Fibrin-binding antibody, fibrinolytic protein containing said antibody, and medicinal preparation containing said protein

Abstract

The present disclosure provides a fibrin-binding antibody, a fibrinolytic protein containing said antibody, and a medicinal preparation containing said protein.

Description

Fibrin-binding antibody, fibrinolytic protein containing said antibody, and pharmaceutical preparation containing said protein

[0001] The present invention relates to an antibody that binds to fibrin, a fibrinolytic protein containing the antibody, and a pharmaceutical preparation containing the protein. In particular, the present invention relates to a fibrinolytic protein suitable for large-scale purification and a pharmaceutical preparation containing the protein. The present invention also relates to a method for stabilizing a fibrinolytic protein and a method for producing a stabilized fibrinolytic protein.

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

[0003] Furthermore, a fusion protein of an antibody that binds to fibrin and a pro-urokinase mutant has been developed, and it has been disclosed that the fusion protein has a thrombolytic activity (Patent Document 3).

[0004] WO2014 / 133093WO2018 / 203517WO2021 / 200922

[0005] The present invention provides antibodies that bind to fibrin, fibrinolytic proteins containing the antibodies, and pharmaceutical preparations containing the proteins. Specifically, WO 2021 / 200922 (Patent Document 3) revealed problems with large-scale production (e.g., limitations on the amount of protein produced in protein-producing cells, the generation of protein degradation products, the need for serum in culture, the need for specific additives for purification, and inadvertent activation). In certain aspects, the present invention can alleviate or resolve any one or more of these problems. Furthermore, the fusion protein disclosed in Patent Document 3 did not bind to mouse urokinase receptor but showed reactivity with human urokinase receptor. In certain aspects, the present invention can reduce or eliminate reactivity with human urokinase receptor.

[0006] The present invention provides the following: [1] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin and a catalytic domain of pro-urokinase having an amino acid sequence corresponding to the amino acid sequence of amino acids 156 to 411 of pro-urokinase as set forth in SEQ ID NO: 6 or 7, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker, wherein the antibody or antigen-binding fragment comprises the heavy chain variable region and the light chain variable region of an antibody that binds to insoluble fibrin, and wherein the phenylalanine in the catalytic domain corresponding to the phenylalanine at position 157 of pro-urokinase as set forth in SEQ ID NO: 6 or 7 is substituted with another amino acid. [2] The fusion protein according to [1] above, wherein the phenylalanine is substituted with isoleucine, glutamic acid, aspartic acid, or cysteine. [3] The fusion protein according to [1] or [2] above, wherein the phenylalanine is substituted with glutamic acid or aspartic acid. [4] The fusion protein according to any one of [1] to [3] above, which has a reduced ability to bind to one or more urokinase receptors selected from the group consisting of human urokinase receptor and mouse urokinase receptor, compared to the fusion protein having the amino acid sequence set forth in SEQ ID NO: 1. [5] The fusion protein according to any one of [1] to [4] above, which does not show significant binding to one or more urokinase receptors selected from the group consisting of human urokinase receptor and mouse urokinase receptor.[6] The fusion protein according to any one of [1] to [3] above, further comprising an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence of amino acids 7 to 43 of prourokinase set forth in SEQ ID NO: 6 or 7 between the antibody or antigen-binding fragment thereof and the catalytic domain, in which the amino acids of the 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 prourokinase set forth in SEQ ID NO: 6 or 7, respectively, are substituted with other amino acids, thereby reducing or eliminating the binding ability to the human urokinase receptor. [7] The fusion protein according to any one of [1] to [4] above, further comprising an EGF-like domain between the antibody or antigen-binding fragment thereof and the catalytic domain, which has an amino acid sequence corresponding to the amino acid sequence from the 7th to the 43rd amino acids of pro-urokinase as set forth in SEQ ID NO: 6 or 7, in which the amino acids of the 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 pro-urokinase as set forth in SEQ ID NO: 6 or 7 are substituted with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing or eliminating the binding ability to the human urokinase receptor. [8] The fusion protein according to any one of [1] to [4] above, further comprising an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence of amino acids 7 to 43 of prourokinase set forth in SEQ ID NO: 6 or 7 between the antibody or antigen-binding fragment thereof and the catalytic domain, wherein the amino acids in the EGF-like domain corresponding to the amino acids from asparagine at position 22 to isoleucine at position 28 of prourokinase set forth in SEQ ID NO: 6 or 7 are deleted (the deleted site may optionally have an insertion of 1 to 7 amino acids (a peptide that has no binding affinity to the human urokinase receptor, for example, a peptide of 2 amino acids, for example, GG)). [9] A pharmaceutical preparation comprising the fusion protein according to any one of [1] to [8] above.

[10] A method for producing the fusion protein according to any one of [1] to [8] above, comprising culturing a protein-producing cell having an expressible nucleic acid encoding the fusion protein to express the fusion protein, and purifying the fusion protein from the resulting culture.

[11] The method according to

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

[12] The method according to

[10] or

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

[13] A method for producing a fusion protein variant (preferably having excellent stability) or a pharmaceutical composition containing said variant, wherein the fusion protein comprises an antibody or antigen-binding fragment thereof that binds to insoluble fibrin and a catalytic domain of pro-urokinase having an amino acid sequence corresponding to the amino acid sequence of amino acids 156 to 411 of pro-urokinase set forth in SEQ ID NO: 6 or 7, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker, and the antibody or antigen-binding fragment comprises the heavy chain variable region and the light chain variable region of an antibody that binds to insoluble fibrin, and the method comprises introducing an F157E or F157D mutation into the fusion protein, thereby conferring excellent stability compared to the fusion protein before modification.

[0007]

[21] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, and a catalytic domain of pro-urokinase having an amino acid sequence corresponding to the amino acid sequence of amino acids 156 to 411 of pro-urokinase as set forth in SEQ ID NO: 6 or 7, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker, wherein the antibody or antigen-binding fragment comprises the heavy-chain variable region and the light-chain variable region of an antibody that binds to insoluble fibrin, and further comprises an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence of amino acids 7 to 43 of pro-urokinase as set forth in SEQ ID NO: 6 or 7, between the antibody or antigen-binding fragment portion and the catalytic domain portion, wherein the amino acids of the EGF-like domain corresponding to asparagine 22, asparagine 27, histidine 29, tryptophan 30, and glutamine 40 of pro-urokinase as set forth in SEQ ID NO: 6 or 7, respectively, are substituted with other amino acids, thereby reducing or eliminating the binding ability to the human urokinase receptor in the fusion protein.

[22] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin and a catalytic domain of pro-urokinase having an amino acid sequence corresponding to the amino acid sequence of amino acids 156 to 411 of pro-urokinase set forth in SEQ ID NO: 6 or 7, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region of an antibody that binds to insoluble fibrin, The fusion protein further comprises an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from the 7th to the 43rd amino acids of prourokinase set forth in SEQ ID NO: 6 or 7 between the antibody or antigen-binding fragment thereof and the catalytic domain, in which the amino acids of the 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 prourokinase set forth in SEQ ID NO: 6 or 7 are substituted with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing or eliminating the ability to bind to the human urokinase receptor.

[23] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of prourokinase having an amino acid sequence corresponding to the 7th to 43rd amino acids of prourokinase set forth in SEQ ID NO: 6 or 7, a kringle domain of prourokinase corresponding to the 49th to 131st amino acids, and a catalytic domain of prourokinase having an amino acid sequence corresponding to the 156th to 411th amino acid sequence, wherein the antibody or antigen-binding fragment and the catalytic domain of prourokinase are linked directly or via a linker, wherein the linker is a non-cleavable linker, and the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region of an antibody that binds to insoluble fibrin, The fusion protein further comprises an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence of the 7th to 43rd amino acids of prourokinase set forth in SEQ ID NO: 6 or 7 between the antibody or antigen-binding fragment thereof and the catalytic domain, in which the amino acids of the 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 prourokinase set forth in SEQ ID NO: 6 or 7 have been substituted with other amino acids, thereby reducing or eliminating the ability to bind to the human urokinase receptor.

[24] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of prourokinase having an amino acid sequence corresponding to the 7th to 43rd amino acids of prourokinase set forth in SEQ ID NO: 6 or 7, a kringle domain of prourokinase corresponding to the 49th to 131st amino acids, and a catalytic domain of prourokinase having an amino acid sequence corresponding to the 156th to 411th amino acid sequence, wherein the antibody or antigen-binding fragment and the catalytic domain of prourokinase are linked directly or via a linker, wherein the linker is a non-cleavable linker, and the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region of an antibody that binds to insoluble fibrin, The fusion protein further comprises an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from the 7th to the 43rd amino acids of prourokinase set forth in SEQ ID NO: 6 or 7 between the antibody or antigen-binding fragment thereof and the catalytic domain, in which the amino acids of the 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 prourokinase set forth in SEQ ID NO: 6 or 7 are substituted with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing or eliminating the ability to bind to the human urokinase receptor.

[25] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of prourokinase having an amino acid sequence corresponding to the 7th to 43rd amino acids of prourokinase set forth in SEQ ID NO: 6 or 7, a kringle domain of prourokinase corresponding to the 49th to 131st amino acids, and a catalytic domain of prourokinase having an amino acid sequence corresponding to the 156th to 411th amino acids, wherein the antibody or antigen-binding fragment and the catalytic domain of prourokinase are linked directly or via a linker, wherein the linker is a non-cleavable linker, and the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region of an antibody that binds to insoluble fibrin, A fusion protein which further comprises an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence of amino acids 7 to 43 of prourokinase set forth in SEQ ID NO: 6 or 7 between the antibody or antigen-binding fragment thereof and the catalytic domain, wherein the amino acid sequence of the EGF-like domain corresponding to the amino acid sequence from asparagine at position 22 to isoleucine at position 28 of prourokinase set forth in SEQ ID NO: 6 or 7 is deleted (the deleted site may have an insertion of 1 to 7 amino acids (a peptide that has no binding affinity to the human urokinase receptor, for example, a peptide of 2 amino acids, for example, GG))), and which has reduced or preferably lost binding ability to the human urokinase receptor.

[26] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of prourokinase having an amino acid sequence corresponding to the 7th to 43rd amino acids of prourokinase set forth in SEQ ID NO: 6 or 7, a kringle domain of prourokinase corresponding to the 49th to 131st amino acids, and a catalytic domain of prourokinase having an amino acid sequence corresponding to the 156th to 411th amino acid sequence, wherein the antibody or antigen-binding fragment and the catalytic domain of prourokinase are linked directly or via a linker, wherein the linker is a non-cleavable linker, and the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region of an antibody that binds to insoluble fibrin, A fusion protein which further comprises an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence of the 7th to 43rd amino acids of prourokinase set forth in SEQ ID NO: 6 or 7 between the antibody or antigen-binding fragment thereof and the catalytic domain, wherein the amino acid sequence of the EGF-like domain corresponding to the amino acid sequence from asparagine at position 22 to isoleucine at position 28 of prourokinase set forth in SEQ ID NO: 6 or 7 is deleted, and 1 to 7 amino acids (a peptide that has no binding affinity to the human urokinase receptor, for example, a peptide of 2 amino acids, for example, GG) are inserted at the deleted site, thereby reducing or preferably eliminating the binding ability to the human urokinase receptor.

[0008] [31A] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin and a catalytic domain of pro-urokinase having an amino acid sequence corresponding to the amino acid sequence of amino acids 156 to 411 of pro-urokinase as set forth in SEQ ID NO: 6 or 7, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker, wherein the linker is a non-cleavable linker, the antibody or antigen-binding fragment comprises the heavy chain variable region and the light chain variable region of an antibody that binds to insoluble fibrin, and wherein the phenylalanine in the catalytic domain corresponding to the phenylalanine at position 157 of pro-urokinase as set forth in SEQ ID NO: 6 or 7 is substituted with another amino acid. [31B] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of pro-urokinase having an amino acid sequence corresponding to amino acids 7 to 43 of pro-urokinase set forth in SEQ ID NO: 6 or 7, and a catalytic domain of pro-urokinase having an amino acid sequence corresponding to amino acids 156 to 411, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker, wherein the linker is a non-cleavable linker, the antibody or antigen-binding fragment comprises the heavy chain variable region and the light chain variable region of an antibody that binds to insoluble fibrin, and wherein the phenylalanine in the catalytic domain corresponding to phenylalanine at position 157 of pro-urokinase set forth in SEQ ID NO: 6 or 7 has been substituted with another amino acid.[31C] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, a kringle domain of pro-urokinase corresponding to amino acids 49 to 131 of pro-urokinase set forth in SEQ ID NO: 6 or 7, and a catalytic domain of pro-urokinase having an amino acid sequence corresponding to amino acids 156 to 411, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker, wherein the linker is a non-cleavable linker, and the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region of an antibody that binds to insoluble fibrin, and wherein the phenylalanine in the catalytic domain corresponding to phenylalanine at position 157 of pro-urokinase set forth in SEQ ID NO: 6 or 7 is substituted with another amino acid. [31D] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of pro-urokinase having an amino acid sequence corresponding to amino acids 7 to 43 of pro-urokinase set forth in SEQ ID NO: 6 or 7, a kringle domain of pro-urokinase corresponding to amino acids 49 to 131, and a catalytic domain of pro-urokinase having an amino acid sequence corresponding to amino acids 156 to 411, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker, wherein the linker is a non-cleavable linker, and the antibody or antigen-binding fragment comprises the heavy chain variable region and the light chain variable region of an antibody that binds to insoluble fibrin, and wherein the phenylalanine in the catalytic domain corresponding to phenylalanine at position 157 of pro-urokinase set forth in SEQ ID NO: 6 or 7 is substituted with another amino acid.

[32] The fusion protein according to

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

[33] The fusion protein according to

[31] or

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

[34] The fusion protein according to any one of

[31] to

[33] above, further comprising an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence of amino acids 7 to 43 of prourokinase set forth in SEQ ID NO: 6 or 7 between the antibody or antigen-binding fragment thereof and the catalytic domain, in which the amino acids of the 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 prourokinase set forth in SEQ ID NO: 6 or 7, respectively, are substituted with other amino acids, thereby reducing or eliminating the binding ability to the human urokinase receptor.

[35] The fusion protein according to any one of

[31] to

[34] above, further comprising an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence of amino acids 7 to 43 of pro-urokinase set forth in SEQ ID NO: 6 or 7 between the antibody or antigen-binding fragment thereof and the catalytic domain, in which the amino acids of the 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 pro-urokinase set forth in SEQ ID NO: 6 or 7 are substituted with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing or eliminating the binding ability to the human urokinase receptor.

[36] The fusion protein according to any one of

[31] to

[34] above, further comprising an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence of amino acids 7 to 43 of prourokinase as set forth in SEQ ID NO: 6 or 7 between the antibody or antigen-binding fragment thereof and the catalytic domain, wherein the amino acid sequence of the EGF-like domain corresponding to the amino acid sequence from asparagine at position 22 to isoleucine at position 28 of prourokinase as set forth in SEQ ID NO: 6 or 7 is deleted (for example, by inserting 1 to 7 amino acids (a peptide that has at least no binding affinity to human urokinase receptor, preferably a peptide that has no binding affinity to human urokinase receptor and mouse urokinase receptor, for example, a peptide of 2 amino acids, for example, GG) at the deleted site), thereby reducing or preferably eliminating the binding ability to the human urokinase receptor.

[37] A pharmaceutical preparation comprising the fusion protein according to any one of

[31] to

[36] above.

[38] A method for producing the fusion protein according to any one of

[31] to

[36] above, comprising culturing a protein-producing cell having an expressible nucleic acid encoding the fusion protein to express the fusion protein, and purifying the fusion protein from the resulting culture.

[39] The method according to

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

[40] The method according to

[38] or

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

[0009]

[41] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of prourokinase having an amino acid sequence corresponding to the 7th to 43rd amino acids of prourokinase set forth in SEQ ID NO: 6 or 7, a kringle domain of prourokinase corresponding to the 49th to 131st amino acids, and a catalytic domain of prourokinase having an amino acid sequence corresponding to the 156th to 411th amino acids, wherein the antibody or antigen-binding fragment and the catalytic domain of prourokinase are linked directly or via a linker, wherein the linker is a non-cleavable linker, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region of an antibody that binds to insoluble fibrin, and wherein the phenylalanine in the catalytic domain corresponding to the 157th phenylalanine of prourokinase set forth in SEQ ID NO: 6 or 7 has been substituted with another amino acid, A fusion protein in which the amino acids in the 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 pro-urokinase set forth in SEQ ID NO: 6 or 7 have been substituted with other amino acids, thereby reducing or eliminating the ability to bind to the human urokinase receptor.

[42] The fusion protein of

[41] above, in which the phenylalanine in the catalytic domain corresponding to phenylalanine at position 157 has been substituted with an amino acid other than tyrosine.

[43] The fusion protein of

[41] above, in which the phenylalanine in the catalytic domain corresponding to phenylalanine at position 157 has been substituted with isoleucine, glutamic acid, aspartic acid, or cysteine.

[44] The fusion protein of

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

[45] The fusion protein of

[41] above, in which the amino acids in the 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 pro-urokinase set forth in SEQ ID NO: 6 or 7 are substituted with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing or eliminating the binding ability to the human urokinase receptor.

[46] The fusion protein of

[42] above, in which the amino acids in the 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 pro-urokinase set forth in SEQ ID NO: 6 or 7 are substituted with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing or eliminating the binding ability to the human urokinase receptor.

[47] The fusion protein of

[43] above, in which the amino acids in the 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 pro-urokinase set forth in SEQ ID NO: 6 or 7 are substituted with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing or eliminating the binding ability to the human urokinase receptor.

[48] The fusion protein of

[44] above, in which the amino acids in the 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 pro-urokinase set forth in SEQ ID NO: 6 or 7 are substituted with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing or eliminating the binding ability to the human urokinase receptor.

[0010]

[61] The fusion protein according to any one of the above, wherein the antibody or antigen-binding fragment thereof specifically binds to insoluble fibrin (particularly, binds weaker to one or more, preferably all, selected from the group consisting of fibrinogen, soluble fibrin, and fibrin degradation products than to insoluble fibrin, and particularly preferably does not bind significantly).

[62] The fusion protein according to any one of the above, wherein the antibody or antigen-binding fragment thereof binds to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 8.

[63] The fusion protein according to any one of the above, wherein the antibody or antigen-binding fragment thereof has, as CDRs, heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3 corresponding to those of an antibody selected from the group consisting of the 102-10 antibody, the 34-105 antibody, and the Fib-0355 antibody disclosed in WO2014 / 133093.

[64] The fusion protein according to any one of the above, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 10, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 11, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 12, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 13, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 14, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 15.

[65] The fusion protein according to any one of the above, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 17.

[66] The fusion protein according to any one of the above, comprising: a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 18, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 19, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 20; and a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 21, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 22, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 23.

[67] The fusion protein according to any one of the above, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 25.

[68] The fusion protein according to any one of the above, wherein the antibody or antigen-binding fragment thereof competes with a reference antibody for binding to insoluble fibrin, the reference antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 17.

[69] The fusion protein according to any one of the above, wherein the antibody or antigen-binding fragment thereof competes with a reference antibody for binding to insoluble fibrin, the reference antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 25.

[70] The fusion protein according to any one of the above, wherein the fusion protein has the amino acid sequence of SEQ ID NO: 107.

[71] The fusion protein according to any one of the above, wherein the antibody or antigen-binding fragment thereof competes with a reference antibody for binding to insoluble fibrin, the reference antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 107. -6 M or less, 10 -7 M or less, or 10 -8 The fusion protein according to any one of the above, having a binding dissociation constant (KD) of not more than M.

[72] The fusion protein according to any one of the above, wherein the antibody or antigen-binding fragment thereof is a Fab fragment or a Fab′ fragment {preferably, the fusion protein may be one in which the C-terminus of the heavy chain of the fragment is linked to the N-terminus of pro-urokinase or a portion thereof, or a mutant thereof}.

[0011]

[81] The fusion protein according to any one of the above, having any one or more, preferably two or more, more preferably three or more, and even more preferably all of the properties selected from the group consisting of (i) binding specificity for insoluble fibrin, (ii) binding affinity for insoluble fibrin, (iii) resistance to inhibition by plasminogen activator inhibitor (PAI-1), and (iv) low inducibility of matrix metalloproteinase (MMP) activity in thrombus or periembolic tissue.

[82] The fusion protein according to any one of the above, linked to an imaging agent.

[0012]

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

[92] The pharmaceutical preparation described in

[91] above for use in treating thromboembolism or diseases accompanied by extravascular or intravascular accumulation of insoluble fibrin (e.g., diseases accompanied by extravascular accumulation of insoluble fibrin, preferably Alzheimer's dementia).

[93] The pharmaceutical preparation described in

[91] above for use in treating a condition or disease for which lysis of fibrin clots is an appropriate therapeutic method.

[94] The pharmaceutical preparation described in

[91] above for use in treating cerebrovascular disorders and cardiovascular disorders accompanied by thrombus or embolism formation.

[95] The pharmaceutical preparation described in

[91] above for use in treating a condition or symptom associated with thrombus or embolism formation in cerebrovascular disorders and cardiovascular disorders.

[96] The pharmaceutical preparation described in

[91] above for use in treating a condition or symptom of cerebrovascular disorders and cardiovascular disorders.

[97] The pharmaceutical preparation described in

[91] above for use in treating ischemic cerebrovascular disorders.

[98] The pharmaceutical preparation according to

[91] above, for use in treating either cerebral infarction or transient ischemic attack.

[99] The pharmaceutical preparation according to

[91] above, for use in treating ischemic cardiovascular disease.

[100] The pharmaceutical preparation according to

[91] above, for use in treating either angina pectoris or myocardial infarction.

[101] The pharmaceutical preparation according to any of

[91] to

[100] above, for use in treating a disease in the acute phase.

[102] The pharmaceutical preparation according to

[91] above, for use in dissolving fibrin clots in a subject having a fibrin clot.

[103] A pharmaceutical preparation for use in detecting thrombi or embolism in the body, comprising the fusion protein according to

[82] above.

[104] The pharmaceutical preparation according to

[103] above, for use simultaneously with the medical uses specified in any of

[92] to

[102] above.

[0013] The present invention provides the following:

[1001] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin and a catalytic domain of pro-urokinase having an amino acid sequence corresponding to the amino acid sequence from amino acids 156 to 411 of pro-urokinase set forth in SEQ ID NO: 6 or 7, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker, wherein the antibody or antigen-binding fragment comprises the heavy-chain variable region and the light-chain variable region of an antibody that binds to insoluble fibrin, and wherein the arginine in the catalytic domain corresponding to the arginine at position 156 of pro-urokinase set forth in SEQ ID NO: 6 or 7 has been substituted with another amino acid {wherein the fusion protein preferably has significantly increased stability against degradation in human plasma compared to AMU1114, and may have, for example, a mutation of R156I, R156E, or R156D}.

[1002] The fusion protein according to

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

[1003] The fusion protein according to

[1001] or

[1002] above, wherein the binding ability to one or more urokinase receptors selected from the group consisting of human urokinase receptor and mouse urokinase receptor is reduced compared to the fusion protein having the amino acid sequence set forth in SEQ ID NO: 1.

[1004] The fusion protein according to any of

[1001] to

[1003] above, wherein the fusion protein does not exhibit significant binding to one or more urokinase receptors selected from the group consisting of human urokinase receptor and mouse urokinase receptor.

[1005] The fusion protein according to any one of

[1001] to

[1003] above, further comprising an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence of amino acids 7 to 43 of prourokinase set forth in SEQ ID NO: 6 or 7 between the antibody or antigen-binding fragment thereof and the catalytic domain, in which the amino acids of the 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 prourokinase set forth in SEQ ID NO: 6 or 7, respectively, are substituted 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, further comprising an EGF-like domain between the antibody or antigen-binding fragment thereof and the catalytic domain, which has an amino acid sequence corresponding to the amino acid sequence from the 7th to the 43rd amino acids of pro-urokinase as set forth in SEQ ID NO: 6 or 7, in which the amino acids of the 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 pro-urokinase as set forth in SEQ ID NO: 6 or 7 are substituted with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing or eliminating the binding ability to the human urokinase receptor.

[1007] The fusion protein according to any one of

[1001] to

[1003] above, further comprising an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence of amino acids 7 to 43 of pro-urokinase set forth in SEQ ID NO: 6 or 7 between the antibody or antigen-binding fragment thereof and the catalytic domain, wherein the amino acids in the EGF-like domain corresponding to the amino acids from asparagine at position 22 to isoleucine at position 28 of pro-urokinase set forth in SEQ ID NO: 6 or 7 are deleted (the deletion site may optionally have an insertion of 1 to 7 amino acids (a peptide that does not have binding affinity to the human urokinase receptor, for example, a peptide of 2 amino acids, for example, GG)).

[1008] The fusion protein according to any of

[1001] to

[1007] above, wherein the amino acid in the catalytic domain corresponding to phenylalanine at position 157 of pro-urokinase as set forth in SEQ ID NO: 6 or 7 is phenylalanine.

[1009] A pharmaceutical preparation comprising the fusion protein according to any of

[1001] to

[1008] above.

[1010] The pharmaceutical preparation according to

[1009] above, for use in a method for dissolving a thrombus or an embolus in a subject with an acute or chronic thrombotic or embolic disease.

[1011] The pharmaceutical preparation according to

[1009] or

[1010] above, for use in a method for dissolving a thrombus or an embolus in a subject with an acute infarct disease.

[1012] A method for producing the fusion protein according to any of

[1001] to

[1008] above, comprising culturing a protein-producing cell capable of expressing a nucleic acid encoding the fusion protein to express the fusion protein, and purifying the fusion protein from the resulting culture.

[1013] The method according to

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

[1014] The method according to

[1012] above or

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

[1015] A method for modifying a modified fusion protein, the fusion protein comprising: an antibody or antigen-binding fragment thereof that binds to insoluble fibrin; and a catalytic domain of pro-urokinase having an amino acid sequence corresponding to amino acids 156 to 411 of pro-urokinase as set forth in SEQ ID NO: 6 or 7, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker; the antibody or antigen-binding fragment comprising the heavy chain variable region and the light chain variable region of an antibody that binds to insoluble fibrin; and introducing a mutation (e.g., a point mutation such as R156I, R156E, or R156D) into the amino acid corresponding to R156 of the fusion protein, thereby conferring superior stability and superior urokinase activity when bound to insoluble fibrin, compared to the fusion protein before modification. A modified fusion protein obtained by the method.

[1016] The method of

[1015] above, which comprises further introducing a mutation (e.g., a point mutation such as F157Y) into the amino acid corresponding to F157 of the fusion protein, and a modified fusion protein obtained by the method.

[1017] The method of

[1015] or

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

[1018] The fusion protein of

[1007] above, wherein the insoluble fibrin-binding antibody or antigen-binding fragment thereof is an insoluble fibrin-binding antibody or antigen-binding Fab or Fab' thereof, and the concatenated heavy chain variable region, EGF-like domain, and catalytic domain consists of the amino acid sequence of SEQ ID NO: 64.

[1019] The fusion protein according to

[1007] above, which can exhibit more rapid urokinase activity than a control protein under the conditions of no pretreatment with plasmin, 5 μg / mL plasminogen, a plasmin substrate (e.g., 15 μL / well of Testteam S PLG, manufactured by Sekisui Medical), and 37°C, wherein the control protein is a complex of a conjugate comprising a heavy chain variable region, an EGF-like domain, and a catalytic domain, the conjugate having the amino acid sequence of SEQ ID NO: 63, and a light chain having the amino acid sequence of SEQ ID NO: 17.

[0014]

[1021] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, and a catalytic domain of pro-urokinase having an amino acid sequence corresponding to the amino acid sequence of amino acids 156 to 411 of pro-urokinase as set forth in SEQ ID NO: 6 or 7, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker, wherein the antibody or antigen-binding fragment comprises the heavy chain variable region and the light chain variable region of an antibody that binds to insoluble fibrin, and further comprises an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence of amino acids 7 to 43 of pro-urokinase as set forth in SEQ ID NO: 6 or 7 between the antibody or antigen-binding fragment portion and the catalytic domain portion, wherein the amino acids of the EGF-like domain corresponding to asparagine 22, asparagine 27, histidine 29, tryptophan 30, and glutamine 40 of pro-urokinase as set forth in SEQ ID NO: 6 or 7, respectively, are substituted 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 pro-urokinase having an amino acid sequence corresponding to the amino acid sequence of amino acids 156 to 411 of pro-urokinase set forth in SEQ ID NO: 6 or 7, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region of an antibody that binds to insoluble fibrin, The fusion protein further comprises an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from the 7th to the 43rd amino acids of prourokinase set forth in SEQ ID NO: 6 or 7 between the antibody or antigen-binding fragment thereof and the catalytic domain, in which the amino acids of the 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 prourokinase set forth in SEQ ID NO: 6 or 7 are substituted with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing or eliminating the ability to bind to the human urokinase receptor.

[1023] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of pro-urokinase having an amino acid sequence corresponding to amino acids 7 to 43 of pro-urokinase set forth in SEQ ID NO: 6 or 7, a kringle domain of pro-urokinase corresponding to amino acids 49 to 131, and a catalytic domain of pro-urokinase having an amino acid sequence corresponding to amino acids 156 to 411, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker, wherein the linker is a non-cleavable linker, and the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region of an antibody that binds to insoluble fibrin, The fusion protein further comprises an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence of the 7th to 43rd amino acids of prourokinase set forth in SEQ ID NO: 6 or 7 between the antibody or antigen-binding fragment thereof and the catalytic domain, in which the amino acids of the 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 prourokinase set forth in SEQ ID NO: 6 or 7 have been substituted with other amino acids, thereby reducing or eliminating the ability to bind 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 pro-urokinase having an amino acid sequence corresponding to amino acids 7 to 43 of pro-urokinase set forth in SEQ ID NO: 6 or 7, a kringle domain of pro-urokinase corresponding to amino acids 49 to 131, and a catalytic domain of pro-urokinase having an amino acid sequence corresponding to amino acids 156 to 411, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker, wherein the linker is a non-cleavable linker, and the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region of an antibody that binds to insoluble fibrin, The fusion protein further comprises an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence from the 7th to the 43rd amino acids of prourokinase set forth in SEQ ID NO: 6 or 7 between the antibody or antigen-binding fragment thereof and the catalytic domain, in which the amino acids of the 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 prourokinase set forth in SEQ ID NO: 6 or 7 are substituted with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing or eliminating the ability to bind to the human urokinase receptor.

[1025] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of pro-urokinase having an amino acid sequence corresponding to amino acids 7 to 43 of pro-urokinase set forth in SEQ ID NO: 6 or 7, a kringle domain of pro-urokinase corresponding to amino acids 49 to 131, and a catalytic domain of pro-urokinase having an amino acid sequence corresponding to amino acids 156 to 411, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker, wherein the linker is a non-cleavable linker, and the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region of an antibody that binds to insoluble fibrin, A fusion protein which further comprises an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence of amino acids 7 to 43 of prourokinase set forth in SEQ ID NO: 6 or 7 between the antibody or antigen-binding fragment thereof and the catalytic domain, wherein the amino acid sequence of the EGF-like domain corresponding to the amino acid sequence from asparagine at position 22 to isoleucine at position 28 of prourokinase set forth in SEQ ID NO: 6 or 7 is deleted (the deleted site may have an insertion of 1 to 7 amino acids (a peptide that has no binding affinity to the human urokinase receptor, for example, a peptide of 2 amino acids, for example, GG))), and which has reduced or preferably lost 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 pro-urokinase having an amino acid sequence corresponding to amino acids 7 to 43 of pro-urokinase set forth in SEQ ID NO: 6 or 7, a kringle domain of pro-urokinase corresponding to amino acids 49 to 131, and a catalytic domain of pro-urokinase having an amino acid sequence corresponding to amino acids 156 to 411, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker, wherein the linker is a non-cleavable linker, and the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region of an antibody that binds to insoluble fibrin, A fusion protein which further comprises an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence of the 7th to 43rd amino acids of prourokinase set forth in SEQ ID NO: 6 or 7 between the antibody or antigen-binding fragment thereof and the catalytic domain, wherein the amino acid sequence of the EGF-like domain corresponding to the amino acid sequence from asparagine at position 22 to isoleucine at position 28 of prourokinase set forth in SEQ ID NO: 6 or 7 is deleted, and 1 to 7 amino acids (a peptide that has no binding affinity to the human urokinase receptor, for example, a peptide of 2 amino acids, for example, GG) are inserted at the deleted site, thereby reducing or preferably eliminating the binding ability to the human urokinase receptor.

[0015] [1031A] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin and a catalytic domain of pro-urokinase having an amino acid sequence corresponding to the amino acid sequence of amino acids 156 to 411 of pro-urokinase as set forth in SEQ ID NO: 6 or 7, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker, wherein the linker is a non-cleavable linker, the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region of an antibody that binds to insoluble fibrin, and wherein the arginine in the catalytic domain corresponding to the arginine at position 156 of pro-urokinase as set forth in SEQ ID NO: 6 or 7 has been substituted with another amino acid. [1031B] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of pro-urokinase having an amino acid sequence corresponding to amino acids 7 to 43 of pro-urokinase set forth in SEQ ID NO: 6 or 7, and a catalytic domain of pro-urokinase having an amino acid sequence corresponding to amino acids 156 to 411, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker, wherein the linker is a non-cleavable linker, the antibody or antigen-binding fragment comprises the heavy chain variable region and the light chain variable region of an antibody that binds to insoluble fibrin, and wherein the phenylalanine in the catalytic domain corresponding to arginine at position 156 of pro-urokinase set forth in SEQ ID NO: 6 or 7 has been substituted with another amino acid.[1031C] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, a kringle domain of pro-urokinase corresponding to amino acids 49 to 131 of pro-urokinase set forth in SEQ ID NO: 6 or 7, and a catalytic domain of pro-urokinase having an amino acid sequence corresponding to amino acids 156 to 411, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker, wherein the linker is a non-cleavable linker, and the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region of an antibody that binds to insoluble fibrin, and wherein the arginine in the catalytic domain corresponding to arginine at position 156 of pro-urokinase set forth in SEQ ID NO: 6 or 7 has been substituted with another amino acid. [1031D] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of pro-urokinase having an amino acid sequence corresponding to amino acids 7 to 43 of pro-urokinase set forth in SEQ ID NO: 6 or 7, a kringle domain of pro-urokinase corresponding to amino acids 49 to 131, and a catalytic domain of pro-urokinase having an amino acid sequence corresponding to amino acids 156 to 411, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker, wherein the linker is a non-cleavable linker, and the antibody or antigen-binding fragment comprises the heavy chain variable region and the light chain variable region of an antibody that binds to insoluble fibrin, and wherein arginine in the catalytic domain corresponding to arginine at position 156 of pro-urokinase set forth in SEQ ID NO: 6 or 7 is substituted with another amino acid.

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

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

[1033] The fusion protein according to

[1031] or

[1032] above, which has a reduced ability to bind to one or more urokinase receptors selected from the group consisting of human urokinase receptor and mouse urokinase receptor, as compared to the fusion protein having the amino acid sequence set forth in SEQ ID NO: 1.

[1034] The fusion protein according to any of

[1031] to

[1033] above, which does not show significant binding to one or more urokinase receptors selected from the group consisting of human urokinase receptor and mouse urokinase receptor.

[1035] The fusion protein according to any one of

[1031] to

[1034] above, further comprising an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence of amino acids 7 to 43 of prourokinase set forth in SEQ ID NO: 6 or 7 between the antibody or antigen-binding fragment thereof and the catalytic domain, in which the amino acids of the 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 prourokinase set forth in SEQ ID NO: 6 or 7, respectively, are substituted 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, further comprising an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence of amino acids 7 to 43 of pro-urokinase set forth in SEQ ID NO: 6 or 7 between the antibody or antigen-binding fragment thereof and the catalytic domain, in which the amino acids of the 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 pro-urokinase set forth in SEQ ID NO: 6 or 7 are substituted with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing or eliminating the binding ability to the human urokinase receptor.

[1037] The fusion protein according to any one of

[1031] to

[1034] above, further comprising an EGF-like domain having an amino acid sequence corresponding to the amino acid sequence of amino acids 7 to 43 of prourokinase as set forth in SEQ ID NO: 6 or 7 between the antibody or antigen-binding fragment thereof and the catalytic domain, wherein the amino acid sequence of the EGF-like domain corresponding to the amino acid sequence from asparagine at position 22 to isoleucine at position 28 of prourokinase as set forth in SEQ ID NO: 6 or 7 is deleted (for example, by inserting 1 to 7 amino acids (a peptide that has at least no binding affinity to human urokinase receptor, preferably a peptide that has no binding affinity to human urokinase receptor and mouse urokinase receptor, e.g., a peptide of 2 amino acids, e.g., GG) at the deletion site), thereby reducing or preferably eliminating the binding ability to the human urokinase receptor.

[1038] The fusion protein according to any of

[1031] to

[1037] above, wherein the amino acid in the catalytic domain corresponding to phenylalanine at position 157 of pro-urokinase as set forth in SEQ ID NO: 6 or 7 is phenylalanine.

[1039] A pharmaceutical preparation comprising the fusion protein according to any of

[1031] to

[1036] above.

[1040] The pharmaceutical preparation according to

[1039] above, for use in a method for dissolving thrombi or emboli in a subject with acute infarction disease or chronic thrombotic or embolic disease.

[1041] The pharmaceutical preparation according to

[1039] or

[1040] above, for use in a method for dissolving thrombi or emboli in a subject with acute infarction disease.

[1042] A method for producing the fusion protein according to any of

[1031] to

[1036] above, comprising culturing a protein-producing cell capable of expressing a nucleic acid encoding the fusion protein to express the fusion protein, and purifying the fusion protein from the resulting culture.

[1043] The method according to

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

[1044] The method according to

[1042] above or

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

[0016]

[1051] A fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, an EGF-like domain of pro-urokinase having an amino acid sequence corresponding to amino acids 7 to 43 of pro-urokinase set forth in SEQ ID NO: 6 or 7, a kringle domain of pro-urokinase corresponding to amino acids 49 to 131, and a catalytic domain of pro-urokinase having an amino acid sequence corresponding to amino acids 156 to 411, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker, wherein the linker is a non-cleavable linker, the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region of an antibody that binds to insoluble fibrin, and wherein arginine in the catalytic domain corresponding to arginine at position 156 of pro-urokinase set forth in SEQ ID NO: 6 or 7 has been substituted with another amino acid, A fusion protein in which the amino acids in the 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 prourokinase set forth in SEQ ID NO: 6 or 7 have been substituted with other amino acids, thereby reducing or eliminating the ability to bind to the human urokinase receptor.

[1052] The fusion protein of

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

[1053] The fusion protein of

[1051] above, in which the amino acids in the 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 prourokinase set forth in SEQ ID NO: 6 or 7 have been substituted with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing or eliminating the ability to bind to the human urokinase receptor.

[1054] The above-mentioned

[1052] fusion protein, in which the amino acids in the 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 pro-urokinase set forth in SEQ ID NO: 6 or 7 are substituted with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, and the binding ability to the human urokinase receptor is reduced or lost.

[0017]

[1061] The fusion protein according to any one of the above, wherein the antibody or antigen-binding fragment thereof specifically binds to insoluble fibrin (particularly, binds weaker to one or more, preferably all, selected from the group consisting of fibrinogen, soluble fibrin, and fibrin degradation products than to insoluble fibrin, and particularly preferably does not bind significantly).

[1062] The fusion protein according to any one of the above, wherein the antibody or antigen-binding fragment thereof binds to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 8.

[1063] The fusion protein according to any one of the above, wherein the antibody or antigen-binding fragment thereof has, as CDRs, heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3 corresponding to those of an antibody selected from the group consisting of the 102-10 antibody, the 34-105 antibody, and the Fib-0355 antibody disclosed in WO2014 / 133093.

[1064] The fusion protein according to any one of the above, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 10, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 11, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 12, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 13, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 14, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 15.

[1065] The fusion protein according to any one of the above, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 17.

[1066] The fusion protein of any of the above, comprising a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 18, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 19, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 20; and a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 21, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 22, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 23.

[1067] The fusion protein of any of the above, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region having the amino acid sequence of SEQ ID NO: 25.

[1068] The fusion protein of any of the above, wherein the antibody or antigen-binding fragment thereof competes with a reference antibody for binding to insoluble fibrin, the reference antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16 and a light chain variable region having the amino acid sequence of SEQ ID NO: 17.

[1069] The fusion protein of any of the above, wherein the antibody or antigen-binding fragment thereof competes with a reference antibody for binding to insoluble fibrin, the reference antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 24 and a light chain variable region having the amino acid sequence of SEQ ID NO: 25.

[1070] The fusion protein of any of the above, having the amino acid sequence of SEQ ID NO: 107.

[1071] An antibody or antigen-binding fragment thereof, -6 M or less, 10 -7 M or less, or 10 -8 The fusion protein according to any one of the above, having a binding dissociation constant (KD) of not more than M.

[1072] The fusion protein according to any one of the above, wherein the antibody or antigen-binding fragment thereof is a Fab fragment or a Fab' fragment {preferably, the fusion protein may be one in which the C-terminus of the heavy chain of the fragment is linked to the N-terminus of pro-urokinase or a portion thereof, or a mutant thereof}.

[0018]

[1081] The fusion protein according to any one of the above, having any one or more, preferably two or more, more preferably three or more, and even more preferably all of the properties selected from the group consisting of (i) binding specificity for insoluble fibrin, (ii) binding affinity for insoluble fibrin, (iii) resistance to inhibition by plasminogen activator inhibitor (PAI-1), and (iv) low induction of matrix metalloproteinase (MMP) activity in tissues surrounding a thrombus or embolism.

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

[0019]

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

[1092] The pharmaceutical formulation or pharmaceutical composition described in

[1091] above for use in treating a thromboembolic disease (e.g., a thrombotic disease, an embolic disease, thrombosis, or embolism).

[1093] The pharmaceutical formulation or pharmaceutical composition described in

[1091] above for use in treating a condition or disease for which lysis of a fibrin clot is a suitable therapeutic modality.

[1094] The pharmaceutical formulation or pharmaceutical composition described in

[1091] above for use in treating cerebrovascular disorders and cardiovascular disorders accompanied by thrombus or embolism formation.

[1095] The pharmaceutical formulation or pharmaceutical composition described in

[1091] above for use in treating a condition or symptom associated with thrombus or embolism formation in cerebrovascular disorders and cardiovascular disorders.

[1096] The pharmaceutical formulation or pharmaceutical composition described in

[1091] above for use in treating a condition or symptom of cerebrovascular disorders and cardiovascular disorders.

[1097] The pharmaceutical preparation or pharmaceutical composition according to

[1091] above, for use in treating an ischemic cerebrovascular disorder.

[1098] The pharmaceutical preparation or pharmaceutical composition according to

[1091] above, for use in treating either cerebral infarction or transient ischemic attack.

[1099] The pharmaceutical preparation or pharmaceutical composition according to

[1091] above, for use in treating an ischemic cardiovascular disorder.

[1100] The pharmaceutical preparation or pharmaceutical composition according to

[1091] above, for use in treating either angina pectoris or myocardial infarction.

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

[1091] to

[1100] above, for use in treating a disease in the acute or chronic phase.

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

[1091] to

[1100] above, for use in treating a disease in the acute phase.

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

[1091] to

[1100] above, for use in treating a disease in the chronic phase.

[1104] The pharmaceutical preparation or pharmaceutical composition according to

[1091] above, for use in dissolving a fibrin clot in a subject having a fibrin clot.

[1105] A pharmaceutical preparation or composition for use in detecting thrombus or embolism in the body, comprising the fusion protein of

[1082] above.

[1106] The pharmaceutical preparation or composition of

[1105] above for simultaneous use in any of the medical uses specified in

[1092] to

[1104] above.

[0020]

[1201] A protein complex of a heavy chain having the amino acid sequence set forth in SEQ ID NO: 64 and a light chain having the amino acid sequence set forth in SEQ ID NO: 17.

[1202] A pharmaceutical preparation or pharmaceutical composition comprising the protein complex set forth in

[1201] above.

[1203] The pharmaceutical preparation or pharmaceutical composition set forth in

[1202] above for use in treating thrombotic diseases, embolic diseases, thrombosis, or embolism.

[1204] The pharmaceutical preparation or pharmaceutical composition set forth in

[1202] above for use in treating a condition or disease for which lysis of fibrin clots is an appropriate therapeutic method.

[1205] The pharmaceutical preparation or pharmaceutical composition set forth in

[1202] above for use in treating cerebrovascular disorders and cardiovascular disorders accompanied by thrombus or embolism formation.

[1206] The pharmaceutical preparation or pharmaceutical composition set forth in

[1202] above for use in treating a condition or symptom associated with thrombus or embolism formation in cerebrovascular disorders and cardiovascular disorders.

[1207] The pharmaceutical formulation or pharmaceutical composition according to

[1202] above, for use in treating the conditions or symptoms of cerebrovascular disorders and cardiovascular disorders.

[1208] The pharmaceutical formulation or pharmaceutical composition according to

[1202] above, for use in treating ischemic cerebrovascular disorders.

[1209] The pharmaceutical formulation or pharmaceutical composition according to

[1202] above, for use in treating either cerebral infarction or transient ischemic attack.

[1210] The pharmaceutical formulation or pharmaceutical composition according to

[1202] above, for use in treating ischemic cardiovascular disorders.

[1211] The pharmaceutical formulation or pharmaceutical composition according to

[1202] above, for use in treating either angina pectoris or myocardial infarction.

[1212] The pharmaceutical formulation or pharmaceutical composition according to any of

[1202] to

[1211] above, for use in treating a disease in the acute or chronic phase.

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

[1202] to

[1211] above, for use in treating a disease in the acute phase.

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

[1202] to

[1211] above, for use in treating a disease in the chronic phase.

[1215] The pharmaceutical preparation or pharmaceutical composition according to

[1202] above, for use in dissolving a fibrin clot in a subject having a fibrin clot.

[1216] A pharmaceutical formulation or composition for use in detecting thrombi or embolism in the body, comprising the fusion protein of

[1201] linked to an imaging agent.

[1217] The pharmaceutical formulation or composition of

[1216] for simultaneous use in any of the medical uses specified in

[1203] to

[1215] .

[0021]

[1301] A method for treating a thrombotic disease, embolic disease, infarction, or thromboembolism (preferably, acute thrombotic disease, embolic disease, infarction, or thromboembolism) in a subject in need thereof, comprising administering a therapeutically effective amount of an active ingredient to the subject, wherein the active ingredient comprises a fusion protein comprising an insoluble fibrin-binding domain (preferably, an antigen-binding fragment of an antibody that binds to insoluble fibrin and does not substantially bind to fibrinogen, particularly a Fab fragment), and the EGF domain, kringle domain, and catalytic domain of pro-urokinase (wherein the amino acid region corresponding to amino acid positions 22 to 28 in SEQ ID NO: 1 is deleted, and the deleted site may contain an inactive amino acid sequence of several amino acids), preferably a protein comprising the amino acid sequence of SEQ ID NO: 1 or 67.

[1302] The method according to

[1301] above, wherein the thrombotic disease, embolic disease, infarction, or thromboembolism is acute infarction or thromboembolism.

[1303] The method of

[1301] or

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

[1304] The method of any of

[1301] to

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

[1305] The method of any of

[1301] to

[1304] above, wherein a higher thrombus patency rate or embolism patency rate is achieved than alteplase or preferably netecteplase when administered in equimolar amounts.

[1306] The method of any of

[1301] to

[1305] above, wherein a lower treatment-related bleeding frequency is achieved than alteplase and netecteplase when administered in equimolar amounts.

[1307] The method according to any one of

[1301] to

[1306] above, wherein the administration of an equimolar amount of the protein shows (i) a higher thrombus patency rate or embolism patency rate and (ii) a lower incidence of treatment-related bleeding than alteplase or preferably necteteplase.

[1308] A protein having the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 67, or a composition comprising the protein, for use in the methods according to

[1301] to

[1307] above.

[1309] Any of the methods described above, wherein the protein further comprises a protein having the amino acid sequence set forth in SEQ ID NO: 17 or 25.

[1310] A protein having the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 67, or a composition comprising said protein, for use in the method described in

[1309] above.

[1311] The composition described in

[1310] above, further comprising a protein having the amino acid sequence set forth in SEQ ID NO: 17 or 25.

[0022]

[1321] A method for treating a disease accompanied by extravascular or intravascular accumulation or deposition of insoluble fibrin in a subject in need thereof (e.g., a disease accompanied by extravascular accumulation or deposition of insoluble fibrin, preferably Alzheimer's dementia), comprising administering a therapeutically effective amount of an active ingredient to the subject, wherein the active ingredient comprises a fusion protein comprising an insoluble fibrin-binding domain (preferably an antigen-binding fragment of an antibody that binds to insoluble fibrin and does not substantially bind to fibrinogen, particularly a Fab fragment), and the EGF domain, kringle domain, and catalytic domain of pro-urokinase (wherein the amino acid region corresponding to amino acid positions 22 to 28 in SEQ ID NO: 1 is deleted, and the deleted site may contain an inactive amino acid sequence of several amino acids), preferably a protein comprising the amino acid sequence of SEQ ID NO: 1 or 67.

[1322] The method according to the above-mentioned

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

[1323] The method according to

[1321] or

[1322] above, wherein the subject suffers from accumulation of β-amyloid.

[1324] The method according to any of

[1321] to

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

[1325] A composition for use in the method according to any of

[1321] to

[1324] above, comprising a protein having the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 67.

[1326] The composition according to

[1325] above, further comprising a protein having the amino acid sequence set forth in SEQ ID NO: 17 or 25.

[0023] FIG. 1 shows the reactivity of AMU1114 variants (particularly those with a modified F157 in the urokinase moiety) toward substrates. The vertical axis represents absorbance at 405 nm. FIG. 2 shows the reactivity of AMU1114 variants (particularly those with a modified F157 in the urokinase moiety) toward substrates after plasmin treatment. The vertical axis represents absorbance at 405 nm. FIG. 3A shows the results of SDS-PAGE of AMU1114 variants (particularly those with a modified F157 in the urokinase moiety) after incubation in PBS at 37°C for 0 hours. FIG. 3B shows the results of SDS-PAGE of AMU1114 variants (particularly those with a modified F157 in the urokinase moiety) after incubation in PBS at 37°C for 24 hours. Figure 3C shows the results of SDS-PAGE of AMU1114 variants (particularly variants of F157 in the urokinase moiety) after 120 hours of incubation in PBS at 37°C. Figure 4 shows the reactivity of AMU1114 variants with substrates after plasmin treatment. The vertical axis represents absorbance at 405 nm. Figure 5 shows the reactivity of AMU1114 variants with substrates for human urokinase receptor or mouse urokinase receptor. The vertical axis represents absorbance at 405 nm. Figure 6A shows gel filtration chromatograms of urokinase expressed in Chinese hamster ovary (CHO) cells in the presence of various concentrations of fetal bovine serum (FBS) and purified. Figure 6B shows the results of electrophoresis of urokinase expressed in Chinese hamster ovary (CHO) cells in the presence of various concentrations of fetal bovine serum (FBS) and purified. Figure 7A shows the results of electrophoresis of AMU1114 expressed in Chinese hamster ovary (CHO) cells in the presence or absence of 20% FBS and purified. Figure 7B shows the results of electrophoresis of AMU1114 variants expressed in Chinese hamster ovary (CHO) cells in the absence of serum (serum-free medium) and purified. Figure 8A shows a gel filtration chromatogram of AMU1114 expressed in Chinese hamster ovary (CHO) cells in the absence of serum (serum-free medium) and purified. Figure 8B shows a gel filtration chromatogram of AMU1114 variants expressed in Chinese hamster ovary (CHO) cells in the absence of serum (serum-free medium) and purified. Figure 9 shows an alignment of AMU1114 and its variants.Figure 10 shows the reactivity of AMU1114 variants to substrates after plasmin treatment. The vertical axis represents absorbance at 405 nm. Figure 11 shows the reactivity of AMU1114 variants to substrates for human or mouse urokinase receptors. The vertical axis represents absorbance at 405 nm. Figure 12A shows histological staining images of lungs from thrombin thrombosis model mice administered PBS, AMU1114 (Δ22-28, F157E) (0.08 mg / kg body weight or 0.32 mg / kg body weight; designated Amu 0.08 and Amu 0.32, respectively), or tenecteplase (0.05 mg / kg body weight or 0.2 mg / kg body weight; designated Tene 0.05 and Tene 0.2, respectively). Figure 12B shows a graph of the total signal area corresponding to thrombi. Figure 13 shows the alignment of the heavy chain amino acid sequences of AMU1114, AMU1114 (Δ22-28, F157E), AMU1114 (Δ22-28, R156E), AMU1114 (Δ22-28, R156I, F157Y), and AMU1114 (Δ22-28, P155G, R156I, F157Y) with AMU1114 and each of their variants. Each of the variants lacks the amino acid sequence of the EGF-like domain corresponding to the amino acid sequence from asparagine at position 22 to isoleucine at position 28 of pro-urokinase set forth in SEQ ID NO: 6 or 7 in urokinase, with two glycine residues inserted in place of the deleted site. Figure 14 shows the stability results for AMU1114 and its various variants. Figure 15 shows the resistance to plasmin of AMU1114 and its respective variants. Figure 16A shows the urokinase activity of AMU1114 and its respective variants with plasmin pretreatment. Figure 16B shows the urokinase activity of AMU1114 and its respective variants without plasmin pretreatment. Figure 17 shows the urokinase activity of AMU1114 and AMU1114(Δ22-28, F157E) with and without plasmin pretreatment. Figure 18 shows the urokinase activity of AMU1114 and various variants with and without plasmin pretreatment.In Figure 18, AMU1114(Δ22-28, R156E), AMU1114(Δ22-28, R156I, F157Y), and AMU1114(Δ22-28, P155G, R156I, F157Y) are designated as variants A, B, and C, respectively. Figure 19 shows the degradation stability of AMU1114 and AMU1114(Δ22-28, F157E). Figure 20 shows the degradation stability of AMU1114 and various variants. In Figure 20, AMU1114 (Δ22-28, R156E), AMU1114 (Δ22-28, R156I, F157Y), and AMU1114 (Δ22-28, P155G, R156I, F157Y) are designated as variants A, B, and C, respectively. Figure 21 shows a thrombus formation experiment for the preparation of a rat cerebral infarction model. Figure 22 shows the process of placing the prepared thrombus at the bifurcation of the left middle cerebral artery using a catheter. Panel A is a frontal image, and panel B is a lateral image. Figure 23 shows angiography results before and after placement of the thrombus. After placement of the thrombus, blood flow to the middle major artery (MCA) ceased, and no image of the MCA was observed. Figure 24 shows the results of angiography taken 1 hour, 3 hours, 6 hours, and 24 hours after intravenous injection of the thrombolytic agent. The upper panel shows the negative control, and the lower panel shows the AMU1114 (Δ22-28) administration group. The lower panel shows that the thrombus was dissolved one hour after intravenous injection. Figure 25 shows the results of an experiment in which AMU1114 (Δ22-28) was administered to a mouse model of Alzheimer's dementia. Specific Description of the Invention

[0024] As used herein, a "subject" is a mammal, and may be, for example, a dog, cat, cow, horse, pig, primate (e.g., monkey, gorilla, orangutan, bonobo, chimpanzee, and human), and may be, for example, a human.

[0025] As used herein, "fibrin" refers to an insoluble clot formed by cleavage of the C-terminus of the three polypeptide chains (Aα chain, Bβ chain, and γ chain) that constitute fibrinogen. Herein, fibrin may also be referred to as insoluble fibrin. More specifically, cleavage of the C-terminus of fibrinogen results in a state called fibrin monomer, which polymerizes with calcium to form a poorly soluble fibrin polymer. The fibrin polymers are cross-linked by the action of factor XIII to form stabilized fibrin (insoluble fibrin or fibrin gel, as defined herein). Insoluble fibrin is degraded by plasmin. Plasmin is contained in plasma as its precursor, plasminogen. When plasminogen is degraded at the Arg-Val peptide by a plasminogen activator (e.g., urokinase, tissue plasminogen activator, and streptokinase), plasmin is produced. Plasmin can be inhibited, limiting its action, by proteins called plasmin inhibitors.

[0026] The fibrinogen Aα chain may be a human fibrinogen Aα chain, including the human fibrinogen Aα chain having the amino acid sequence registered under GenBank accession number AAI01936.1 and the human fibrinogen Aα chain having an amino acid sequence corresponding to the amino acid sequence.

[0027] The fibrinogen Bβ chain may be a human fibrinogen Bβ chain, including a human fibrinogen β chain having an amino acid sequence registered under NCBI reference number NP_005132.2 and a human fibrinogen β chain having an amino acid sequence corresponding to the amino acid sequence.

[0028] The fibrinogen gamma chain may be a human fibrinogen gamma chain, including a human fibrinogen gamma chain having an amino acid sequence registered under GenBank accession number AAH07044.1 and a human fibrinogen gamma chain having an amino acid sequence corresponding to the amino acid sequence.

[0029] As used herein, "urokinase" refers to a type of serine protease 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 peptide bond between Lys158 and Ile159 is cleaved to form active urokinase (the cleaved chains are linked to each other by disulfide bonds). Urokinase has three domains: an EGF-like domain, a kringle domain, and a catalytic domain. Furthermore, cleavage between Lys135 and Lys136 converts urokinase to low-molecular-weight urokinase. The amino acid numbering of prourokinase is determined based on the amino acid sequence of prourokinase after cleavage of the signal peptide (e.g., the amino acid sequence set forth in SEQ ID NO: 6). Examples of prourokinase include human prourokinase. Examples of human pro-urokinase include human pro-urokinase having the amino acid sequence registered under GenBank Registration No. AAA61253.1 and human pro-urokinase having an amino acid sequence corresponding to said amino acid sequence. In the amino acid sequence registered under GenBank Registration No. AAA61253.1, the sequence of amino acids 1 to 20 corresponds to the signal peptide. The EGF-like domain may have a pro-urokinase sequence corresponding to amino acids 7 to 43 of the amino acid sequence set forth in SEQ ID NO: 6, the kringle domain may have a pro-urokinase sequence corresponding to amino acids 49 to 131 of the amino acid sequence set forth in SEQ ID NO: 6, and the catalytic domain may have a pro-urokinase sequence corresponding to amino acids 136 to 403 of the amino acid sequence set forth in SEQ ID NO: 6. Various pro-urokinases can be used as long as they are activated as two-chain prokinases by plasmin or kallikrein and have the ability to convert plasminogen into plasmin by degrading it.

[0030] As used herein, the term "corresponding" refers to a second amino acid sequence having a sequence corresponding to a 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 cleave plasminogen and convert it to plasmin).

[0031] As used herein, "antibody" refers to an immunoglobulin. The antibody may be of various isotypes, such as IgG. The antibody is preferably a monoclonal antibody. The antibody may be a human chimeric antibody, a humanized antibody, or a human antibody. A human chimeric antibody may be produced by replacing the constant region of a non-human antibody with the constant region of a human antibody. A humanized antibody may be produced by replacing the six CDRs of a human antibody with the six corresponding CDRs of a non-human antibody. A human antibody can be produced using an animal (e.g., a mouse) in which at least the heavy chain variable region of an immunoglobulin has been replaced with the corresponding region of a human locus. 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. As used herein, the antibody is preferably a humanized antibody. As used herein, the antibody is preferably a human antibody. When produced intracellularly, an antibody has a signal peptide, but when secreted extracellularly, the signal peptide is cleaved. Therefore, when administered as a pharmaceutical, a signal peptide is not required for antibodies.

[0032] As used herein, "CDR" refers to a complementarity determining region present in the heavy chain variable region and light chain variable region of an antibody. There are three CDRs in each of the heavy and light chain variable regions, and they are designated CDR1, CDR2, and CDR3 from the N-terminus. CDRs can be determined, for example, according to the numbering system 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.).

[0033] As used herein, "antigen-binding fragment of an antibody" refers to a fragment of an antibody that maintains its ability to bind to an antigen. Examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv, scFv (single-chain Fv), diabody, 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 this. Antigen-binding fragments of other antibodies can also be prepared by methods well known to those skilled in the art. Such antigen-binding fragments of antibodies can be used in the present invention.

[0034] As used herein, the term "fusion protein" refers to a protein formed by linking peptides derived from two or more different proteins via peptide bonds. A fusion protein of a first peptide and a second peptide may contain a third peptide and an additional peptide, or may contain only the first peptide and the second peptide, as long as the function of the invention is not significantly impaired. As used herein, a fusion protein of a first peptide and a second peptide may be a fusion protein containing the first peptide and the second peptide in this order or a fusion protein containing the peptides in a different order, as long as the function of the invention is not significantly impaired. Preferably, the fusion protein is a fusion protein containing the first peptide and the second peptide in this order. In a fusion protein, the peptides derived from two or more different proteins may be linked via a linker or without a linker, as long as the function of the invention is not significantly impaired. When a linker is used, the linker may be a flexible linker.

[0035] As used herein, "thrombotic disease" refers to a disease in which a blood clot (thrombus) clogs a blood vessel, obstructing blood flow and causing organ damage and other disorders. The term "thrombotic disease" is used in a broad sense herein to encompass embolic diseases in which a blood clot migrates from its original site and blocks a blood vessel through the bloodstream. Therefore, the term "thrombotic disease" is used to encompass thromboembolism. Thrombi and embolisms include those that partially obstruct blood flow (e.g., about 50% or more, about 60% or more, about 70% or more, about 80% or more, or about 90% or more), and those that completely obstruct blood flow (e.g., about 100%). As used herein, "infarction" refers to a condition in which tissue necrosis occurs as a result of blood vessel blockage by a thrombus or embolism.

[0036] <Modified Fusion Protein of the Present Invention> (A) The present invention provides a fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin and a catalytic domain of pro-urokinase having an amino acid sequence corresponding to the amino acid sequence of amino acids 136 to 403 of pro-urokinase as set forth in SEQ ID NO: 6, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker. In this case, in the amino acid sequence corresponding to the amino acid sequence of amino acids 136 to 403 of pro-urokinase as set forth in SEQ ID NO: 6, either or both (preferably both) of lysine 135 and lysine 136 are substituted with other amino acids, thereby suppressing cleavage of the catalytic domain from pro-urokinase. In a preferred embodiment, both of the amino acids corresponding to lysine 135 and lysine 136 of pro-urokinase as set forth in SEQ ID NO: 6 are substituted with glycine. Thus, in a particularly preferred embodiment, the pro-urokinase or a portion thereof in the fusion protein may have an amino acid sequence corresponding to the amino acid sequence of a mutant of pro-urokinase as set forth in SEQ ID NO: 7, or a portion thereof.

[0037] The pro-urokinase portion of the fusion protein of the present invention is inactive until it binds to insoluble fibrin, and upon binding to insoluble fibrin, the pro-urokinase portion is cleaved by nearby plasmin to generate and activate double-chain urokinase, which then cleaves nearby plasminogen to produce large amounts of plasmin, thereby effectively dissolving insoluble fibrin.

[0038] According to the present invention, the pro-urokinase in the fusion protein may further comprise a kringle domain. The pro-urokinase may further comprise an EGF-like domain and a kringle domain. In a preferred embodiment, the fusion protein comprises an EGF-like domain, a kringle domain, and a catalytic domain, in that order. In a more preferred embodiment, the fusion protein comprises a region of pro-urokinase corresponding to amino acids 7 to 403 of the amino acid sequence set forth in SEQ ID NO: 6 or 7. In a most preferred embodiment, the fusion protein may comprise a pro-urokinase portion having an amino acid sequence corresponding to the amino acid sequence set forth in SEQ ID NO: 6 or 7.

[0039] According to the present invention, the fusion protein comprises an antibody or antigen-binding fragment thereof that binds to insoluble fibrin, which may comprise the heavy chain variable region and the light chain variable region of an antibody or antigen-binding fragment thereof that binds to insoluble fibrin.

[0040] According to the present invention, the antibody or antigen-binding fragment thereof that binds to insoluble fibrin of 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 the Fab' fragment is linked to pro-urokinase or a portion thereof as described above. In one embodiment, the light chain variable region of the Fab fragment or the Fab' fragment is linked to pro-urokinase or a portion thereof as described above. The antigen-binding fragment may comprise a complex of the heavy chain variable region and the light chain variable region that are configured as separate peptides (e.g., in the case of a Fab fragment or a Fab' fragment), or may be a single fusion peptide in which the heavy chain variable region and the light chain variable region are linked (e.g., in the case of an scFv fragment).

[0041] As used herein, when referring to an antigen-binding fragment, the terms "heavy chain" and "light chain" indicate whether the fragment is derived from the "heavy chain" or "light chain" of the antibody from which it was derived, and do not indicate whether the molecular weight of the antigen-binding fragment is large or small.

[0042] In a preferred embodiment of the present invention, in the fusion protein, the arginine in the catalytic domain corresponding to arginine at position 156 of pro-urokinase set forth in SEQ ID NO: 6 is substituted 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 phenylalanine at position 157 of pro-urokinase set forth in SEQ ID NO: 6 is preferably phenylalanine, but may be substituted with another amino acid.

[0043] (B) Therefore, the present invention provides a fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin and a catalytic domain of pro-urokinase having an amino acid sequence corresponding to the amino acid sequence of amino acids 136 to 403 of pro-urokinase set forth in SEQ ID NO:7, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker, in which the arginine in the catalytic domain corresponding to the arginine at position 156 of pro-urokinase set forth in SEQ ID NO:7 is substituted with glutamic acid or aspartic acid (preferably glutamic acid). In this embodiment, the phenylalanine in the catalytic domain corresponding to the phenylalanine at position 157 of pro-urokinase set forth in SEQ ID NO:6 is preferably phenylalanine, but may be substituted with another amino acid.

[0044] (C) In a preferred embodiment, the present invention provides a fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin and a region of pro-urokinase having an amino acid sequence corresponding to the amino acid sequence from positions 7 to 403 of pro-urokinase as set forth in SEQ ID NO:7, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker, in which the arginine in the catalytic domain corresponding to the arginine at position 156 of pro-urokinase as set forth in SEQ ID NO:7 is substituted with glutamic acid or aspartic acid (preferably glutamic acid). In a more preferred embodiment, the region of pro-urokinase may comprise a region of pro-urokinase having an amino acid sequence corresponding to the amino acid sequence from positions 1 to 411 of pro-urokinase as set forth in SEQ ID NO:7. In this embodiment, the phenylalanine in the catalytic domain corresponding to the phenylalanine at position 157 of pro-urokinase as set forth in SEQ ID NO:6 is preferably phenylalanine, but may be substituted with another amino acid.

[0045] According to the present invention, the phenylalanine or arginine is substituted with various amino acids (e.g., amino acids other than tyrosine). This preferably improves the cleavage resistance of the fusion protein of the present invention to proteolytic enzymes (e.g., plasmin). In a preferred embodiment, the phenylalanine or arginine is substituted with alanine, lysine, cysteine, serine, isoleucine, glycine, glutamic acid, or aspartic acid. In a more preferred embodiment, the phenylalanine or arginine is substituted with isoleucine, glycine, glutamic acid, or aspartic acid. This can suppress the degradation and subsequent activation of the fusion protein during fusion protein expression, fusion protein purification, and / or fusion protein storage. In an even more preferred embodiment, the phenylalanine or arginine is substituted with glutamic acid or aspartic acid. This can suppress degradation and subsequent activation of the fusion protein during expression, purification, and / or storage. Furthermore, activation in the presence of plasmin can exhibit plasminogen degradation activity equivalent to that of a fusion protein without the phenylalanine or arginine substitution. Furthermore, when a fusion protein without the phenylalanine or arginine substitution is produced from a protein-expressing cell, fragments of the fusion protein are generated. However, by substituting an amino acid other than tyrosine for the phenylalanine or arginine in the fusion protein, preferably with glutamic acid or aspartic acid, the amount of fragmented fusion protein produced can be reduced or prevented from increasing, even when the protein-expressing cell expressing the fusion protein is cultured in a low-serum medium (e.g., containing 5% or less serum) or serum-free medium. Furthermore, when a fusion protein without the above-mentioned phenylalanine or arginine substitution is produced from protein-expressing cells, the amount of the fusion protein produced continues to increase for 5 to 7 days, but beyond this period the amount of the fusion protein produced does not increase and the proportion of the light chain in the produced protein increases.In contrast, the proportion of light chains in the protein produced can be reduced by substituting the phenylalanine or arginine in the fusion protein with an amino acid other than tyrosine, preferably with glutamic acid or aspartic acid. Furthermore, by substituting the phenylalanine or arginine in the fusion protein with an amino acid other than tyrosine, preferably with glutamic acid or aspartic acid, the production of the fusion protein continues to increase even after 7 days, thereby significantly increasing the production of the fusion protein (e.g., 3- to 5-fold) compared to the production of a fusion protein without the substitution. Reducing or avoiding the use of serum can be beneficial because it can reduce variations in protein production due to variations in serum quality and reduce culture costs.

[0046] Therefore, in the fusion protein, substitution of the arginine in the catalytic domain corresponding to arginine at position 156 of prourokinase set forth in SEQ ID NO:7 with glutamic acid or aspartic acid (preferably glutamic acid) substituted with another amino acid, particularly substitution with glutamic acid or aspartic acid, is important in terms of increased production yield, increased purity, increased urokinase activity in the presence of fibrin, and / or cost reduction. In this embodiment, the phenylalanine in the catalytic domain corresponding to phenylalanine at position 157 of prourokinase set forth in SEQ ID NO:6 is preferably phenylalanine, but may be substituted with another amino acid.

[0047] (D) In ​​a preferred embodiment of the present invention, there is provided a fusion protein comprising an antibody or antigen-binding fragment thereof that binds to insoluble fibrin and a region of pro-urokinase having an amino acid sequence corresponding to the amino acid sequence from positions 7 to 403 of pro-urokinase as set forth in SEQ ID NO:7, wherein the antibody or antigen-binding fragment and the catalytic domain of pro-urokinase are linked directly or via a linker, in which the arginine in the catalytic domain corresponding to the arginine at position 156 of pro-urokinase as set forth in SEQ ID NO:7 is substituted with another amino acid, glutamic acid or aspartic acid (preferably glutamic acid). In a preferred embodiment, the pro-urokinase region has an amino acid sequence corresponding to the amino acid sequence from positions 1 to 411 of pro-urokinase as set forth in SEQ ID NO:7. In this embodiment, the phenylalanine in the catalytic domain corresponding to the phenylalanine at position 157 of pro-urokinase as set forth in SEQ ID NO:6 is preferably phenylalanine, but may be substituted with another amino acid.

[0048] <Further Modified Fusion Protein of the Present Invention> According to the present invention, there is provided a modified fusion protein having a lower binding affinity to the urokinase receptor than the above-mentioned fusion protein.

[0049] Specifically, in the further variants of the present invention, the amino acid in the EGF-like domain corresponding to tryptophan at position 30 of pro-urokinase set forth in SEQ ID NO: 6 or 7 in the amino acid sequence of any of the above fusion proteins (for example, any of (A) to (D)) is substituted with another amino acid (particularly arginine), thereby reducing the binding affinity of the further variants of the present invention to the human urokinase receptor compared to the above fusion proteins.

[0050] Furthermore, in the further variants of the present invention, in the amino acid sequence of any of the above fusion proteins (for example, any of (A) to (D)), the amino acids in the 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 pro-urokinase set forth in SEQ ID NO: 6 or 7 are substituted with other amino acids, thereby making the further variants of the present invention have a lower binding affinity to the human urokinase receptor than the above fusion proteins.

[0051] In one embodiment, in the amino acid sequence of any of the above fusion proteins (e.g., any of (A) to (D)), the amino acids in the 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 pro-urokinase set forth in SEQ ID NO: 6 or 7 are substituted with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing the binding affinity of the further variant of the present invention to the human urokinase receptor compared to the above fusion protein.

[0052] In one embodiment, in the amino acid sequence of the fusion protein (A) of the present invention, the amino acids in the 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 pro-urokinase set forth in SEQ ID NO: 6 or 7 are substituted with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing the binding affinity of the further variant of the present invention to the human urokinase receptor compared to the fusion protein.

[0053] In one embodiment, in the further variant of the present invention, the amino acids in the 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 pro-urokinase set forth in SEQ ID NO: 6 or 7 in the amino acid sequence of the fusion protein (B) are substituted with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing the binding affinity of the further variant of the present invention to the human urokinase receptor compared to the fusion protein.

[0054] In one embodiment, in the further variant of the present invention, the amino acids in the 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 pro-urokinase set forth in SEQ ID NO: 6 or 7 in the amino acid sequence of the fusion protein (C) are substituted with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing the binding affinity of the further variant of the present invention to the human urokinase receptor compared to the fusion protein.

[0055] In one embodiment, in the further variant of the present invention, the amino acids in the 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 pro-urokinase set forth in SEQ ID NO: 6 or 7 in the amino acid sequence of the fusion protein (D) are substituted with tyrosine, serine, arginine, arginine, and glutamic acid, respectively, thereby reducing the binding affinity of the further variant of the present invention to the human urokinase receptor compared to the fusion protein.

[0056] In a preferred embodiment, in the amino acid sequence of any of the above fusion proteins (e.g., any of (A) to (D)), the amino acid sequence of the EGF-like domain corresponding to the amino acid sequence from asparagine 22 to isoleucine 28 of pro-urokinase as set forth in SEQ ID NO: 6 or 7 is deleted. This is thought to result in the mutant losing its ability to bind to the urokinase receptor. In a preferred embodiment, in the amino acid sequence of any of the above fusion proteins (e.g., any of (A) to (D)), the amino acid sequence of the EGF-like domain corresponding to the amino acid sequence from asparagine 22 to isoleucine 28 of pro-urokinase as set forth in SEQ ID NO: 6 or 7 is deleted, and 1 to 7 amino acids (a peptide that has no binding affinity to at least the human urokinase receptor, preferably a peptide that has no binding affinity to the human urokinase receptor and the mouse urokinase receptor, for example, a peptide of 2 to 7 amino acids, e.g., 2 amino acids, such as GG) are inserted at the deleted site. The insertion of an amino acid sequence can be performed to alleviate shortening of the protein length due to deletion. The inserted amino acid sequence does not need to exhibit any particular activity and can consist of or contain neutral amino acids such as G and S. As a result, the further variant of the present invention has significantly no binding affinity to human urokinase receptor (particularly human urokinase receptor and mouse urokinase receptor) or has a lower binding affinity than the above-mentioned fusion protein to the receptor, and preferably has significantly no binding affinity to human urokinase receptor and mouse urokinase receptor.

[0057] In a particularly preferred embodiment, the further variant of the present invention comprises an antibody that binds to insoluble fibrin (anti-insoluble fibrin antibody) or an antigen-binding fragment thereof, and a region of pro-urokinase corresponding to amino acids 7 to 403 of the amino acid sequence of SEQ ID NO:6 or 7, in which the amino acid sequence of the EGF-like domain corresponding to the amino acid sequence from asparagine at amino acid 22 to isoleucine at amino acid 28 of pro-urokinase of SEQ ID NO:6 or 7 is deleted, and the arginine in the catalytic domain corresponding to arginine at amino acid 156 of pro-urokinase of SEQ ID NO:7 is substituted with another amino acid, preferably glutamic acid or aspartic acid (more preferably glutamic acid). In a more preferred embodiment, the amino acid in the catalytic domain corresponding to phenylalanine at amino acid 157 of pro-urokinase of SEQ ID NO:6 is phenylalanine. Preferably, both amino acids 135 and 136 of pro-urokinase of SEQ ID NO:6 or 7 are substituted with glycine. In further variants of the present invention, one or more of the amino acids in the catalytic domain corresponding to amino acids 155 to 157 of pro-urokinase set forth in SEQ ID NO:7 (e.g., amino acid 156, amino acids 156 and 157, and amino acids 155 to 157) are substituted with another amino acid (preferably glycine at position 155, glutamic acid or aspartic acid (more preferably glutamic acid) at position 156, or tyrosine at position 157), and these variants preferably have superior degradation stability in human plasma compared to AMU1114. Representative examples of these variants include AMU1114(Δ22-28), AMU1114(Δ22-28, R156E), AMU1114(Δ22-28, R156I, F157Y), and AMU1114(Δ22-28, P155G, R156I, F157Y). Δ22-28 may lack amino acid positions 22 to 28 and instead contain several inactive amino acids (eg, GG) at those positions.Furthermore, these variants preferably express urokinase activity in human plasma earlier than AMU1114(Δ22-28,F157E) having the amino acid sequence of SEQ ID NO: 63 under conditions without pretreatment with plasmin (i.e., they are early-activating variants). These early-activating variants are preferably administered to patients with, for example, chronic thrombotic diseases, embolic diseases, thromboembolic diseases, or infarction diseases, or preferably acute thrombotic diseases, embolic diseases, thromboembolic diseases, or infarction diseases, and more preferably to patients with acute infarction diseases. In contrast, AMU1114(Δ22-28,F157E) is more preferably administered to patients with chronic thrombotic diseases or infarction diseases. The further variants of the present invention may have additional mutations (e.g., mutations, particularly point mutations), and may have sequence identity with AMU1114 of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, and have superior degradation stability in human plasma compared to AMU1114.

[0058] In a preferred embodiment, the antibody that binds to insoluble fibrin (anti-insoluble fibrin antibody) or antigen-binding fragment thereof is a humanized or human antibody or antigen-binding fragment thereof.

[0059] In a preferred embodiment, an antibody that binds to insoluble fibrin (anti-insoluble fibrin antibody) or an antigen-binding fragment thereof comprises a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 10, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 11, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 12, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 13, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 14, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 15.

[0060] In a preferred embodiment, an antibody that binds to insoluble fibrin (anti-insoluble fibrin antibody) or an antigen-binding fragment thereof comprises a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 18, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 19, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 20, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 21, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 22, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 23.

[0061] In a preferred embodiment, the antibody that binds to insoluble fibrin (anti-insoluble fibrin antibody) or its antigen-binding fragment comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 17.

[0062] In a preferred embodiment, the antibody that binds to insoluble fibrin (anti-insoluble fibrin antibody) or its antigen-binding fragment comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 25.

[0063] In a preferred embodiment, in a further variant of the present invention, the heavy chain has the amino acid sequence set forth in SEQ ID NO: 64, 65, or 66. In a preferred embodiment, in a further variant of the present invention, the light chain has the amino acid sequence set forth in SEQ ID NO: 17. In a preferred embodiment, in a further variant of the present invention, the heavy chain has the amino acid sequence set forth in SEQ ID NO: 64, 65, or 66, and the light chain has the amino acid sequence set forth in SEQ ID NO: 17. In a preferred embodiment, in a further variant of the present invention, the heavy chain has the amino acid sequence set forth in SEQ ID NO: 64, and the light chain has the amino acid sequence set forth in SEQ ID NO: 17. This variant can be used for the treatment of thrombotic diseases, embolic diseases, thromboembolic diseases, preferably acute infarction diseases, etc. In a preferred embodiment, the further variant of the present invention does not have a mutation that confers resistance to cleavage by plasmin. This variant can be used for the treatment of thrombotic diseases, embolic diseases, thromboembolic diseases, or infarction diseases, preferably chronic thrombotic diseases, more preferably acute thrombotic diseases, embolic diseases, thromboembolic diseases, preferably acute infarction diseases, etc. In a preferred embodiment, in a further variant of the present invention, the heavy chain has the amino acid sequence set forth in SEQ ID NO: 63, and the light chain has the amino acid sequence set forth in SEQ ID NO: 17. This variant can be used for the treatment of thrombotic diseases, embolic diseases, thromboembolic diseases, or infarction diseases, preferably chronic thrombotic diseases, more preferably acute thrombotic diseases, embolic diseases, thromboembolic diseases, preferably acute infarction diseases, etc.

[0064] In these variants, cleavage occurs between the amino acids corresponding to lysine 158 and isoleucine 159 in the amino acid sequence set forth in SEQ ID NO: 6 or 7, and it is preferred that the peptides before and after the cleavage site are linked by a disulfide bond (i.e., forming active urokinase).

[0065] The binding affinity to human urokinase receptor may be at least 2 / 3, 4 / 5, 6 / 7, 8 / 9, 10 / 10, 20 / 30, 40 / 50, 60 / 70, 80 / 90, or 100 / 10 compared to the fusion protein set forth in SEQ ID NO: 1. Binding affinity can be determined by ELISA assay, which measures the amount of fusion protein bound to a plate on which urokinase receptor has been immobilized, and can be calculated by subtracting the value (background) obtained from a negative control (e.g., phosphate-buffered saline (PBS)). In ELISA assays, the amount of fusion protein bound to the urokinase receptor can be detected using an enzyme-labeled antibody. The enzyme-labeled antibody can be an antibody that binds to the fusion protein (e.g., an anti-human heavy chain or light chain antibody), the enzyme can be, for example, horseradish peroxidase (HRP), and the substrate can be, for example, 3,3',5,5'-tetramethylbenzidine (TMB).

[0066] As a result, even if the fusion protein of the present invention leaks around a thrombus or embolism, it will not bind to the urokinase receptor in the surrounding tissue, inhibiting the production of MMP enzymes in the tissue and preventing tissue damage, which is thought to prevent the occurrence of undesirable side effects caused by binding to the urokinase receptor.

[0067] In a variant of the fusion protein of the present invention, the antibody or antigen-binding fragment thereof binds to insoluble fibrin. In a preferred embodiment, in a variant of the fusion protein of the present invention, the antibody or antigen-binding fragment thereof specifically binds to insoluble fibrin. Here, "specifically binds to insoluble fibrin" means that it binds to insoluble fibrin but binds weaker to one or more, preferably all, selected from the group consisting of fibrinogen, soluble fibrin, and fibrin degradation products than to insoluble fibrin, and particularly preferably does not bind significantly.

[0068] In the present invention, an antibody that binds to insoluble fibrin is an antibody that binds to insoluble fibrin with stronger affinity than to fibrinogen (i.e., an antibody that binds to insoluble fibrin with a lower dissociation constant K D An antibody that binds to insoluble fibrin with stronger affinity than to fibrinogen may be, for example, an antibody that binds to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 8 or 9 (see WO 2014 / 133093). The amino acid sequence set forth in SEQ ID NO: 8 corresponds to the amino acid sequence set forth in SEQ ID NO: 1 of WO 2014 / 133093, and the amino acid sequence set forth in SEQ ID NO: 9 corresponds to the amino acid sequence set forth in SEQ ID NO: 2 of WO 2014 / 133093. The peptide portion consisting of the amino acid sequence set forth in SEQ ID NO: 8 or 9 is not exposed on the protein surface in fibrinogen, but is exposed on the protein surface only in insoluble fibrin and is accessible to antibodies. Therefore, an antibody that binds to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 8 or 9 may have binding selectivity for insoluble fibrin.

[0069] <Antibody or antigen-binding fragment thereof> Examples of antibodies or antigen-binding fragments thereof that bind to insoluble fibrin with stronger affinity than to fibrinogen include antibodies specific to insoluble fibrin (for example, antibodies that bind to insoluble fibrin and do not substantially bind to fibrinogen), such as the 102-10 antibody (having a heavy chain variable region having heavy chain CDR1 to 3 set forth in SEQ ID NOS: 27 to 29, respectively, and a light chain variable region having light chain CDR1 to 3 set forth in SEQ ID NOS: 30 to 32, respectively) and the 34-105 antibody (having a heavy chain variable region having heavy chain CDR1 to 3 set forth in SEQ ID NOS: 31 to 32, respectively) disclosed in WO2014 / 133093. and Fib-0355 antibody (having a heavy chain variable region having heavy chain CDR1 to 3 set forth in SEQ ID NOS: 39 to 41, respectively, and a light chain variable region having light chain CDR1 to 3 set forth in SEQ ID NOS: 42 to 44, respectively), or an antigen-binding fragment thereof, which have, as corresponding CDRs, heavy chain CDR1 to 3 and light chain CDR1 to 3 of an antibody selected from the group consisting of Fib-0355 antibody (having a heavy chain variable region having heavy chain CDR1 to 3 set forth in SEQ ID NOS: 39 to 41, respectively, and a light chain variable region having light chain CDR1 to 3 set forth in SEQ ID NOS: 42 to 44, respectively).

[0070] Examples of antibodies or antigen-binding fragments thereof that bind to insoluble fibrin with stronger affinity than to fibrinogen also include antibodies or antigen-binding fragments thereof that have, as corresponding CDRs, heavy chain CDR1 to 3 and light chain CDR1 to 3 of an antibody selected from the group consisting of the 99 antibody (having a heavy chain variable region having heavy chain CDR1 to 3 set forth in SEQ ID NOs: 45 to 47, respectively, and a light chain variable region having light chain CDR1 to 3 set forth in SEQ ID NOs: 48 to 50, respectively), the 1101 antibody (having a heavy chain variable region having heavy chain CDR1 to 3 set forth in SEQ ID NOs: 51 to 53, respectively, and a light chain variable region having light chain CDR1 to 3 set forth in SEQ ID NOs: 54 to 56, respectively), and the 0211 antibody (having a heavy chain variable region having heavy chain CDR1 to 3 set forth in SEQ ID NOs: 57 to 59, respectively, and a light chain variable region having light chain CDR1 to 3 set forth in SEQ ID NOs: 60 to 62, respectively) disclosed in WO 2018 / 203517 . Antibodies or antigen-binding fragments thereof that bind to insoluble fibrin with stronger affinity than to fibrinogen include anti-insoluble fibrin antibodies that do not bind to fibrinogen.

[0071] Further examples of antibodies or antigen-binding fragments thereof that bind to insoluble fibrin with stronger affinity than to fibrinogen include antibodies or antigen-binding fragments thereof comprising a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 10, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 11, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 12, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 13, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 14, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 15. The antibody may be a human chimeric antibody or a humanized antibody. The present invention also provides humanized antibodies comprising the above-mentioned heavy chain variable region and light chain variable region.

[0072] An example of an antibody or antigen-binding fragment thereof that binds to insoluble fibrin with stronger affinity than to fibrinogen is an antibody or antigen-binding fragment thereof comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 16 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 17. In addition to the heavy chain variable region, the antibody may further comprise a heavy chain constant region or a portion thereof in a Fab fragment. The antibody may be a human chimeric antibody or a humanized antibody. The present invention also provides a humanized antibody having the above-described heavy chain variable region and light chain variable region.

[0073] Further examples of antibodies or antigen-binding fragments thereof that bind to insoluble fibrin with stronger affinity than to fibrinogen include antibodies or antigen-binding fragments thereof comprising a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 18, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 19, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 20, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 21, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 22, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 23. The antibody may be a human chimeric antibody or a humanized antibody. The present invention also provides humanized antibodies comprising the above-mentioned heavy chain variable region and light chain variable region.

[0074] An example of an antibody or antigen-binding fragment thereof that binds to insoluble fibrin with stronger affinity than to fibrinogen is an antibody or antigen-binding fragment thereof comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 24 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 25. In addition to the heavy chain variable region, the antibody may further comprise a heavy chain constant region or a portion thereof within a Fab fragment. The antibody may be a human chimeric antibody or a humanized antibody. The present invention also provides a humanized antibody comprising the above-described heavy chain variable region and light chain variable region.

[0075] The present invention provides the following: [1] An antibody or antigen-binding fragment thereof that binds to insoluble fibrin, comprising: (1) a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 74, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 75, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 76, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 77, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 78, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 79; or (2) a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 82, a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 83, and a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 84, and a light chain variable region comprising a light chain CDR1 having the amino acid sequence set forth in SEQ ID NO: 85, a light chain CDR2 having the amino acid sequence set forth in SEQ ID NO: 86, and a light chain CDR3 having the amino acid sequence set forth in SEQ ID NO: 87. [2] An insoluble fibrin antibody or an antigen-binding fragment thereof, comprising: (3) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 80 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 81; or (4) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 88 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 89.

[0076] According to the present invention, there can be provided an antibody that competes with the antibody described in (3) above for binding to insoluble fibrin, preferably antibodies that compete with each other, wherein the antibody is preferably a humanized antibody.

[0077] According to the present invention, there can be provided an antibody that competes with the antibody described in (4) above for binding to insoluble fibrin, preferably antibodies that compete with each other, wherein the antibody is preferably a humanized antibody.

[0078] According to the present invention, the antibody described in (1) above may be a humanized antibody and an insoluble fibrin-specific antibody. According to the present invention, the antibody described in (2) above may be a humanized antibody and an insoluble fibrin-specific antibody. According to the present invention, the antibody described in (3) above may be a humanized antibody and an insoluble fibrin-specific antibody. According to the present invention, the antibody described in (4) above may be a humanized antibody and an insoluble fibrin-specific antibody. According to the present invention, an antibody that competes with the antibody described in (3) above for binding to insoluble fibrin, preferably an antibody that competes with each other, may be a humanized antibody and an insoluble fibrin-specific antibody. According to the present invention, an antibody that competes with the antibody described in (4) above for binding to insoluble fibrin, preferably an antibody that competes with each other, may be a humanized antibody and an insoluble fibrin-specific antibody.

[0079] The antibody of the present invention and its antigen-binding fragment portion can bind to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 8. Here, the antibody of the present invention may be a humanized antibody.

[0080] In one aspect, a variant of the fusion protein of the present invention has the amino acid sequence set forth in SEQ ID NO: 1. In one aspect, a variant of the fusion protein of the present invention has the amino acid sequence set forth in SEQ ID NO: 2. In one aspect, a variant of the fusion protein of the present invention has the amino acid sequence set forth in SEQ ID NO: 3. In one aspect, a variant of the fusion protein of the present invention has the amino acid sequence set forth in SEQ ID NO: 26. In one aspect, a variant of the fusion protein of the present invention has the amino acid sequence set forth in SEQ ID NO: 4. In one aspect, a variant of the fusion protein of the present invention has the amino acid sequence set forth in SEQ ID NO: 5. In one aspect, a variant of the fusion protein of the present invention has the amino acid sequence set forth in SEQ ID NO: 63.

[0081] <Characteristics of the Modified Form> The modified form of the fusion protein of the present invention may have one or more, preferably two or more, more preferably three or more, and even more preferably all of the following characteristics selected from the group consisting of: (i) binding specificity for insoluble fibrin; (ii) binding affinity for insoluble fibrin; (iii) resistance to inhibition by plasminogen activator inhibitor (PAI-1); and (iv) low inducibility of matrix metalloproteinase (MMP) activity in perithrombus tissue. This may provide a safer and more effective treatment than previous tissue plasminogen activator (tPA) therapy. It may also provide a new technological innovation for thrombolytic therapy using urokinase. In particular, it may exhibit significant manufacturing advantages over the fusion protein disclosed in WO 2021 / 200922A and may reduce side effects.

[0082] <Method for producing a variant of the fusion protein of the present invention> The present invention provides a method for producing a variant of the fusion protein of the present invention. The variant of the fusion protein of the present invention can be produced from protein-producing cells. For example, the variant can be produced from protein-producing cells by transiently or preferably stably introducing a gene encoding the variant into the protein-producing cells and culturing the protein-producing cells under conditions suitable for producing the variant.

[0083] Thus, the present invention provides protein-producing cells expressibly carrying a gene encoding a variant of the fusion protein of the present invention. The protein-producing cells may be contained in a cell cryoprotectant solution or may be frozen. Thus, the present invention provides frozen stocks, as well as working cell banks, master cell banks, and research cell banks, containing protein-producing cells expressibly carrying a gene encoding a variant of the fusion protein of the present invention. The variant of the fusion protein of the present invention may be operably linked, in particular, to a regulatory sequence, thereby enabling its expression in the protein-producing cells.

[0084] According to the present invention, the fusion protein of the present invention can be prepared by a method well 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, E. coli, yeast, and mammalian cells), preferably mammalian cells (e.g., cells suitable for protein expression, e.g., mammalian cells suitable for protein expression, such as Chinese hamster ovary cells (CHO cells) and human cells such as 293 cells and their derivatives). Protein-producing cells also include CHO cells (e.g., ExpiCHO cells). TM Cell, ExpiCHO-S TM Cell, Free Style TM Expression can be carried out using, for example, an expression vector containing a nucleic acid encoding the fusion protein of the present invention operably linked to a promoter that can be driven in the expression cells. If the fusion protein requires a light chain, the light chain can be co-expressed in the expression cells.

[0085] The variants of the fusion proteins of the present invention can provide a solution to the problem of low expression levels of fusion proteins in protein-producing cells. With conventional fusion proteins, increasing the incubation time does not improve the production of the fusion protein, but rather increases the production of fragments. In contrast, the variants of the fusion proteins of the present invention increase their production levels by increasing the incubation time. Furthermore, their plasminogen degradation activity in the presence of plasmin can be equivalent to that of conventional fusion proteins.

[0086] In one aspect, a method for producing a fusion protein variant with excellent stability or a pharmaceutical composition containing the variant is provided, the method comprising introducing an F157E or F157D mutation into the fusion protein of the present invention, thereby conferring superior stability compared to the fusion protein before modification. In one aspect, a method for improving the stability of a fusion protein variant is provided, the method comprising introducing an F157E or F157D mutation into the fusion protein of the present invention, thereby conferring superior stability compared to the fusion protein before modification. The modification can be made to the DNA encoding the variant. Those skilled in the art will be able to introduce the desired modification into DNA and proteins as appropriate, referring to codons in animals such as humans.

[0087] The medium may be a serum-containing medium, a low-serum medium (e.g., a serum concentration of less than 5%), a serum-free medium, a xeno-free medium, or a chemically defined medium. From the viewpoint of preventing impurity contamination, a serum-free medium, a xeno-free medium, or a chemically defined medium is preferably used. The medium can be replaced as appropriate during culture. Furthermore, from the viewpoint of reducing production costs, a serum-free medium such as a serum-free medium, a xeno-free medium, or a chemically defined medium is preferably used. The modified fusion protein of the present invention (including the R156 modification) has suppressed fragmentation in the absence of serum, and therefore has an advantage in production compared to the unmodified form.

[0088] By adding a signal peptide to the fusion protein, the fusion protein can be produced extracellularly. Therefore, in this case, the cell culture supernatant can be collected and the fusion protein can be recovered from the supernatant. The fusion protein can be purified using various purification techniques well known to those skilled in the art, such as gel filtration and affinity purification. The fusion protein variants of the present invention (including the R156 variant) are resistant to fragmentation during purification, facilitating the management of purification conditions, including temperature and storage conditions. For example, while conventional fusion proteins required purification in the presence of L-arginine, the variants can be purified in the absence of L-arginine. Furthermore, due to their high storage stability, the formulation composition can be flexibly designed. In particular, the formulation does not need to contain L-arginine.

[0089] The resulting fusion protein variants can be immobilized on the bottom of a plate, activated with plasmin, and then washed away to assess their plasminogen degradation activity. For example, the activated mutants can be reacted with a substrate such as HD-valyl-L-leucyl-L-lysyl-p-nitroanilide dihydrochloride in the presence of plasminogen on the plate, and the color developed upon substrate degradation is determined by colorimetric quantification of p-nitroaniline, which is then detected.

[0090] <Compositions and Pharmaceutical Preparations> The present invention may provide compositions or pharmaceutical preparations comprising variants of the fusion proteins of the present invention. The compositions and pharmaceutical preparations may further comprise pharmaceutically acceptable additives in addition to the variants of the fusion proteins of the present invention. Examples of additives include pH buffers, isotonicity agents, salts, preservatives, and analgesics. Pharmaceutical preparations may be prepared for intravenous or intraarterial administration. In one aspect, the pharmaceutical preparations of the present invention may be lyophilized. In one aspect, the present invention provides a kit (i.e., a kit for preparation at the time of use) comprising the lyophilized pharmaceutical preparation and water for injection.

[0091] The pharmaceutical preparation can be used to dissolve thrombi formed in the body. Therefore, the pharmaceutical preparation can be used to treat thrombosis. The pharmaceutical preparation can also be used to treat conditions or diseases caused by thrombi. The pharmaceutical preparation can also be used to treat conditions or diseases in which dissolution of fibrin clots is an appropriate therapeutic method. The pharmaceutical preparation can also be used to treat cerebrovascular disorders and cardiovascular disorders accompanied by thrombus formation. For example, the pharmaceutical preparation can treat conditions or symptoms associated with thrombus formation in cerebrovascular disorders and cardiovascular disorders, or thereby treat conditions or symptoms of cerebrovascular disorders and cardiovascular disorders. Treatment includes therapeutic treatment and prophylactic treatment. Therapeutic treatment includes slowing the rate of deterioration of a condition or symptom, slowing deterioration, stopping deterioration, or ameliorating a condition or symptom, while prophylactic treatment includes preventing the onset of a condition or symptom or reducing the incidence. Cerebrovascular disorders include ischemic cerebrovascular disorders. Examples of ischemic cerebrovascular disorders include 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 cerebral blood vessel, and cerebral embolism is a condition in which a thrombus formed in a blood vessel other than the brain is carried to the brain and blocks a cerebral blood vessel. Cardiovascular disorders include ischemic heart disease. Ischemic heart disease includes angina pectoris and myocardial infarction. Pharmaceutical preparations can be preferably used in the acute phase of these diseases. This is because use in the acute phase can prevent tissue damage caused by the formation of thrombi 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 disease, it is recommended to administer a thrombolytic agent within 4.5 hours from the onset, and for ischemic cardiovascular disease, it is recommended to administer a thrombolytic agent within 6 hours from the onset. The pharmaceutical preparation of the present invention can be used in the same manner.

[0092] According to the present invention, the variants of the fusion proteins of the present invention are capable of lysing fibrin clots. In one embodiment, the fibrin clot is a thrombus.

[0093] <Imaging Agent> According to the present invention, a variant of the fusion protein of the present invention may be linked (including covalently) to an imaging agent. The variant and the imaging agent may be directly linked, with or without a linker. A variant linked to an imaging agent may enable imaging of fibrin clots (particularly thrombi) in vivo, and may be used, for example, to observe thrombi in vivo. A contrast agent may be used as the imaging agent. The contrast agent may be an imaging agent for magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), or the like. Thus, the present invention provides a formulation or composition for use in in vivo imaging, comprising a variant of the fusion protein of the present invention linked to an imaging agent.

[0094] According to the present invention, there is provided a method for treating thrombosis or dissolving a fibrin clot in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a variant of the fusion protein of the present invention.

[0095] According to the present invention, there is provided a pharmaceutical formulation comprising a therapeutically effective amount of a variant of the fusion protein of the present invention for use in a method for treating thrombosis or dissolving a fibrin clot in a subject in need thereof.

[0096] The present invention provides variants of the fusion proteins of the present invention for use in methods of treating thrombosis or dissolving fibrin clots in a subject in need thereof.

[0097] According to the present invention there is provided the use of a variant of the fusion protein of the present invention in the manufacture of a medicament for use in a method of treating thrombosis or a method of lysing a fibrin clot in a subject in need thereof.

[0098] According to the present invention, there is provided a method for treating a condition or symptom of cerebrovascular and cardiovascular disorders in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a variant of the fusion protein of the present invention.

[0099] According to the present invention, there is provided a pharmaceutical formulation comprising a variant of the fusion protein of the present invention for use in a method for treating cerebrovascular and cardiovascular conditions or symptoms in a subject in need thereof.

[0100] The present invention provides variants of the fusion proteins of the present invention for use in methods of treating cerebrovascular and cardiovascular conditions or symptoms in a subject in need thereof.

[0101] There is provided the use of a variant of the fusion protein of the invention in the manufacture of a medicament for use in treating a cerebrovascular or cardiovascular condition or symptom in a subject in need thereof.

[0102] Example 1: Preparation of a fusion protein that dissolves fibrin clots In WO 2021 / 200922A, a fusion protein (hereinafter referred to as "AMU1114") containing an antibody fragment that specifically binds to insoluble fibrin and a pro-urokinase mutant was synthesized. AMU1114 is a complex of a fusion protein (heavy chain) (SEQ ID NO: 1) in which the Fab region of an antibody and a pro-urokinase mutant are linked, and an antibody light chain (SEQ ID NO: 17).

[0103] When AMU1114 was expressed in Chinese hamster ovary cells (CHO cells), the expression level reached a plateau 5 to 7 days after the start of culture, and continued culture did not increase the expression level of AMU1114. More specifically, as shown in Figure 8A, the production amount of AMU1114 did not increase, and only the recovery amount of light chain (L chain) increased.

[0104] When culture conditions were examined, as shown in Figures 8A and 8B, expressing AMU1114 in the CHO cells in serum-free medium increased the yield of the light chain and decreased the yield of AMU1114, whereas expressing AMU1114 in the CHO cells in the presence of 20% fetal bovine serum (FBS) increased the yield of AMU1114 and decreased the yield of the light chain. Furthermore, when purifying AMU1114 from CHO cells, it was essential to purify it in the presence of L-arginine. The yield of AMU1114 recovered after purification was 25-35 mg / L.

[0105] Example 2: Preparation of a fusion protein variant In this example, a variant of AMU1114 was prepared. Specifically, a site-specific amino acid substitution mutation was introduced into F396 (corresponding to F157 in urokinase) in the heavy chain of AMU1114. The amino acid substitution mutation was introduced by PCR using a mutation introduction primer and DNA encoding AMU1114 as a template. PrimeStar HS DNA polymerase (Takara) was used as the PCR enzyme.

[0106] AMU1114 integrated into pcDNA3.4 (ThermFisher) and each PCR product 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 FastGene Gel / PCR Extraction Kit (Nippon Genetics). Competent High DH5α (Toyobo) was transformed and cultured on an LB medium (50 μg / mL) plate for 16 hours. Plasmids were extracted from the transformed E. coli using the FastGene Plasmid Mini Kit (Nippon Genetics) and sequenced. This confirmed that the mutations had been introduced as intended.

[0107] AMU1114 and various mutants were expressed in CHO cells. Specifically, AMU1114 and various mutants were transiently expressed using an ExpiCHO Expression System (Thermo Fisher). After 7 days of culture, the culture supernatant was collected. FBS was added to the medium to a concentration of 20%. The transiently expressed culture supernatant was added to a CaptoL (Cytiva) column equilibrated with PBS, washed with the same buffer, and then eluted with 100 mM glycine buffer, pH 3.0, to obtain the final product.

[0108] Each mutant was adjusted to 10 μg / mL in PBS and dispensed at 100 μL / well into a 96-well plate. The plate was then left to stand at room temperature for 90 minutes for immobilization. 300 μL / well of 1% BSA in TBS-T was dispensed into the plate and blocked by leaving the plate to stand at room temperature for 60 minutes. After washing the blocked plate with TBS-T, 150 μL / well of a reaction solution prepared with PBS containing 5 μg / mL plasminogen and 15 μL / well of substrate (Test Team S PLG, Sekisui Medical) was added. Absorbance at 405 nm and 505 nm was measured every 5 minutes for 2 hours.

[0109] The results are shown in Figure 1. As shown in Figure 1, all mutants except for the F157Y mutant showed reduced reactivity to the substrate, and some mutants (F157A, F157K, F157C, and F157S) showed significantly reduced reactivity to the substrate, with some mutants (F157I, F157G, F157D, and F157E) showing even greater reductions in reactivity to the substrate. This is thought to be because these mutants are resistant to plasmin.

[0110] Each variant was adjusted to 10 μg / mL with PBS, dispensed at 100 μL / well into a 96-well plate, and left to stand at room temperature for 90 minutes for immobilization. 300 μL / well of 1% BSA TBS-T was dispensed into the immobilized plate, and the plate was blocked by leaving it at room temperature for 60 minutes. Plasmin solution adjusted to 5 μg / mL was added to the blocked plate at 100 μL / well and allowed to react at 37°C for 60 minutes. After washing with TBS-T, 150 μL / well of a reaction solution prepared with PBS containing 5 μg / mL plasminogen and 15 μL / well of substrate (Test Team S PLG, Sekisui Medical) was added, and the absorbance at 405 nm and 505 nm was measured every 5 minutes for 2 hours.

[0111] The results are shown in Figure 2. As shown in Figure 2, when treated with plasmin, all mutants showed high reactivity to the substrate. In particular, the activity of the F157E and F157D mutants was equivalent to that of AMU1114.

[0112] Each purified F157 variant was adjusted to 1 mg / mL with PBS and incubated at 37°C. Samples taken after 0 hours (Figure 3A), 24 hours (Figure 3B), and 120 hours (Figure 3C) were analyzed by SDS-PAGE electrophoresis. In Figures 3A-3C, each variant is represented by the single-letter code for the amino acid at the F157 position. The F157D and F157E variants were shown to be particularly stable. The yields of the F157E and F157D variants were 3- to 5-fold higher than those of AMU1114.

[0113] Example 3: Binding ability to urokinase receptor In this example, the binding ability of AMU1114 to the human urokinase receptor was confirmed. After the binding ability was confirmed, N22Y, N27S, H29R, W30R, and Q40E mutations were further introduced into the F157D mutant and F157E mutant, 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).

[0114] Additionally, an AMU1114 mutant (N22G, K23G, Y24_I28del, F157E) was created by site-directed mutagenesis. 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 set forth in SEQ ID NO: 63. A multiple alignment of the amino acid sequences of the created mutants is shown in Figure 9. The calculated yield was 100 mg / L.

[0115] The urokinase receptors used were a mutant of human urokinase having the amino acid sequence set forth in SEQ ID NO: 6 and a mouse urokinase receptor having the amino acid sequence set forth in SEQ ID NO: 7. A 6xHis tag was added to the C-terminus of each urokinase, followed by affinity purification.

[0116] Specifically, the PCR product of pcDNA3.3 (ThermFisher) and urokinase receptor was reacted with EcoRI (Takara) and XhoI (Takara) for 2 hours according to the manufacturer's protocol, followed by 1% agarose gel electrophoresis and purification using FastGene Gel / PCR Extraction Kit (Nippon Genetics). The vector (pcDNA3.3) and insert were reacted for 30 minutes using Ligation High (Takara). Competent High DH5α (Toyobo) was transformed and cultured on an LB medium (50 μg / mL) plate for 16 hours. Plasmids were extracted from transformed E. coli using a FastGene Plasmid Mini Kit (Nippon Genetics) and sequenced. Urokinase mutants were transiently expressed using an ExpiCHO Expression System (Thermo Fisher). Cultures were performed for 12 days, and the culture supernatant was collected. The transiently expressed culture supernatant was dialyzed against 50 mM Tris-HCl pH 8.5, 300 mM NaCl, loaded onto a Ni column (Cytiva) equilibrated with the same buffer, washed with the same buffer, and eluted with PBS containing 400 mM imidazole to obtain the final product.

[0117] The mutants were purified. The AMU1114 (N22Y, N27S, H29R, W30R, Q40E, F157D) mutant, AMU1114 (N22Y, N27S, H29R, W30R, Q40E, F157E) mutant, and AMU1114 (Δ22-28, F157E) mutant were transiently expressed in serum-free medium using only an ExpiCHO Expression System (Thermo Fisher). Culture was performed for 12 days, and the culture supernatant was collected. The transiently expressed culture supernatant was loaded onto CaptoL (Cytiva) equilibrated with PBS, washed with the same buffer, and then eluted with 100 mM glycine buffer, pH 3.0. The eluted sample was purified by gel filtration using Superdex 200 pg (Cytiva) to obtain the final product.

[0118] Each variant was adjusted to 10 μg / mL with PBS, dispensed at 100 μL / well into a 96-well plate, and left to stand at room temperature for 90 minutes for immobilization. 300 μL / well of 1% BSA TBS-T was dispensed into the immobilized plate, and the plate was blocked by leaving it at room temperature for 60 minutes. Plasmin solution adjusted to 5 μg / mL was added to the blocked plate at 100 μL / well and allowed to react at 37°C for 60 minutes. After washing with TBS-T, 150 μL / well of a reaction solution prepared with PBS containing 5 μg / mL plasminogen and 15 μL / well of substrate (Test Team S PLG, Sekisui Medical) was added, and the absorbance at 405 nm and 505 nm was measured every 5 minutes for 2 hours.

[0119] The results are shown in Figures 4 and 10. As shown in Figures 4 and 10, all three mutants exhibited reactivity equivalent to that of AMU1114 in the presence of plasmin.

[0120] Human and mouse urokinase receptors were adjusted to 1 μg / mL in PBS, dispensed into a 96-well plate at 50 μL / well, and left to stand at room temperature for 90 minutes for immobilization. 300 μL / well of TBS-T containing 1% BSA was dispensed into the plate and left to stand at room temperature for 60 minutes for blocking. Each sample (mutant) adjusted to 1000 ng / mL, 111.1 ng / mL, or 12.3 ng / mL was added to the blocked plate at 50 μL / well and allowed to react at room temperature for 60 minutes. After washing with TBS-T, 50 μL / well of HRP-conjugated anti-human Kappa chain antibody (Bethyl) diluted 1 / 50,000 with TBS-T containing 1% BSA was dispensed and incubated at room temperature for 60 minutes. After washing, the plate was incubated with TMB (3,3',5,5'-tetramethylbenzidine) color reagent for 15 minutes. The reaction was stopped with 30 μL / well of 2N H2SO4, and the absorbance at 450 nm was measured.

[0121] The results are shown in Figure 5. As shown in Figure 5, it was revealed for the first time that AMU1114 has binding ability to the human urokinase receptor. Therefore, the binding ability of the above three mutants to the human urokinase receptor was tested. As shown in Figures 5 and 11, all three mutants lost their binding ability to the mutant human urokinase. Furthermore, as shown in Figure 11, the AMU1114 (Δ22-28, F157E) mutant did not show significant binding to either the human or mouse urokinase receptor, as can be seen when compared with the negative control PBS. Furthermore, when an AMU1114 mutant with F157E / D and W30R was prepared, this mutant also showed extremely reduced binding ability to the human urokinase receptor.

[0122] C-terminal His-tagged urokinase was expressed in ExpiCHO containing 5%, 10%, or 20% FBS. Culture supernatants from 12-day cultures were dialyzed against 50 mM Tris-HCl pH 8.5, 300 mM NaCl, loaded onto a Ni column (Cytiva) equilibrated with the same buffer, washed with the same buffer, and eluted with PBS containing 400 mM imidazole. The eluted sample was subjected to gel filtration on Superdex 75 pg (Cytiva) to obtain the final sample.

[0123] The results are shown in Figures 6A and 6B. As shown in Figures 6A and 6B, low-molecular-weight uPA was observed when cells were cultured in the presence of 5% FBS, and a decrease in the amount of low-molecular-weight uPA was observed when cells were cultured in the presence of 20% FBS. This indicates that cell culture in the presence of FBS is necessary for uPA expression.

[0124] AMU1114 was similarly expressed in ExpiCHO cells in the presence of 20% FBS or in the absence of serum. Purification of AMU1114 as described above and electrophoresis confirmed the presence of various fragments in the absence of serum (see Figure 7A), but the amounts of these fragments were significantly reduced in the presence of 20% FBS (see Figure 7A).

[0125] Next, four variants of AMU1114 were similarly expressed in ExpiCHO cells under serum-free conditions. When the variants were purified and electrophoresed as described above, these mutants were not fragmented even when cultured under serum-free conditions (see Figure 7B).

[0126] AMU1114 and AMU1114 variants were analyzed by gel filtration after affinity purification. The results are shown in Figures 8A and 8B. As shown in Figures 8A and 8B, for AMU1114, a strong light chain peak was observed relative to the AMU1114 peak, whereas for the AMU1114 variants, the appearance of the light chain was suppressed and the AMU1114 variant peak was relatively increased.

[0127] From the above results, it was found that the F157 mutant of AMU1114 exhibits physiological activity equivalent to that of AMU1114 in the presence of plasmin, but is more stable than AMU1114, has higher purity due to less contamination by free light chains, and is produced in approximately 3 to 5 times higher yield than AMU1114.

[0128] AMU1114 (Δ22-28, F157E) was administered to thrombin thrombosis model mice, and the degree of thrombus lysis was compared with that of a tenecteplase-treated group and a negative control group. Specifically, thrombin thrombosis model mice were generated by intravenous injection of 600 U / kg of thrombin into female Balb / c mice. Thirty minutes after the intravenous injection, PBS (negative control), tenecteplase (positive control), and AMU1114 (Δ22-28, F157E) were administered intravenously. Sixty minutes later, lungs were removed from the mice under deep anesthesia, and 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 thrombolysis, the smaller the total area. As a result, as shown in Figures 12A and 12B and Table 1, an extremely large number of thrombi were observed in the negative control group, whereas almost no thrombi were observed in the positive control group or the AMU1114 (Δ22-28, F157E)-administered group.

[0129]

[0130] Example 4: Construction and functional analysis of AMU1114(Δ22-28, R156E), AMU1114(Δ22-28, R156I, F157Y), and AMU1114(Δ22-28, P155G, R156I, F157Y) AMU1114(Δ22-28, R156E), AMU1114(Δ22-28, R156I, F157Y), and AMU1114(Δ22-28, P155G, R156I, F157Y) were constructed (see FIG. 13 ). The heavy chains of AMU1114(Δ22-28, R156E), AMU1114(Δ22-28, R156I, F157Y), and AMU1114(Δ22-28, P155G, R156I, F157Y) were designed to have the amino acid sequences of SEQ ID NOs: 64, 65, and 66, respectively. The light chain had the amino acid sequence of SEQ ID NO: 17. Hereinafter, AMU1114(Δ22-28, R156E), AMU1114(Δ22-28, R156I, F157Y), and AMU1114(Δ22-28, P155G, R156I, F157Y) may be referred to as variants A, B, and C, respectively.

[0131] PCR Reaction PCR amplification was performed on the gene sequence of AMU1114 (Δ22-28, Phe157Glu) inserted into pcDNA3.4 (ThermoFisher) using the following primers.

[0132] PCR enzyme: PrimeStar HS DNA polymerase (Takara) AGGAGGCTCGAGTAGCAATGAACTTC (SEQ ID NO: 70) GTTTCAATGCGGCCAGAAAACATTGAGACCGGAATTTAAGATCATAGGCGGGGAGTTCACGAC (SEQ ID NO: 71) TCTCAATGTTTTCTGGCCGCATTGAAAC (SEQ ID NO: 72) CAAACTCATTACTAACCGGTAGGGATCG (SEQ ID NO: 73)

[0133] Restriction enzyme treatment: AMU1114 integrated into pcDNA3.4 (ThermFisher) and each PCR product 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 FastGene Gel / PCR Extraction Kit (Nippon Genetics).

[0134] Ligation Reaction The vector (pcDNA3.4) and insert were reacted for 30 minutes using Ligation High (Takara).

[0135] Transformation Competent High DH5α (Toyobo) was used for transformation, and the transformants were cultured on an LB medium (50 μg / mL) plate for 16 hours.

[0136] Gene Confirmation Plasmid was extracted from the transformed E. coli using FastGene Plasmid Mini Kit (Nippon Genetics) and subjected to sequence analysis to confirm the production of AMU1114 (Δ22-28, R156E) having the amino acid sequence set forth in SEQ ID NO: 64.

[0137] AMU1114 variants were transiently expressed using the ExpiCHO Expression System (Thermo Fisher). Cultures were performed for 12 days and the culture supernatants were collected. Cultures were performed in serum-free medium. The transiently expressed culture supernatants were loaded onto CaptoL (Cytiva) equilibrated with PBS, washed with the same buffer, and eluted with 100 mM glycine buffer, pH 3.0. The eluted sample was subjected to gel filtration using Superdex 200pg 16 / 600 (Cytiva) equilibrated with PBS, 150 mM L-Arg, pH 7.4, to obtain the final product. The addition of L-arginine improved the solubility of the variants. The yields of AMU1114 (Δ22-28, R156E) and AMU1114 (Δ22-28, F157E) were approximately 80 mg / L and 85 mg / L, respectively, which were comparable. The light chains of AMU1114(Δ22-28, R156E) and AMU1114(Δ22-28, F157E) had the amino acid sequence set forth in SEQ ID NO: 17. The heavy chain of AMU1114(Δ22-28, F157E) had the amino acid sequence set forth in SEQ ID NO: 63.

[0138] Urokinase activity comparison experiment - without plasmin pretreatment. AMU1114, AMU1114(Δ22-28, Phe157Glu), and AMU1114(Δ22-28, Arg156Glu) were adjusted to 10 μg / mL in PBS and dispensed at 100 μL / well into a 96-well plate. The plate was then immobilized at room temperature for 90 minutes. 300 μL / well of 1% BSA in TBS-T was dispensed and the plate was blocked by incubating at room temperature for 60 minutes. After washing with TBS-T, 150 μL / well of a reaction solution containing 5 μg / mL plasminogen and 15 μL / well of substrate (Test Team S PLG, Sekisui Medical) in PBS was added. 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 degrades fibrin to generate fibrin degradation products. In this experiment, urokinase activity is estimated based on the amount of substrate reacted.

[0139] Urokinase activity comparison experiment - with plasmin pretreatment. AMU1114, AMU1114(Δ22-28, Phe157Glu), and AMU1114(Δ22-28, Arg156Glu) were adjusted to 10 μg / mL in PBS and dispensed at 100 μL / well into a 96-well plate. The plate was then immobilized at room temperature for 90 minutes. 1% BSA in TBS-T was dispensed at 300 μL / well and the plate was then blocked at room temperature for 60 minutes. Plasmin solution adjusted to 5 μg / mL was added at 100 μL / well to the blocked plate, and the plate was incubated at 37°C for 60 minutes. After washing with TBS-T, 150 μL / well of a reaction solution prepared with PBS containing 5 μg / mL plasminogen and 15 μL / well of substrate (Test Team S PLG, Sekisui Medical) was added, and the absorbance at 405 nm and 505 nm was measured every 5 minutes for 2 hours.

[0140] Stability experiment Purified AMU1114, AMU1114(Δ22-28, Phe157Glu), and AMU1114(Δ22-28, Arg156Glu) were adjusted to 1 mg / mL with PBS and left to stand at 37°C. After 48 hours, the samples were examined by SDS-PAGE electrophoresis.

[0141] Plasmin resistance experiment: Purified AMU1114, AMU1114(Δ22-28, Phe157Glu), and AMU1114(Δ22-28, Arg156Glu) were adjusted to 1 mg / mL, and 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). These were mixed 1:1 and incubated at 37°C for 60 minutes. The reaction samples were confirmed by SDS-PAGE electrophoresis.

[0142] Results The results of a stability experiment on AMU1114 and its two variants are shown in Figure 14. AMU1114 was degraded by incubation at 37°C for 48 hours, and H-chain fragments were clearly observed. 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, and no H-chain fragments were detected. Next, the resistance of AMU1114 and its two variants to plasmin was tested. AMU1114 and its variants were reacted with 1 / 10, 1 / 50, or 1 / 250 of the amount of plasmin under the above conditions. The obtained samples were analyzed by electrophoresis, and it was found that AMU1114 was almost completely degraded by 1 / 10 the amount of plasmin, as shown in Figure 15. AMU1114 (Δ22-28, F157E) exhibited strong resistance to plasmin, while AMU1114 (Δ22-28, R156E) exhibited moderate resistance to plasmin. Next, the urokinase activity of AMU1114 and its variants was examined under conditions with and without plasmin pretreatment. Under conditions with plasmin pretreatment, AMU1114 and its variants exhibited equivalent urokinase activity, as shown in Figure 16A. In contrast, under conditions without plasmin pretreatment (conditions closer to physiological conditions), AMU1114(Δ22-28, R156E) exhibited the highest urokinase activity, as shown in Figure 16B, which exceeded that of AMU1114(Δ22-28, F157E) and was comparable to that of AMU1114. These results demonstrate that AMU1114(Δ22-28, R156E) is relatively stable in blood and exhibits moderate resistance to plasmin, but is more easily activated than AMU1114(Δ22-28, F157E) when bound to fibrin in the presence of fibrin. These properties of AMU1114(Δ22-28, R156E) suggest its suitability for use in thrombolytic therapy.Furthermore, AMU1114(Δ22-28, R156E) is thought to exhibit no significant binding to the human urokinase receptor due to its Δ22-28 structure. Furthermore, while AMU1114 unintentionally fragments when expressed in cells under serum-free conditions, AMU1114(Δ22-28, F157E) did not fragment when expressed in cells under serum-free conditions. Similar to AMU1114(Δ22-28, F157E), AMU1114(Δ22-28, R156E) did not fragment when expressed in cells under serum-free conditions, and it was recovered in a yield equivalent to that of AMU1114(Δ22-28, F157E). Thus, AMU1114(Δ22-28, R156E) also demonstrated advantages in terms of production.

[0143] Furthermore, as shown in Figure 17, AMU1114(Δ22-28, F157E) requires longer activation time than AMU1114 (see the right panel of Figure 17I). When the reaction time was short, AMU1114(Δ22-28, F157E) showed almost no thrombolytic activity (see Figure 17II). On the other hand, AMU1114(Δ22-28, F157E) maintained stability even after incubation in PBS at 37°C for 48 hours (see Figure 19). This suggests that AMU1114(Δ22-28, F157E) is expected to exert a stable thrombolytic activity over the long term. For example, AMU1114(Δ22-28, F157E) may be more suitable for chronic thrombotic diseases than acute infarction diseases.

[0144] In contrast, AMU1114(Δ22-28, R156E), AMU1114(Δ22-28, R156I, F157Y), and AMU1114(Δ22-28, P155G, R156I, F157Y) require a slightly longer time for activation than AMU1114 as shown in Figure 18 (see the right panel of Figure 18(I)), but are quickly activated and exhibit high activity equivalent to that of AMU1114 even when the reaction time is short (see Figure 18(II)). Furthermore, AMU1114(Δ22-28, R156E), AMU1114(Δ22-28, R156I, F157Y), and AMU1114(Δ22-28, P155G, R156I, F157Y) maintained stability after incubation in PBS at 37°C for 48 hours (see Figure 20). This suggests that AMU1114(Δ22-28, R156E), AMU1114(Δ22-28, R156I, F157Y), and AMU1114(Δ22-28, P155G, R156I, F157Y) exert a rapid-acting thrombolytic effect, which is expected to last for a long period of time. For example, AMU1114 (Δ22-28, R156E), AMU1114 (Δ22-28, R156I, F157Y), and AMU1114 (Δ22-28, P155G, R156I, F157Y) can be preferably applied to acute infarction diseases and the like.

[0145] Example 5: Treatment experiment in a rat cerebral infarction model (1) Generation of UK-active human AMU1114 (Δ22-28) Human AMU1114 (Δ22-28) was transiently expressed using the ExpiCHO Expression System (Thermo Fisher). Culture was performed for 7 days, and the culture supernatant was collected. The culture supernatant was loaded onto CaptoL (Cytiva) equilibrated with PBS, washed with the same buffer, and then eluted with 100 mM glycine buffer, pH 3.0. The eluted target substance was collected and purified by gel filtration using Superdex 200pg (Cytiva) to obtain the final product.

[0146] (2) Preparation of a Rat Cerebral Infarction Model A method for preparing a cerebral infarction model has been developed in which a thrombus prepared from rat whole blood is transferred to the rat middle cerebral artery using a microcatheter (Komatsu T, Translational Stroke Res, 2024). It has been found that thrombi prepared from rat whole blood are hardly dissolved by conventional thrombolytic agents. In this example, we attempted to prepare a model more similar to human thrombus. Specifically, 75 μL, 50 μL, or 25 μL of 50 mg / mL human fibrinogen in PBS (Arg) was added to 1 mL of rat whole blood, and 25 μL, 50 μL, or 75 μL of PBS (Arg) was added to each to bring the total volume to 100 μL. Since the fibrinogen concentration in rat blood is 2.5 mg / mL, the human fibrinogen content in the prepared solution corresponds to 60%, 50%, or 33% of that concentration. Plasmin (86 μg / ml and 8.6 μg / ml) was added to these solutions for 1 hour at 37°C (see Figure 21). In future cerebral infarction models, we will prepare rat cerebral infarction models by adding 60% human fibrinogen to rat blood to obtain thrombi.

[0147] A microcatheter was inserted into the rat's tail artery, and then guided to the left internal carotid artery using an angiography device in the angiography room. The thrombus obtained above, measuring 0.861 mm in diameter and 3 mm in length, was placed in the left middle cerebral artery bifurcation via the microcatheter to induce cerebral infarction. The angiography device confirmed that the thrombus had been placed in the appropriate location (see Figures 22 and 23).

[0148] Five minutes after the creation of cerebral infarction, thrombolytic agents (tPA, tenecteplase, and a modified AMU1114) were intravenously administered via the rat tail vein. The modified AMU in this case was the UK-activated AMU1114(Δ22-28). Thrombolysis was monitored using an angiography system 1, 3, 6, and 24 hours after intravenous administration (see Figure 24). Tenecteplase did not demonstrate thrombolysis, and two of three animals in the 46 nmol / animal group died of convulsions within four hours. These convulsive deaths were thought to be due to cerebral hemorrhage. In contrast, two of three animals in the 46 nmol / animal AMU1114(Δ22-28) group showed thrombus lysis and infarction reversal in two of the three animals. Furthermore, no convulsive deaths were observed in the AMU1114(Δ22-28) group.

[0149] Example 6: Treatment experiment on an Alzheimer's dementia model Alzheimer's dementia is said to be caused by the accumulation of β-amyloid, which is thought to accumulate using insoluble fibrin as a scaffold. In this example, we investigated the effect of AMU1114 (Δ22-28) administration on β-amyloid accumulation in an Alzheimer's dementia mouse model.

[0150] A murine version of AMU1114 (Δ23-29) was generated for administration to a mouse model. Amino acid positions 23-29 in the mouse version correspond to amino acid positions 22-28 of pro-urokinase in the amino acid sequence set forth in SEQ ID NO: 1. The heavy chain had the amino acid sequence set forth in SEQ ID NO: 68, and the light chain had the amino acid sequence set forth in SEQ ID NO: 69. The heavy and light chains were transiently expressed in the ExpiCHO Expression System (Thermo Fisher) to express murine version AMU1114 (Δ23-29). Culture was performed for 7 days, and the culture supernatant was collected. FBS was added to the medium to a concentration of 20% to suppress enzyme activity. The culture supernatant was mixed with 50 mM sodium phosphate buffer, pH 6.0, at a ratio of 1:2, and the mixture was applied to a HiTrap SP column (Cytiva) equilibrated with 50 mM sodium phosphate buffer, pH 6.0. The column was washed with 50 mM sodium phosphate buffer, pH 6.0, and a 9:1 mixture of 50 mM sodium phosphate buffer, pH 6.0, and 500 mM NaCl, followed by gradient elution down to 0:10. The eluted target product was collected and purified by gel filtration on Superdex 200 pg (Cytiva) to obtain the final product.

[0151] Eight-week-old Alzheimer's disease mouse models (APP NL-GF mice) were intraperitoneally administered with either mouse-type AMU1114 (Δ22-28) or saline (negative control) once a week for eight doses. The mice were sacrificed under anesthesia one week after the final dose. After perfusion with PBS, the right brain was removed and formalin-fixed, while the left brain was removed and cryopreserved.

[0152] Beta-amyloid was extracted from brain tissue and quantified. (1) Brain tissue and 400 μL of TBS / protease inhibitors were placed in a 1.5 mL tube and homogenized using a homogenizer. The homogenizer was then washed with 400 μL of the same solution. The washed solution was spun down and combined with the first tube, and the mixture was vortexed briefly. The homogenate was centrifuged at 217,000 × g (50,000 rpm) at 4°C for 20 minutes. (2) After removing the supernatant, 800 μL of TBS / protease inhibitors (containing 1.0 mol / L sucrose) was added to the pellet, which was then suspended (vortexed) and centrifuged at 50,000 rpm at 4°C for 20 minutes. (3) After removing the supernatant, 400 μL of 1% Triton X-100 / TBS / protease inhibitors was added to the pellet and homogenized. The homogenizer was washed with 500 μL of the same solution. After washing, the homogenate was spun down and combined with the first tube and gently vortexed. This homogenate was incubated at 37°C for 15 minutes and centrifuged at 50,000 rpm for 60 minutes. (4) After removing the supernatant, 400 μL of 2% SDS / TBS / protease inhibitors was added to the precipitate and homogenized. This was combined with the 500 μL of the same solution that had been washed, and gently vortexed. This homogenate was incubated at 37°C for 15 minutes and centrifuged at 50,000 rpm for 40 minutes. (5) After removing the supernatant, 400 μL of 2% SDS / TBS / protease inhibitors was added to the precipitate and homogenized. This was combined with the 500 μL of the same solution that had been washed, and gently vortexed. The homogenate was incubated at 37°C for 15 minutes and centrifuged at 50,000 rpm for 40 minutes. (6) After removing the supernatant, 250 μL of 70% formic acid was added to the precipitate and sonicated. After sonication, the homogenate was centrifuged at 50,000 rpm for 20 minutes at 4°C. Four sets of ultrasonic waves were performed: 10 cycles at OUTPUT 3 and DUTY 40% with a 1-minute break on ice. (7) The supernatant was collected, dried in an evaporator, and suspended in 100 μL of DMSO after 20 minutes of sonication. (8) The samples were stored at -80°C until ELISA was performed. For ELISA, the samples were removed from the freezer and centrifuged at 20,000 × g for 20 minutes at 4°C.The supernatant was collected and diluted with the kit diluent.

[0153] Experiments were performed on five mouse models (three males and two females), and β-amyloid accumulation was reduced in all individuals. In the negative control group, β-amyloid concentrations averaged 116,606 pM (negative control), while in the AMU1114(Δ23-29)-treated group, they decreased to an average of 77,431 pM. The results, tabulated separately for males and females, are shown in Figure 25. Furthermore, the AMU1114(Δ23-29)-treated group showed no adverse reactions, such as bleeding or inflammation in the brain or elsewhere, no significant difference in body weight compared to the control group, and no behavioral abnormalities. By not conferring plasmin resistance, AMU1114 or its modified forms activated pro-urokinase, which induced early degradation of insoluble fibrin, likely contributing to these remarkable effects. This indicates that AMU1114 or its variants that do not confer plasmin resistance are beneficial for acute thrombotic and embolic diseases. Furthermore, AMU1114 or its variants that do not confer plasmin resistance are also beneficial for Alzheimer's dementia. While AMU1114 variants that have been hypothetically conferred plasmin resistance are also thought to be effective for Alzheimer's dementia, it is clear that the introduction of a mutation to confer plasmin resistance is not essential for AMU1114 or its variants.

[0154] Contents of the Sequence Listing SEQ ID NO: 1: Amino acid sequence of the heavy chain of AMU1114 EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRKAHYGNYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGGGGGSGGGGSGGSSSNELHQV PSNCDCLNGGTCVSNKYFSNIHWCNCPKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATTVLQQTYHAHRS DALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGGGPSSPPEELKFQCGQKTLRPRFKIIIGGEFTTIENQP WFAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHH NDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLALSAGGAS SEQ ID NO: 2: Amino acid sequence of the heavy chain of AMU1114 F157DEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYLQ WSSLKASDTAMYYCTRKAHYGNYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGGGGGSGGGGSGGSSSNELHQV PSNCDCLNGGTCVSNKYFSNIHWCNCPKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATTVLQQTYHAHRS DALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGGGPSSPPEELKFQCGQKTLRPRDKIIIGGEFTTIENQP WFAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHH NDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLALSAGGAS SEQ ID NO: 3: Amino acid sequence of the heavy chain of AMU1114 F157EEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRKAHYGNYGFAYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCGSGGGGSGGGGSGGSSSNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATTVLQQTYHAHRSDA LQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGGGPSSPPEELKFQCGQKTLRPREKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVIS ATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLALSAGGAS SEQ ID NO: 4: Amino acid sequence of the heavy chain of AMU1114 F157D, further modifiedEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYLQ WSSLKASDTAMYYCTRKAHYGNYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGGGGGSGGGGSGGSSSNELHQV PSNCDCLNGGTCVSYKYFSSIRRCNCPKKFGGEHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRS DALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGGGPSSPPEELKFQCGQKTLRPRDKIIIGGEFTTIENQP WFAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHH NDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLALSAGGAS SEQ ID NO: 5: Amino acid sequence of the heavy chain of AMU1114 F157E, further modifiedEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYLQ WSSLKASDTAMYYCTRKAHYGNYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGGGGGSGGGGSGGSSSNELHQV PSNCDCLNGGTCVSYKYFSSIRRCNCPKKFGGEHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRS DALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGGGPSSPPEELKFQCGQKTLRPREKIIGGEFTTIENQP WFAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHH NDIALKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEV TTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLALSAGGAS SEQ ID NO: 6: Amino acid sequence of pro-urokinase SNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGKKPSSPPEELKFQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLAL SEQ ID NO: 7: Amino acid sequence of pro-urokinase K135G, K136G SNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYC RNPDNRRRPWCYVQVGLKPLVQECMVHDCADGGGPSSPPEELKFQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVISATHC FIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLY PEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLAL SEQ ID NO: 8: Amino acid sequence of Fibrinogen Bbeta 231-246 CNIPVVSGKECEEIIR SEQ ID NO: 9: Amino acid sequence of Fibrinogen gamma 232-246 KNWIQYKEGFGHLSP SEQ ID NO: 10: Amino acid sequence of Humanized 1101 HCDR1 SYWMH SEQ ID NO: 11: Amino acid sequence of Humanized 1101 HCDR2 AIYPGNSDTRYSPSFQG SEQ ID NO: 12: Amino acid sequence of Humanized 1101 HCDR3 KAHYGNYGFAY ​​SEQ ID NO: 13: Amino acid sequence of Humanized 1101 LCDR1 RASQHINNWLASEQ ID NO: 14: Amino acid sequence of Humanized 1101 LCDR2 GATSLQS SEQ ID NO: 15: Amino acid sequence of Humanized 1101 LCDR3 QQYWSTPLT SEQ ID NO: 16: Amino acid sequence of Humanized 1101 VH MGSTAILALLLAVLQGVCAEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRKAHYGNYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 17: Humanized 1101 Amino acid sequence of VL MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCRASQHINNWLAWYQQKPGKAPKLLISGATSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYWSTPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 18: Amino acid sequence of Humanized 99 HCDR1 NYGMN SEQ ID NO: 19: Amino acid sequence of Humanized 99 HCDR2 WINTKIG EPTYAQKFQG SEQ ID NO: 20: Amino acid sequence of Humanized 99 HCDR3 LLDY SEQ ID NO: 21: Amino acid sequence of Humanized 99 LCDR1 RASQSVLYSSNQKNYLA SEQ ID NO: 22: Amino acid sequence of Humanized 99 LCDR2 WASSLQS SEQ ID NO: 23: Amino acid sequence of Humanized 99 LCDR3 HQYLSSYT SEQ ID NO: 24: Amino acid sequence of Humanized 99 VHQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTKIGEPTYAQKFQGRVTMTRDKSSSTAYMELSSLRSEDTAVYYCARLLDYWGQGTLVTVSS SEQ ID NO: 25: Amino acid sequence of Humanized 99 VL DIQMTQSPSSLSASVGDRVTITCRASQSVLYSSNQKNYLAWYQQKPGKSPKLLIYWASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYLSSYTFGQGTKVEIKR SEQ ID NO: 26: Amino acid sequence of the heavy chain of AMU1114 F157E / D, further modified EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRKAHYGNYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGGGGGSGGGGSGGSSSNELHQV PSNCDCLNGGTCVSYKYFSSIRRCNCPKKFGGEHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRS DALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGGGPSSPPEELKFQCGQKTLRPRXKIIGGEFTTIENQP WFAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHH NDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLALSAGGAS SEQ ID NO: 27:102-10 Amino acid sequence of HCDR1 FTNYGMNSEQ ID NO: 28:102-10 Amino acid sequence of HCDR2 WINTYTGEATYA SEQ ID NO: 29:102-10 Amino acid sequence of HCDR3 LMDY SEQ ID NO: 30:102-10 Amino acid sequence of LCDR1 KASQDINKYIA SEQ ID NO: 31:102-10 Amino acid sequence of LCDR2 YTSTLQP SEQ ID NO: 32:102-10 Amino acid sequence of LCDR3 LQYDNLTW SEQ ID NO: 33:34-105 Amino acid sequence of HCDR1 KSVSTSGYSY SEQ ID NO: 34:34-105 Amino acid sequence of HCDR2 LVS SEQ ID NO: 35:34-105 Amino acid sequence of HCDR3 QHIRELTR SEQ ID NO: 36:34-105 Amino acid sequence of LCDR1 GFTFSSYA SEQ ID NO: 37: 34-105 Amino acid sequence of LCDR2 ISSGGTT SEQ ID NO: 38: 34-105 Amino acid sequence of LCDR3 VRGGTIGAY SEQ ID NO: 39: Fib-0355 Amino acid sequence of HCDR1 QSVLYSSNQK SEQ ID NO: 40: Fib-0355 Amino acid sequence of HCDR2 YWASTRES SEQ ID NO: 41: Fib-0355 Amino acid sequence of HCDR3 YLSS SEQ ID NO: 42: Fib-0355 Amino acid sequence of LCDR1 YTFTNYG SEQ ID NO: 43: Fib-0355 Amino acid sequence of LCDR2 NTNTGE SEQ ID NO: 44: Fib-0355 Amino acid sequence of LCDR3 RLLDY SEQ ID NO: 45: 99 Amino acid sequence of HCDR1 NYGMN SEQ ID NO: 46:99 Amino acid sequence of HCDR2 WINTKIGEPTYAEEFKG SEQ ID NO: 47:99 Amino acid sequence of HCDR3 LLDY SEQ ID NO: 48:99 Amino acid sequence of LCDR1 RASESVDSYGNSFMH SEQ ID NO: 49:99 Amino acid sequence of LCDR2 RASNLES SEQ ID NO: 50:99 Amino acid sequence of LCDR3 QQSNEDPRT SEQ ID NO: 51:1101 Amino acid sequence of HCDR1 SYWMH SEQ ID NO: 52:1101 Amino acid sequence of HCDR2 AIYPGNSDTRNNQKFKG SEQ ID NO: 53:1101 Amino acid sequence of HCDR3 KAHYGNYGFAY ​​SEQ ID NO: 54:1101 Amino acid sequence of LCDR1 KASDHINNWLA SEQ ID NO: 55:1101 Amino acid sequence of LCDR2 GATSLETSEQ ID NO: 56:1101 Amino acid sequence of LCDR3 QQYWSTPLT SEQ ID NO: 57:0211 Amino acid sequence of HCDR1 SYAMS SEQ ID NO: 58:0211 Amino acid sequence of HCDR2 AISSGGTTYYPDSVKG SEQ ID NO: 59:0211 Amino acid sequence of HCDR3 GGTIGAYW SEQ ID NO: 60:0211 Amino acid sequence of LCDR1 KSSQSVLYSSNQKNYLA SEQ ID NO: 61:0211 Amino acid sequence of LCDR2 WASTRES SEQ ID NO: 62:0211 Amino acid sequence of LCDR3 HQYLSSWT SEQ ID NO: 63: AMU1114 (Δ22-28, F157E) heavy chain amino acid sequence EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRKAHYGNYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGGGGSGGG GSGGSSSNELHQVPSNCDCLNGGTCVSGGHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATTVLQQT YHAHRSDALQLGLGKHNYCRNPDNRRRRPWCYVQVGLKPLVQECMVHDCADGGGPSSPPEELKFQCGQKTLRPREKIIGGEFTT IENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTL AHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLALSAGGAS SEQ ID NO: 64: Amino acid sequence of the heavy chain of AMU1114 (Δ22-28, R156E)EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYL QWSSLKASDTAMYYCTRKAHYGNYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGGGGGSGGGGSGGSSSNEL HQVPSNCDCLNGGTCVSGGHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDA LQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGGGPSSPPEELKFQCGQKTLRPEFKIIIGGEFTTIENQPW FAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHN DIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLALSAGGAS SEQ ID NO: 65: Amino acid sequence of the heavy chain of AMU1114 (Δ22-28, R156I, F157Y)EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYL QWSSLKASDTAMYYCTRKAHYGNYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGGGGGSGGGGSGGSSSNEL HQVPSNCDCLNGGTCVSGGHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDA LQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGGGPSSPPEELKFQCGQKTLRPIYKIIGGEFTTIENQPW FAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHN DIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLALSAGGAS SEQ ID NO: 66: Amino acid sequence of the heavy chain of AMU1114 (Δ22-28, P155G, R156I, F157Y)EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYL QWSSLKASDTAMYYCTRKAHYGNYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGGGGGSGGGGSGGSSSNEL HQVPSNCDCLNGGTCVSGGHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDA LQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGGGPSSPPEELKFQCGQKTLRGIYKIIGGEFTTIENQPW FAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHN DIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLALSAGGAS SEQ ID NO: 67: Amino acid sequence of AMU1114 (Δ22-28) heavy chainEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYL QWSSLKASDTAMYYCTRKAHYGNYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSGGGGGSGGGGSGGSSSNEL HQVPSNCDCLNGGTCVSGGHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDA LQLGLGKHNYCRNPDNRRRRPWCYVQVGLKPLVQECMVHDCADGGGPSSPPEELKFQCGQKTLRPRFKIIGGEFTTIENQPW FAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHN DIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLALSAGGAS SEQ ID NO: 68 (Mouse AMU1114 (Δ23-29) heavy chain)EVQLQQSGTVLARPGASVKMSCKASGFSFTSYWMHWVKQRPGQGLEWIGAIYPGNSDTRNNQKFKGKAKLTAVTSANTAY MELSSLTNEDSAVYYCTRKAHYGNYGFAYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTW NSGSLSSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGSGGGGGSGGGGSGGSSGSVL GAPDESNCGCQNGGVCVSGGRRCSCPRKFQGEHCEIDASKTCYHGNGDSYRGKANTDTKGRPCLAWNAPAVLQKPYNAHRP DAISLGLGKHNYCRNPDNQKRPWCYVQIGLRQFVQECMVHDCSLSGGPSSSSVDQQGFQCGQKALRPRFKIVGGEFTEVEN QPWFAAIYQKNKGGSPPSFKCGGSLISPCWVASAAAHCFIQLPKKENYVVYLGQSKESSYNPGEMKFEVEQLILHEYYREDS LAYHNDIALKIRTSTGQCAQPSRSIQTICLPPRFTDAPFGSDCEITGFGKESESDYLYPKNLKMSVVKLVSHEQCMQPHY YGSEINYKMLCAADPEWKTDSCKGDSGGPLICNIEGRPTLSGIVSWGRGCAEKNKPGVYTRVSHFLDWIQSHIGEEKGLAF SEQ ID NO: 69 (Mouse AMU1114 (Δ23-29) light chain) DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 74 (Humanized 1101 antibody HCDR1): SYWMH SEQ ID NO: 75 (humanized 1101 antibody HCDR2): AIYPGNSDTRYSPSFQG SEQ ID NO: 76 (humanized 1101 antibody HCDR3): KAHYGNYGFAYSEQ ID NO: 77 (humanized 1101 antibody LCDR1): RASQHINNWLA SEQ ID NO: 78 (humanized 1101 antibody LCDR2): GATSLQS SEQ ID NO: 79 (humanized 1101 antibody LCDR3): QQYWSTPLT SEQ ID NO: 80 (humanized 1101 antibody heavy chain variable region): MGSTAILALLLAVLQGVCAEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMHWVRQMPGKGLEWIGAIYPGNSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRKAHYGNYGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 80 (Humanized 1101 antibody light chain variable region): MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCRASQHINNWLAWYQQKPGKAPKLLISGATSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYWSTPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 81 (Humanized 99 antibody HCDR1): NYGMN SEQ ID NO: 82 (humanized 99 antibody HCDR2): WINTKIGEPTYAQKFQG SEQ ID NO: 83 (humanized 99 antibody HCDR3): LLDY SEQ ID NO: 84 (humanized 99 antibody LCDR1): RASQSVLYSSNQKNYLA SEQ ID NO: 85 (humanized 99 antibody LCDR2): WASSLQS SEQ ID NO: 86 (humanized 99 antibody LCDR3): HQYLSSYT SEQ ID NO: 87 (humanized 99 antibody heavy chain variable region): QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTKIGEPTYAQKFQGRVTMTRDKSSSTAYMELSSLRSEDTAVYYCARLLDYWGQGTLVTVSSSEQ ID NO: 88 (humanized 99 antibody light chain variable region): DIQMTQSPSSLSASVGDRVTITCRASQSVLYSSNQKNYLAWYQQKPGKSPKLLIYWASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYLSSYTFGQGTKVEIKR

Claims

1. A method of treating a disease associated with the accumulation or deposition of insoluble fibrin, a thrombotic disease, an embolic disease, an infarct disease, or a thromboembolic disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an active ingredient, said active ingredient comprising a fusion protein comprising an insoluble fibrin-binding domain and the EGF domain, kringle domain, and catalytic domain of pro-urokinase.

2. The method according to claim 1, wherein the thrombotic disease, embolic disease, infarction disease or thromboembolism is an acute thrombotic disease, embolic disease, infarction disease or thromboembolism.

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

4. The method of any one of claims 1 to 3, for opening a thrombus or embolism in a subject.

5. The method according to any one of claims 1 to 4, which shows a higher thrombus patency rate or embolism patency rate than alteplase or preferably necteplase when administered in equimolar amounts.

6. The method according to any one of claims 1 to 5, which exhibits a lower incidence of treatment-related bleeding than alteplase and necteplase when administered in equimolar amounts.

7. The method according to any one of claims 1 to 6, which, when administered in equimolar amounts, exhibits (i) a higher thrombus or embolus patency rate and (ii) a lower incidence of treatment-related bleeding than alteplase or preferably necteplase.

8. The method according to claim 1, wherein the disease associated with the accumulation or deposition of insoluble fibrin is Alzheimer's disease.

9. The method of claim 5, which reduces the accumulation of beta amyloid.

10. A protein having the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 67, or a composition containing said protein, for use in the method according to any one of claims 1 to 9.

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