infarction model was developed using a microcatheter to move thrombi made from rat whole blood to the middle cerebral artery of rats (Komatsu T Translational Stroke Res 2024). Knowing that thrombi made from rat whole blood are rarely dissolved by conventional thrombolytic agents, this example attempts to create a thrombus model more closely resembling that of humans. Specifically, 75 μL, 50 μL, and 25 μL of human fibrinogen (Arg) in PBS were added to 1 ml of rat whole blood, and 25 μL, 50 μL, and 75 μL of PBS (Arg) were added respectively to adjust the solution to 100 μL. The fibrinogen concentration in rat blood is 2.5 mg / ml, therefore the human fibrinogen content in the prepared solutions is equivalent to 60%, 50%, and 33% of that concentration. Fibrinolytic enzymes (86 μg / ml and 8.6 μg / ml) were then applied to the solutions at 37°C for 1 hour (see reference). Figure 21 In subsequent cerebral infarction models, thrombi were generated by adding 60% human fibrinogen to the blood of rats to create a rat cerebral infarction model. A microcatheter was inserted into the rat's caudal artery, and the microcatheter was guided to the left internal carotid artery in the angiography chamber using an angiography device. A thrombus, 0.861 mm in diameter and 3 mm in length, obtained through the aforementioned method, was placed via the microcatheter into a branch of the left middle cerebral artery in the rat to induce a cerebral infarction. The angiography device was used to confirm that the thrombus was placed at the appropriate site (see [reference]). Figure 22 and 23 ). Five minutes after cerebral infarction was induced, a thrombolytic agent (as a modified form of tPA, tenepase, and AMU1114) was administered intravenously via the tail vein of rats. The AMU modifier used at this time was the UK activator of AMU1114 (Δ22-28). Thrombolysis was confirmed using an angiography device at 1, 3, 6, and 24 hours after the intravenous injection of the thrombolytic agent (see [reference]). Figure 24In the case of tenecteplase, thrombolysis could not be confirmed, and in the 46 nmol / animal administration group, 2 out of 3 patients died from convulsions within 4 hours. This convulsion-induced death was considered to be caused by cerebral hemorrhage. In contrast, in the 46 nmol / animal AMU1114(Δ22-28) administration group, 2 out of 3 patients experienced thrombolysis and infarction recanalization. Furthermore, no convulsion-induced death was confirmed in the AMU1114(Δ22-28) administration group. Example 6: Treatment Experiment of an Alzheimer's Dementia Model Alzheimer's disease is thought to be caused by the accumulation of β-amyloid protein, which is believed to accumulate on a scaffold of insoluble fibrin. In this study, the effect of AMU1114 (Δ22-28) on the accumulation of β-amyloid protein was investigated in an Alzheimer's disease mouse model. A mouse-type AMU1114 (Δ23-29) was prepared for administration to a mouse model. Amino acid positions 23-29 in the mouse form correspond to amino acid positions 22-28 of prourokinase in the amino acid sequence described in Serial No. 1. The heavy chain has the amino acid sequence described in Serial No. 68, and the light chain has the amino acid sequence described in Serial No. 69. The heavy and light chains were transiently expressed in the ExpiCHO expression system (Thermo Fisher) to express mouse-type AMU1114 (Δ23-29). After 7 days of culture, the culture supernatant was recovered. To inhibit enzyme activity, FBS was added to the culture medium at a concentration of 20%. The culture supernatant and 50 mM sodium phosphate buffer (pH 6.0) were mixed at a ratio of 1:2 and added to a HiTrap SP column (Cytiva) equilibrated with 50 mM sodium phosphate buffer (pH 6.0). The sample was washed with a buffer solution consisting of 50 mM sodium phosphate buffer (pH 6.0) and 500 mM NaCl in a 9:1 ratio, followed by gradient elution down to 0:10. The eluted target analyte was recovered and purified by gel filtration using a Superdex 200 pg (Cytiva) apparatus to obtain the final product. Eight-week-old Alzheimer's disease mouse models (APP NL-GF mice) were administered mouse-type AMU1114 (Δ22-28) or saline (negative control) intraperitoneally once a week. After eight administrations, the mice were sacrificed under anesthesia one week after the last administration. The right brain was removed after PBS perfusion and fixed in formalin, while the left brain was cryopreserved after removal. β-amyloid protein was extracted from brain tissue and quantified. (1) Add brain tissue and 400 μL of TBS / protease inhibitor to a 1.5 mL tube and homogenize using a homogenizer. Wash the homogenizer with 400 μL of the same solution. Let the wash solution settle by vortexing, combine it with the original tube, and gently vortex. Centrifuge the homogenate at 217000 × g (50000 rpm) at 4 °C for 20 minutes. (2) After removing the supernatant, add 800 μL of TBS / protease inhibitor (containing 1.0 mol / L sucrose) to the precipitate, suspend (vortex), and centrifuge at 50,000 rpm and 4°C for 20 minutes. (3) After removing the supernatant, add 400 μL of 1% Triton X-100 / TBS / protease inhibitor to the precipitate and homogenize. Wash the homogenizer with 500 μL of the same solution. After washing, rotate the sediment and combine it with the original tube, then gently vortex. Incubate the homogenate at 37°C for 15 minutes and centrifuge at 50,000 rpm for 60 minutes. (4) After removing the supernatant, add 400 μL of 2% SDS / TBS / protease inhibitor to the precipitate and homogenize. Combine with the liquid obtained from washing with 500 μL of the same solution and gently vortex. Incubate the homogenate at 37°C for 15 minutes and centrifuge at 50,000 rpm for 40 minutes. (5) After removing the supernatant, add 400 μL of 2% SDS / TBS / protease inhibitor to the precipitate and homogenize. Combine with the liquid obtained from washing with 500 μL of the same solution and gently vortex. Incubate the homogenate at 37°C for 15 minutes and centrifuge at 50,000 rpm for 40 minutes. (6) After removing the supernatant, add 250 μL of 70% formic acid to the precipitate and perform ultrasonic disruption. After disruption, centrifuge at 50,000 rpm and 4°C for 20 minutes. Perform 4 sets of ultrasonic cycles at OUTPUT 3 and DUTY 40% with a 1-minute rest on ice. (7) Collect the supernatant, dry it with an evaporator, add 100uL of DMSO, and suspend it by ultrasonic disruption for 20 minutes. (8) Store at -80°C until ELISA is performed. For ELISA, the sample taken from the refrigerator is centrifuged at 20000×g and 4°C for 20 minutes. The supernatant is collected and diluted with the kit's diluent. The experiment was conducted using a mouse model consisting of 3 males and 2 females (5 mice in total). β-amyloid protein accumulation was reduced in all individuals. In the negative control group, the average β-amyloid protein concentration was 116,606 pM, while in the AMU1114(Δ23-29) treatment group, the average concentration was reduced to 77,431 pM. Results separated by sex are shown below. Figure 25 As shown. Furthermore, no adverse reactions such as hemorrhage and inflammation in the brain or other areas were observed in the AMU1114 (Δ23-29) administration group, no difference in weight compared to the control group, and no behavioral abnormalities were observed. It is believed that by not conferring plasmin resistance, AMU1114 or its modified form activates prourokinase, inducing early breakdown of insoluble fibrin, thus exerting these significant effects. Therefore, AMU1114 or its modified form without plasmin resistance is beneficial for acute thrombotic and embolic diseases. Additionally, AMU1114 or its modified form without plasmin resistance is also beneficial for Alzheimer's dementia. It is believed that even AMU1114 modified forms with plasmin resistance are effective for Alzheimer's dementia, but it is not necessary to introduce mutations for conferring plasmin resistance into AMU1114 or its modified form. The contents of the sequence list Amino acid sequence of the heavy chain of sequence number 1: AMU1114 EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRKAHYGNYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGGGGSGGGGSGGSSSNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGGGPSSPPEELKFQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLALSAGGAS Sequence 2: Amino acid sequence of the heavy chain of AMU1114 F157D EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRKAHYGNYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGGGGSGGGGSGGSSSNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGGGPSSPPEELKFQCGQKTLRPRDKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLALSAGGAS SEQ ID NO: 3: Amino acid sequence of the heavy chain of AMU1114 F157E EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRKAHYGNYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGGGGSGGGGSGGSSSNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGGGPSSPPEELKFQCGQKTLRPREKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVIS ATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLALSAGGAS Sequence 4: Amino acid sequence of AMU1114 F157D, further modified heavy chain EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRKAHYGNYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGGGGSGGGGSGGSSSNELHQVPSNCDCLNGGTCVSYKYFSSIRRCNCPKKFGGEHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGGGPSSPPEELKFQCGQKTLRPRDKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLALSAGGAS SEQ ID NO: 5: Amino acid sequence of the further engineered heavy chain of AMU1114 F157E EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRKAHYGNYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGGGGSGGGGSGGSSSNELHQVPSNCDCLNGGTCVSYKYFSSIRRCNCPKKFGGEHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGGGPSSPPEELKFQCGQKTLRPREKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLALSAGGAS SEQ ID NO: 6: Amino acid sequence of prourokinase SNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGKKPSSPPEELKFQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVI SATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTD YLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLAL Serial number 7: Amino acid sequence of prourokinase K135G and K136G SNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGGGPSSPPEELKFQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVISATHC FIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLAL Serial number 8: Amino acid sequence of fibrinogen Bβ231-246 CNIPVVSGKECEEIIR Serial number 9: Amino acid sequence of fibrinogen γ232-246: KNWIQYKEGFGHLSP Serial number 10: Amino acid sequence of humanized HCDR1 SYWMH Serial number 11: Amino acid sequence of humanized HCDR2 1101 AIYPGNSDTRYSPSFQG Serial number 12: Amino acid sequence of humanized HCDR3 KAHYGNYGFAY Serial number 13: Amino acid sequence of humanized 1101 LCDR1 RASQHINNWLA Serial number 14: Amino acid sequence of humanized 1101 LCDR2 GATSLQS Serial number 15: Amino acid sequence of humanized 1101 LCDR3 QQYWSTPLT Serial number 16: Amino acid sequence of humanized 1101 VH MGSTAILALLLAVLQGVCAEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRKAHY GNYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Serial number 17: Amino acid sequence of humanized 1101 VL MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSSLSASVGDRVTITCRASQHINNWLAWYQQKPGKAPKLLISGATSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYWSTPL TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Serial number 18: Amino acid sequence NYGMN of humanized 99 HCDR1 Serial number 19: Amino acid sequence of humanized 99 HCDR2 (WINTKIG) EPTYAQKFQG Serial number 20: Amino acid sequence of humanized 99 HCDR3 LLDY Serial number 21: Amino acid sequence of humanized 99 LCDR1: RASQSVLYSSNQKNYLA Serial number 22: Amino acid sequence of humanized 99 LCDR2 WASSLQS Serial number 23: Amino acid sequence of humanized 99 LCDR3 HQYLSSYT Serial number 24: Amino acid sequence of humanized 99 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTKIGEPTYAQKFQGRVTMTRDKSSSTAYMELSSLRSEDTAVYYCARLLDYWGQGTLVTVSS Serial No. 25: Amino acid sequence of humanized 99 VL DIQMTQSPSSLSASVGDRVTITCRASQSVLYSSNQKNYLAWYQQKPGKSPKLLIYWASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYLSSYTFGQGTKVEIKR Serial No. 26: Amino acid sequence of further modified heavy chain of AMU1114 F157E / D EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRKAHYGNYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGGGGSGGGGSGGSSSNELHQVPSNCDCLNGGTCVSYKYFSSIRRCNCPKKFGGEHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGGGPSSPPEELKFQCGQKTLRPRXKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLALSAGGAS The amino acid sequence of HCDR1, serial number 27:102-10, is FTNYGMN. The amino acid sequence of HCDR2, sequence number 28:102-10, is WINTYTGEATYA. The amino acid sequence of HCDR3, sequence number 29:102-10, is LMDY. The amino acid sequence of LCDR1, sequence number 30:102-10, is KASQDINKYIA. Serial number 31: 102-10 The amino acid sequence of LCDR2 is YTSTLQP Serial number 32: 102-10 The amino acid sequence of LCDR3 is LQYDNLTW The amino acid sequence of HCDR1, sequence number 33:34-105, is KSVSTSGYSY. Serial number 34: 34-105 HCDR2 amino acid sequence LVS The amino acid sequence of HCDR3, sequence number 35:34-105, is QHIRELTR. The amino acid sequence of LCDR1, sequence number 36:34-105, is GFTFSSYA. The amino acid sequence of LCDR2, sequence number 37:34-105, is ISSGGTT. Serial number 38:34-105 The amino acid sequence of LCDR3 VRGGTIGAY Serial number 39: The amino acid sequence of HCDR1, Fib-0355, is QSVLYSSNQK. Serial number 40: Fib-0355 HCDR2 amino acid sequence YWASTRES Serial number 41: The amino acid sequence of HCDR3 (Fib-0355) YLSS Serial number 42: The amino acid sequence of Fib-0355 LCDR1 is YTFTNYG. Serial number 43: Amino acid sequence of Fib-0355 LCDR2 NTNTGE Serial number 44: Fib-0355, amino acid sequence of LCDR3 RLLDY The amino acid sequence NYGMN of HCDR1, sequence number 45:99 The amino acid sequence of HCDR2, sequence number 46:99, is WINTKIGEPTYAEEFKG. The amino acid sequence of HCDR3, sequence number 47:99, is LLDY. The amino acid sequence of LCDR1, sequence number 48:99, is RASESVDSYGNSFMH. Serial number 49:99 The amino acid sequence of LCDR2 RASNLES Serial number 50:99 Amino acid sequence of LCDR3 QQSNEDPRT The amino acid sequence of HCDR1, sequence number 51:1101, is SYWMH. The amino acid sequence of HCDR2, sequence number 52:1101, is AIYPGNSDTRNNQKFKG. The amino acid sequence of HCDR3, sequence number 53:1101, is KAHYGNYGFAY. The amino acid sequence of LCDR1, sequence number 54:1101, is KASDHINNWLA. The amino acid sequence of LCDR2, sequence number 55:1101, is GATSLET. The amino acid sequence of LCDR3, sequence number 56:1101, is QQYWSTPLT. SYAMS amino acid sequence of HCDR1, sequence number 57:0211 The amino acid sequence of HCDR2, sequence number 58:0211, is AISSGGTTYYPDSVKG. The amino acid sequence of HCDR3, sequence number 59:0211, is GGTIGAYW. The amino acid sequence of LCDR1, serial number 60:0211, is KSSQSVLYSSNQKNYLA. The amino acid sequence of LCDR2, serial number 61:0211, is WASTRES. The amino acid sequence of LCDR3, sequence number 62:0211, is HQYLSSWT. Amino acid sequence of the heavy chain of sequence number 63: AMU1114 (Δ22-28, F157E) Amino acid sequence of the heavy chain of sequence number 64: AMU1114 (Δ22-28, R156E) EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRKAHYGNYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGGGGSGGGGSGGSSSNELHQVPSNCDCLNGGTCVSGGHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGGGPSSPPEELKFQCGQKTLRPEFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLALSAGGAS SEQ ID NO: 65: Amino acid sequence of the heavy chain of AMU1114 (Δ22-28, R156I, F157Y) EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRKAHYGNYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGGGGSGGGGSGGSSSNELHQVPSNCDCLNGGTCVSGGHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGGGPSSPPEELKFQCGQKTLRPIYKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLALSAGGAS SEQ ID NO: 66: Amino acid sequence of the heavy chain of AMU1114 (Δ22-28, P155G, R156I, F157Y) EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRKAHYGNYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGGGGSGGGGSGGSSSNELHQVPSNCDCLNGGTCVSGGHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGGGPSSPPEELKFQCGQKTLRGIYKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLALSAGGAS SEQ ID NO: 67: Amino acid sequence of heavy chain of AMU1114 (Δ22-28) EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRKAHYGNYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGGGGSGGGGSGGSSSNELHQVPSNCDCLNGGTCVSGGHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGGGPSSPPEELKFQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLALSAGGAS SEQ ID NO: 68 (Heavy chain of murine AMU1114(Δ23-29)) EVQLQQSGTVLARPGASVKMSCKASGFSFTSYWMHWVKQRPGQGLEWIGAIYPGNSDTRNNQKFKGKAKLTAVTSANTAYMELSSLTNEDSAVYYCTRKAHYGNYGFAYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGSGGGGSGGGGSGGSSGSVLGAPDESNCGCQNGGVCVSGGRRCSCPRKFQGEHCEIDASKTCYHGNGDSYRGKANTDTKGRPCLAWNAPAVLQKPYNAHRPDAISLGLGKHNYCRNPDNQKRPWCYVQIGLRQFVQECMVHDCSLSGGPSSSVDQQGFQCGQKALRPRFKIVGGEFTEVENQPWFAAIYQKNKGGSPPSFKCGGSLISPCWVASAAHCFIQLPKKENYVVYLGQSKESSYNPGEMKFEVEQLILHEYYREDSLAYHNDIALLKIRTSTGQCAQPSRSIQTICLPPRFTDAPFGSDCEITGFGKESESDYLYPKNLKMSVVKLVSHEQCMQPHYYGSEINYKMLCAADPEWKTDSCKGDSGGPLICNIEGRPTLSGIVSWGRGCAEKNKPGVYTRVSHFLDWIQSHIGEEKGLAF SEQ ID NO: 69 (Murine AMU1114 (Δ23-29) light chain) DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 74 (Humanized 1101 antibody HCDR1): SYWMH Serial number 75 (humanized 1101 antibody HCDR2): AIYPGNSDTRYSPSFQG Serial number 76 (humanized 1101 antibody HCDR3): KAHYGNYGFAY Serial number 77 (humanized 1101 antibody LCDR1): RASQHINNWLA Serial number 78 (humanized 1101 antibody LCDR2): GATSLQS Serial number 79 (humanized 1101 antibody LCDR3): QQYWSTPLT Serial number 80 (humanized 1101 antibody heavy chain variable region): MGSTAILALLLAVLQGVCAEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRKAHY GNYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Serial number 80 (humanized 1101 antibody light chain variable region): MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSSLSASVGDRVTITCRASQHINNWLAWYQQKPGKAPKLLISGATSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYWSTPL TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Serial number 81 (humanized 99 antibody HCDR1): NYGMN Serial number 82 (humanized 99 antibody HCDR2): WINTKIGEPTYAQKFQG Serial number 83 (humanized 99 antibody HCDR3): LLDY Serial number 84 (humanized 99 antibody LCDR1): RASQSVLYSSNQKNYLA Serial number 85 (humanized 99 antibody LCDR2): WASSLQS Serial number 86 (humanized 99 antibody LCDR3): HQYLSSYT Serial number 87 (humanized 99 antibody heavy chain variable region): QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTKIGEPTYAQKFQGRVTMTRDKSSSTAYMELSSLRSEDTAVYYCARLLDYWGQGTLVTVSS Serial number 88 (humanized 99 antibody light chain variable region): DIQMTQSPSSLSASVGDRVTITCRASQSVLYSSNQKNYLAWYQQKPGKSPKLLIYWASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYLSSYTFGQGTKVEIKR

Claims

1. A method for treating a disease, thrombotic disease, embolic disease, infarct disease, or thromboembolism in a subject with accumulation or deposition of insoluble fibrin, comprising the step of administering a therapeutically effective amount of an active ingredient to the subject. The active ingredient comprises a fusion protein containing a domain that binds to insoluble fibrin, as well as the EGF domain, kringle domain, and catalytic domain of prourokinase.

2. The method according to claim 1, wherein, Thrombotic diseases, embolic diseases, infarct diseases, or thromboembolic diseases refer to thromboembolic diseases, embolic diseases, infarct diseases, or thromboembolic diseases in the acute phase.

3. The method according to claim 1 or 2, wherein, Thrombotic diseases, embolic diseases, infarct diseases, or thromboembolism are selected from the group consisting of cerebral infarction, myocardial infarction, pulmonary embolism, peripheral arterial embolism, and mesenteric infarction.

4. The method according to any one of claims 1 to 3, wherein, Open the blood clot or embolism in the object.

5. The method according to any one of claims 1 to 4, wherein, Equimolar doses showed higher thrombus recanalization or embolization rates than alteplase, or preferably higher than tenecteplase.

6. The method according to any one of claims 1 to 5, wherein, Equimolar doses showed a lower frequency of treatment-related bleeding compared to alteplase and tenecteplase.

7. The method according to any one of claims 1 to 6, wherein, By administering equimolar amounts, (i) thrombus recanalization or embolization recanalization rates were observed to be higher than alteplase or preferably higher than tenecteplase, and (ii) treatment-related bleeding frequencies were observed to be lower than alteplase or preferably lower than tenecteplase.

8. The method according to claim 1, wherein, Diseases accompanied by the accumulation or deposition of insoluble fibrin are called Alzheimer's disease.

9. The method according to claim 5, wherein, It reduces the accumulation of β-amyloid protein.

10. A protein having the amino acid sequence described in Serial No. 1 or Serial No. 67, or a composition containing the protein, for use in any one of claims 1 to 9.

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