Ligand-binding molecules with adjustable ligand-binding activity

Abstract

The present invention provides a ligand-binding molecule whose ligand-binding activity is reduced by cleavage of its cleavage site, a method for producing the same, a complex, a fusion protein, and a pharmaceutical composition. [Solution] A ligand-binding molecule comprising antibody VH and antibody VL, wherein the ligand-binding molecule is capable of binding to a ligand and contains at least one protease cleavage sequence, the antibody VL and antibody VH in the ligand-binding molecule are associated, the association is resolved when the protease cleavage sequence is cleaved by a protease, and the binding of the ligand-binding molecule to the ligand when the protease cleavage sequence is cleaved is weaker than the binding of the ligand-binding molecule to the ligand when the protease cleavage sequence is not cleaved.

Description

[Technical Field]

[0001] The present invention provides a ligand-binding molecule having at least one cleavage site, wherein the binding to a ligand is weakened when the cleavage site is cleaved; a method for producing the ligand-binding molecule; and a pharmaceutical composition containing the ligand-binding molecule. [Background technology]

[0002] Antibodies are attracting attention as pharmaceuticals due to their high stability in plasma and low incidence of side effects. Among them, IgG-type antibody drugs have been launched in large numbers, and many more antibody drugs are currently under development (Non-Patent Documents 1 and 2).

[0003] To date, antibody drugs such as rituxan for the CD20 antigen, cetuximab for the EGFR antigen, and herceptin for the HER2 antigen have been approved as cancer treatments (Non-Patent Literature 3). These antibody molecules bind to antigens expressed on cancer cells and exert toxic activity against cancer cells through ADCC or signaling inhibition.

[0004] Furthermore, a method is known in which ligands with physiological activity, such as cytokines, are fused to antibody molecules that bind to cancer antigens highly expressed on cancer cells, thereby delivering ligands to solid tumors using immunocytokines. Cytokines delivered to solid tumors by immunocytokines exert an antitumor effect by activating the immune system. Because cytokines such as IL-2, IL-12, and TNF are highly toxic, it is hoped that delivering these cytokines to the tumor site using antibodies will reduce side effects while enhancing their effectiveness (Non-patent documents 4, 5, 6). However, all of these methods have challenges such as not showing sufficient clinical effect with systemic administration, having a narrow therapeutic window, and being too toxic to be administered systemically, and therefore have not yet been approved as pharmaceuticals.

[0005] A major reason for this is that even immunocytokines, when administered systemically, are exposed to the entire body, potentially exerting toxicity through systemic action, or they can only be administered at extremely low doses to avoid toxicity. There are also reports that the antitumor effect was the same between immunocytokines in which IL-2 was fused to antibodies that bind to cancer antigens and immunocytokines in which IL-2 was fused to antibodies that do not bind to cancer antigens (Non-patent Literature 7).

[0006] As a way to avoid the above problems, molecules have been reported in which cytokines and cytokine receptors are linked by a linker that is cleaved by a protease highly expressed in cancer. Cytokines are inhibited by cytokine receptors linked by the linker, but when the linker is cleaved by the protease, the cytokine is released from the cytokine receptor and becomes active. For example, a molecule in which TNF-alpha and TNF-R are linked by a linker cleaved by uPA (Non-Patent Literature 8) has been reported, and a molecule in which IL-2 and IL-2R are linked by a linker cleaved by MMP-2 (Non-Patent Literature 9) has been reported. However, in these molecules, the cytokines are active even before linker cleavage, and the activity only increases by about 10 times after linker cleavage. In addition, a molecule in which anti-IL-2 scFv is linked to IL-2 via a linker cleaved by MMP-2 instead of IL-2R (Non-Patent Literature 9) has been reported. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Monoclonal antibody successes in the clinic. Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Nat. Biotechnol. (2005) 23, 1073-1078 [Non-Patent Document 2] [ PubMed ] Pavlou AK, Belsey MJ, Eur. J. Pharm. Biopharm. (2005) 59(3), 389–396

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[0008] The present invention has been made in view of the above circumstances, and one of its objectives is to provide a ligand-binding molecule that selectively activates ligands such as cytokines or chemokines in target tissue, a pharmaceutical composition containing the ligand-binding molecule, and a method for producing the pharmaceutical composition and the active ingredient. [Means for solving the problem]

[0009] The inventors diligently conducted research to achieve the above objectives and created a ligand-binding molecule whose ligand-binding activity is weakened when its cleavage site is cleaved. Furthermore, the inventors found that the ligand-binding molecule or a pharmaceutical composition containing the ligand-binding molecule is useful for treating diseases using the ligand, as well as for treating diseases by administering the ligand-binding molecule, and for manufacturing pharmaceuticals for treating diseases. In addition, the inventors created a method for producing the ligand-binding molecule, thus completing the present invention.

[0010] The present invention is based on these findings and specifically includes the embodiments described below as illustrative examples. (1) A ligand-binding molecule that is capable of binding to a ligand, wherein the molecule is a polypeptide having at least one cleavage site, and the binding to the ligand is weakened when the molecule is cleaved at at least one cleavage site. (2) The ligand-binding molecule according to (1), wherein the ligand is released from the ligand-binding molecule when the cleavage site is cleaved. (3) The ligand-binding molecule according to (1) or (2), wherein the cleavage site includes a protease cleavage sequence. (4) The ligand-binding molecule described in (3), wherein the protease is a target tissue-specific protease. (5) The ligand-binding molecule according to (4), wherein the target tissue is cancer tissue and the target tissue-specific protease is cancer tissue-specific protease. (6) The ligand-binding molecule according to (4), wherein the target tissue is inflammatory tissue and the target tissue-specific protease is an inflammatory tissue-specific protease. (7) The ligand-binding molecule according to any one of (3) to (6), wherein the protease is at least one protease selected from matryptase, urokinase (uPA), and metalloproteinase. (8) The ligand-binding molecule described in (3), wherein the protease cleavage sequence is a sequence that includes sequences shown in SEQ ID NOs: 3, 34, 66, 70, 71, 72, 73, 35, 75, 76, 335-345, 1161-1180, 1392-1411, and sequences selected from the sequences listed in Table 1. (9) A ligand-binding molecule according to any one of (3) to (8), wherein a first movable linker is further added to one end of the protease cleavage sequence. (10) The ligand-binding molecule according to (9), wherein a second movable linker is further added to the other end of the protease cleavage sequence. (11) The ligand-binding molecule according to (9), wherein the first movable linker is a movable linker made of a glycine-serine polymer. (12) The ligand-binding molecule according to (10), wherein the second movable linker is a movable linker made of a glycine-serine polymer. (13) The ligand-binding molecule includes an antibody VH, an antibody VL, and an antibody constant region, and is the ligand-binding molecule according to any one of (1) to (12). (14) The cleavage site, or the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are located within the antibody constant region, and is the ligand-binding molecule according to (13). (15) The cleavage site, or the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are inserted at an arbitrary position in the sequence from amino acid 118 (EU numbering) to amino acid 140 (EU numbering) of the antibody heavy chain constant region, and is the ligand-binding molecule according to (14). (16) The cleavage site, or the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are inserted at an arbitrary position in the sequence from amino acid 108 (EU numbering) (Kabat numbering 108) to amino acid 131 (EU numbering) (Kabat numbering 131) of the antibody light chain constant region, and is the ligand-binding molecule according to (14). (17) The cleavage site, or the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are located within the antibody VH or the antibody VL, and is the ligand-binding molecule according to (13). (18) The cleavage site, or the protease cleavage sequence, or the protease cleavage sequence and the first mobile linker, or the protease cleavage sequence, the first mobile linker and the second mobile linker are antibody VH7 amino acid (Kabat numbering) to 16 amino acid (Kabat numbering), 40 amino acid (Kabat numbering) to 47 amino acid (Kabat numbering), 55 amino acid (Kabat numbering) to 69 amino acid (Kabat numbering), and 73 A ligand-binding molecule as described in (17), which is inserted at any position in a sequence selected from the group consisting of amino acids (Kabat numbering) from amino acid 101 to 79 (Kabat numbering), amino acid 83 to 89 (Kabat numbering), amino acid 95 to 99 (Kabat numbering), and amino acid 101 to 113 (Kabat numbering). (19) The ligand-binding molecule according to (17), wherein the cleavage site, or the protease cleavage sequence, or the protease cleavage sequence and a first mobile linker, or the protease cleavage sequence, the first mobile linker and the second mobile linker are inserted at any position in a sequence selected from the group consisting of amino acids VL7 (Kabat numbering) to 19 (Kabat numbering), amino acids 39 (Kabat numbering) to 46 (Kabat numbering), amino acids 49 (Kabat numbering) to 62 (Kabat numbering), and amino acids 96 (Kabat numbering) to 107 (Kabat numbering). (20) The ligand-binding molecule according to (13), wherein the cleavage site, or the protease cleavage sequence, or the protease cleavage sequence and the first mobile linker, or the protease cleavage sequence, the first mobile linker and the second mobile linker are located near the boundary between the antibody constant region and the antibody VH, or / and near the boundary between the antibody constant region and the antibody VL. (21) The cleavage site, or the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are inserted at any position in the sequence from amino acid 109 of antibody VH (Kabat numbering) to amino acid 122 of the antibody heavy chain constant region (EU numbering), the ligand-binding molecule according to (20). (22) The cleavage site, or the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are inserted at any position in the sequence from amino acid 104 of antibody VL (Kabat numbering) to amino acid 113 of the antibody light chain constant region (EU numbering, position 113 of Kabat numbering), the ligand-binding molecule according to (20). (23) The antibody VL and the antibody VH in the ligand-binding molecule are associated, and the association is abolished by cleavage of the cleavage site or by cleavage of the protease cleavage sequence with a protease, the ligand-binding molecule according to any one of (13) to (22). (24) The ligand is a molecule having biological activity, and the ligand-binding molecule inhibits the biological activity of the ligand upon binding to the ligand, the ligand-binding molecule according to any one of (1) to (23). (25) The ligand is a cytokine or a chemokine, the ligand-binding molecule according to any one of (1) to (24). (26) The ligand is a ligand selected from interleukin, interferon, hematopoietic factor, TNF superfamily, chemokine, cell growth factor, and TGF-β family, the ligand-binding molecule according to any one of (1) to (24). (27) The ligand is CXCL10, IL-12, PD-1, or IL-6R, the ligand-binding molecule according to any one of (1) to (24). (28) The ligand is CXCL10, and the ligand-binding molecule contains antibody VH and antibody VL, and the ligand-binding molecule is: (a) Having antibody VH containing H-CDR1 (sequence number: 374), H-CDR2 (sequence number: 375), and H-CDR3 (sequence number: 376); antibody VL containing L-CDR1 (sequence number: 377), L-CDR2 (sequence number: 378), and L-CDR3 (sequence number: 379); or (b) Having antibody VH containing H-CDR1 (sequence number: 380), H-CDR2 (sequence number: 381), and H-CDR3 (sequence number: 382); antibody VL containing L-CDR1 (sequence number: 383), L-CDR2 (sequence number: 384), and L-CDR3 (sequence number: 385); or (c) Having antibody VH and antibody VL that compete with (a) or (b); or (d) The ligand-binding molecule according to (27), having antibody VH and antibody VL that bind to the same epitope as (a) or (b). (29) The ligand-binding molecule according to (28), wherein the ligand-binding molecule is an antibody comprising an antibody heavy chain selected from sequences shown in SEQ ID NOs: 4-14, 23-27, 33, 59, 60, and 346-367, or an antibody light chain selected from sequences shown in SEQ ID NOs: 15-22, 1146-1160, 1282-1380, and 1386-1389. (30) The ligand is IL-12, and the ligand-binding molecule comprises antibody VH and antibody VL, and the ligand-binding molecule is: (a) Having antibody VH containing H-CDR1 (sequence number: 386), H-CDR2 (sequence number: 387), and H-CDR3 (sequence number: 388); antibody VL containing L-CDR1 (sequence number: 389), L-CDR2 (sequence number: 390), and L-CDR3 (sequence number: 391); or (b) Having antibody VH and antibody VL that compete with (a); or (c) The ligand-binding molecule according to (27), having antibody VH and antibody VL that bind to the same epitope as in (a). (31) The ligand-binding molecule according to (30), wherein the ligand-binding molecule is an antibody containing the antibody heavy chain shown in Sequence ID No. 146. (32) The ligand is PD-1, and the ligand-binding molecule comprises antibody VH and antibody VL, and the ligand-binding molecule is: (a) Having antibody VH containing H-CDR1 (sequence number: 392), H-CDR2 (sequence number: 393), and H-CDR3 (sequence number: 394); antibody VL containing L-CDR1 (sequence number: 395), L-CDR2 (sequence number: 396), and L-CDR3 (sequence number: 397); or (b) Having antibody VH and antibody VL that compete with (a); or (c) The ligand-binding molecule according to (27), having antibody VH and antibody VL that bind to the same epitope as in (a). (33) The ligand-binding molecule according to (32), wherein the ligand-binding molecule is an antibody comprising an antibody heavy chain selected from the sequences shown in SEQ ID NOs: 304 and 305, or an antibody light chain selected from the sequences shown in SEQ ID NOs: 306 to 315 and 322. (34) The ligand is IL-6R (IL-6 receptor), the ligand-binding molecule comprises antibody VH and antibody VL, and the ligand-binding molecule is: (a) Having antibody VH containing H-CDR1 with SEQ ID NO: 398, H-CDR2 with SEQ ID NO: 399, and H-CDR3 with SEQ ID NO: 400; antibody VL containing L-CDR1 with SEQ ID NO: 401, L-CDR2 with SEQ ID NO: 402, and L-CDR3 with SEQ ID NO: 403; or (b) Having antibody VH and antibody VL that compete with (a); or (c) The ligand-binding molecule according to (27), having antibody VH and antibody VL that bind to the same epitope as in (a). (35) The ligand-binding molecule according to (34), wherein the ligand-binding molecule is an antibody comprising an antibody heavy chain selected from sequences shown in SEQ ID NOs: 153-156, 157-159, and 404-470, or an antibody light chain selected from sequences shown in SEQ ID NOs: 471-535. (36) The ligand-binding molecule according to any one of (1) to (35), wherein the ligand-binding molecule is an IgG antibody. (37) A ligand-binding molecule according to any one of (1) to (36) that is bound to the ligand. (38) A ligand-binding molecule according to any one of (1) to (36) which is fused with the ligand. (39) The ligand-binding molecule according to (38), wherein the ligand-binding molecule does not bind to another ligand when it is fused with a ligand. (40) The ligand-binding molecule according to (38) or (39), wherein the ligand-binding molecule is fused with the ligand via a linker. (41) The ligand-binding molecule according to (40), wherein the linker does not contain a protease cleavage sequence. (42) The ligand-binding molecule according to any one of (38) to (41), wherein the ligand is CXCL10, the ligand-binding molecule comprises an antibody light chain and an antibody heavy chain, and the antibody light chain or the antibody heavy chain is fused with the ligand. (43) The cleavage site is a ligand-binding molecule as described in (42), which is included in the antibody light chain or the antibody heavy chain. (44) The ligand is CXCL10, and the antibody light chain contained in the ligand-binding molecule is fused with the ligand, and the ligand-binding molecule is: (a) Having an antibody heavy chain containing H-CDR1 with SEQ ID NO: 374, H-CDR2 with SEQ ID NO: 375, and H-CDR3 with SEQ ID NO: 376, and an antibody light chain containing L-CDR1 with SEQ ID NO: 377, L-CDR2 with SEQ ID NO: 378, and L-CDR3 with SEQ ID NO: 379; or (b) Having an antibody heavy chain containing H-CDR1 with SEQ ID NO: 380, H-CDR2 with SEQ ID NO: 381, and H-CDR3 with SEQ ID NO: 382, ​​and an antibody light chain containing L-CDR1 with SEQ ID NO: 383, L-CDR2 with SEQ ID NO: 384, and L-CDR3 with SEQ ID NO: 385, The ligand-binding molecule described in (42) or (43). (45) The ligand-binding molecule according to any one of (42) to (44), wherein the ligand is a CXCL10 variant represented by Sequence ID No. 370. (46) The ligand-binding molecule according to any one of (42) to (45), wherein the antibody light chain contained in the ligand-binding molecule is fused with the ligand, and the series of polypeptides in which CXCL10 and the antibody light chain are fused contains the sequence shown in SEQ ID NO: 372. (47) The ligand-binding molecule according to any one of (38) to (41), wherein the ligand is PD-1, the ligand-binding molecule comprises an antibody light chain and an antibody heavy chain, and the antibody light chain or the antibody heavy chain is fused with the ligand. (48) The cleavage site is a ligand-binding molecule as described in (47), which is included in the antibody light chain or the antibody heavy chain. (49) The ligand-binding molecule according to (47) or (48), wherein the ligand is PD-1, the antibody light chain comprises L-CDR1 corresponding to SEQ ID NO: 395, L-CDR2 corresponding to SEQ ID NO: 396, and L-CDR3 corresponding to SEQ ID NO: 397, and the antibody heavy chain comprises H-CDR1 corresponding to SEQ ID NO: 392, H-CDR2 corresponding to SEQ ID NO: 393, and H-CDR3 corresponding to SEQ ID NO: 394. (50) The ligand-binding molecule according to any one of (47) to (49), wherein the ligand is PD-1 represented by Sequence ID No. 320. (51) The ligand-binding molecule according to any one of (47) to (50), wherein the ligand is PD-1, and an antibody heavy chain contained in the ligand-binding molecule is fused with the ligand, and the series of polypeptides in which PD-1 and the antibody heavy chain are fused include a sequence selected from the sequences shown in SEQ ID NOs: 323 and 324. (52) The ligand-binding molecule according to any one of (47) to (50), wherein the ligand is PD-1, and an antibody light chain contained in the ligand-binding molecule is fused with the ligand, and the series of polypeptides in which PD-1 and the antibody light chain are fused include a sequence selected from the sequences shown in SEQ ID NOs: 325 to 334. (53) The ligand-binding molecule according to any one of (38) to (41), wherein the ligand is IL-12, the ligand-binding molecule comprises an antibody light chain and an antibody heavy chain, and the antibody light chain or the antibody heavy chain is fused with the ligand. (54) The cleavage site is a ligand-binding molecule as described in (53), which is included in the antibody light chain or the antibody heavy chain. (55) The ligand-binding molecule according to (53) or (54), wherein the ligand is IL-12, the antibody light chain comprises L-CDR1 of SEQ ID NO: 389, L-CDR2 of SEQ ID NO: 390, and L-CDR3 of SEQ ID NO: 391, and the antibody heavy chain comprises H-CDR1 of SEQ ID NO: 386, H-CDR2 of SEQ ID NO: 387, and H-CDR3 of SEQ ID NO: 388. (56) The ligand-binding molecule according to any one of (38) to (41), wherein the ligand is IL-6R, the ligand-binding molecule comprises an antibody light chain and an antibody heavy chain, and the antibody light chain or the antibody heavy chain is fused with the ligand. (57) The cleavage site is a ligand-binding molecule as described in (56), which is included in the antibody light chain or the antibody heavy chain. (58) The ligand-binding molecule according to (56) or (57), wherein the ligand is IL-6R, the antibody light chain comprises L-CDR1 corresponding to SEQ ID NO: 401, L-CDR2 corresponding to SEQ ID NO: 402, and L-CDR3 corresponding to SEQ ID NO: 403, and the antibody heavy chain comprises H-CDR1 corresponding to SEQ ID NO: 398, H-CDR2 corresponding to SEQ ID NO: 399, and H-CDR3 corresponding to SEQ ID NO: 400. (59) A complex formed of the ligand and a ligand-binding molecule according to any one of (1) to (36) that is bound to the ligand. (60) A fusion protein in which the ligand and any of the ligand-binding molecules described in (1) to (36) are fused. (61) The fusion protein according to (60), wherein the ligand-binding molecule does not bind to any other ligand when it is fused with a ligand. (62) The fusion protein according to (60) or (61), wherein the ligand-binding molecule is fused with the ligand via a linker. (63) The fusion protein according to (62), wherein the linker does not contain a protease cleavage sequence. (64) The fusion protein according to (62) or (63), wherein the linker is a linker made of a glycine-serine polymer. (65) The fusion protein according to any one of (60) to (64), wherein the ligand is CXCL10, the ligand-binding molecule comprises an antibody light chain and an antibody heavy chain, and the antibody light chain or the antibody heavy chain is fused with the ligand. (66) The fusion protein according to (65), wherein the cleavage site is included in the antibody light chain or antibody heavy chain of the ligand-binding molecule. (67) The ligand is CXCL10, and the antibody light chain contained in the ligand-binding molecule is fused with the ligand, and the ligand-binding molecule is: (a) Having an antibody heavy chain containing H-CDR1 with SEQ ID NO: 374, H-CDR2 with SEQ ID NO: 375, and H-CDR3 with SEQ ID NO: 376, and an antibody light chain containing L-CDR1 with SEQ ID NO: 377, L-CDR2 with SEQ ID NO: 378, and L-CDR3 with SEQ ID NO: 379; or (b) Having an antibody heavy chain containing H-CDR1 with SEQ ID NO: 380, H-CDR2 with SEQ ID NO: 381, and H-CDR3 with SEQ ID NO: 382, ​​and an antibody light chain containing L-CDR1 with SEQ ID NO: 383, L-CDR2 with SEQ ID NO: 384, and L-CDR3 with SEQ ID NO: 385, The fusion protein described in (65) or (66). (68) The fusion protein according to any one of (65) to (67), wherein the ligand is a CXCL10 variant represented by Sequence ID No. 370. (69) The fusion protein according to any one of (65) to (68), wherein the antibody light chain contained in the ligand-binding molecule is fused with the ligand, and the series of polypeptides in which CXCL10 and the antibody light chain are fused contains the sequence shown in SEQ ID NO: 372. (70) The fusion protein according to any one of (60) to (64), wherein the ligand is PD-1, the ligand-binding molecule comprises an antibody light chain and an antibody heavy chain, and the antibody light chain or the antibody heavy chain is fused with the ligand. (71) The cleavage site is a ligand-binding molecule according to (70) contained in the antibody light chain or the antibody heavy chain. (72) The fusion protein according to (70) or (71), wherein the ligand is PD-1, the antibody light chain comprises L-CDR1 of SEQ ID NO: 395, L-CDR2 of SEQ ID NO: 396, and L-CDR3 of SEQ ID NO: 397, and the antibody heavy chain comprises H-CDR1 of SEQ ID NO: 392, H-CDR2 of SEQ ID NO: 393, and H-CDR3 of SEQ ID NO: 394. (73) The fusion protein according to any one of (70) to (72), wherein the ligand is PD-1, represented by Sequence ID No. 320. (74) The fusion protein according to any one of (70) to (73), wherein the ligand is PD-1, and an antibody heavy chain contained in the ligand-binding molecule is fused with the ligand, and the series of polypeptides fused with PD-1 and the antibody heavy chain include a sequence selected from the sequences shown in SEQ ID NOs: 323 and 324. (75) The fusion protein according to any one of (70) to (73), wherein the ligand is PD-1, an antibody light chain contained in the ligand-binding molecule is fused with the ligand, and the series of polypeptides fused with PD-1 and the antibody light chain include a sequence selected from the sequences shown in SEQ ID NOs: 325 to 334. (76) The fusion protein according to any one of (60) to (64), wherein the ligand is IL-12, the ligand-binding molecule comprises an antibody light chain and an antibody heavy chain, and the antibody light chain or the antibody heavy chain is fused with the ligand. (77) The cleavage site is included in the antibody light chain or the antibody heavy chain, the fusion protein as described in (76). (78) The fusion protein according to (76) or (77), wherein the ligand is IL-12, the antibody light chain comprises L-CDR1 of SEQ ID NO: 389, L-CDR2 of SEQ ID NO: 390, and L-CDR3 of SEQ ID NO: 391, and the antibody heavy chain comprises H-CDR1 of SEQ ID NO: 386, H-CDR2 of SEQ ID NO: 387, and H-CDR3 of SEQ ID NO: 388. (79) The fusion protein according to any one of (60) to (64), wherein the ligand is IL-6R, the ligand-binding molecule comprises an antibody light chain and an antibody heavy chain, and the antibody light chain or the antibody heavy chain is fused with the ligand. (80) The cleavage site is a fusion protein according to (79) contained in the antibody light chain or the antibody heavy chain. (81) The fusion protein according to (79) or (80), wherein the ligand is IL-6R, the antibody light chain comprises L-CDR1 of SEQ ID NO: 401, L-CDR2 of SEQ ID NO: 402, and L-CDR3 of SEQ ID NO: 403, and the antibody heavy chain comprises H-CDR1 of SEQ ID NO: 398, H-CDR2 of SEQ ID NO: 399, and H-CDR3 of SEQ ID NO: 400. A pharmaceutical composition comprising a ligand-binding molecule as described in any of (82)(1) to (58). A pharmaceutical composition comprising a ligand-binding molecule and a ligand as described in any of (83)(1) to (37). A pharmaceutical composition comprising the complex described in (84)(59). A pharmaceutical composition comprising the fusion protein described in any of (85)(60) to (81). A method for producing a ligand-binding molecule as described in any of (86)(1) to (58). (87) The method for producing a ligand according to (86), comprising introducing a protease cleavage sequence into a molecule that can bind to a ligand. (88) A method for producing a fusion protein according to any one of (60) to (81), comprising fusing a ligand-binding molecule having a protease cleavage sequence with the ligand. A polynucleotide encoding a ligand-binding molecule as described in any of (89)(1) to (58). A vector containing the polynucleotides described in (90)(89). A host cell containing the polynucleotide described in (91)(89) or the vector described in (90). A method for producing a ligand-binding molecule according to any one of (1) to (58), comprising the step of culturing the host cells described in (92) and (91). A polynucleotide encoding a fusion protein as described in any of (93)(60) to (81). A vector containing the polynucleotides described in (94)(93). A host cell containing the polynucleotide described in (95)(93) or the vector described in (94). A method for producing a fusion protein according to any one of (60) to (81), comprising the step of culturing the host cells described in (96) or (95). (97) Protease substrates containing sequences indicated by sequence numbers 1161-1180 and 1392-1411, and sequences selected from those listed in Table 1. (98) The protease substrate according to (97), wherein the protease is a matryptase or urokinase. (99) The protease substrate according to (97) or (98), wherein the protease is MT-SP1 or uPA. (100) A polypeptide comprising one or more sequences selected from the sequences indicated by sequence numbers 1161-1180 and 1392-1411, and the sequences listed in Table 1.

[0011] The present invention may also specifically encompass the embodiments described below as illustrative. (B1) A ligand-binding molecule, wherein the ligand-binding molecule is capable of binding to a ligand, and the molecule is a polypeptide comprising at least one protease cleavage sequence, which includes one or more sequences selected from the sequences shown in SEQ ID NOs: 1161-1180, 1392-1411, and the sequences listed in Table 1, wherein the binding of the ligand-binding molecule to the ligand when the protease cleavage sequence is cleaved is weaker than the binding of the ligand-binding molecule to the ligand when the protease cleavage sequence is not cleaved. (B2) The ligand-binding molecule according to (B1), wherein the ligand is released from the ligand-binding molecule when the protease cleavage sequence is cleaved. (B3) The ligand-binding molecule according to (B1) or (B2), wherein the protease is a target tissue-specific protease. (B4) The ligand-binding molecule according to (B3), wherein the target tissue is cancer tissue and the target tissue-specific protease is a cancer tissue-specific protease. (B5) The ligand-binding molecule according to (B3), wherein the target tissue is inflammatory tissue and the target tissue-specific protease is an inflammatory tissue-specific protease. (B6) The ligand-binding molecule according to any one of (B1) to (B5), wherein the protease is at least one protease selected from matryptase, urokinase (uPA), and metalloproteinase. (B7) A ligand-binding molecule according to any one of (B1) to (B6), wherein a first movable linker is further added to one end of the protease cleavage sequence. (B8) The ligand-binding molecule according to (B7), wherein the first movable linker is a movable linker made of a glycine-serine polymer. (B9) The ligand-binding molecule according to (B7) or (B8), wherein a second movable linker is further added to the other end of the protease cleavage sequence. (B10) The ligand-binding molecule according to (B9), wherein the second movable linker is a movable linker made of a glycine-serine polymer. (B11) The ligand-binding molecule is the ligand-binding molecule according to any one of (B1) to (B10), comprising antibody VH, antibody VL, and the antibody constant region. (B12) The ligand-binding molecule according to (B11), wherein the protease cleavage sequence, or the protease cleavage sequence and a first mobile linker, or the protease cleavage sequence, a first mobile linker, and a second mobile linker are located within the constant region of the antibody. (B13) The ligand-binding molecule described in (B12), wherein the protease cleavage sequence, or the protease cleavage sequence and a first mobile linker, or the protease cleavage sequence, a first mobile linker, and a second mobile linker are introduced at any position in the sequence from amino acid 118 (EU numbering) to amino acid 140 (EU numbering) of the antibody heavy chain constant region. (B14) The ligand-binding molecule described in (B12), wherein the protease cleavage sequence, or the protease cleavage sequence and a first mobile linker, or the protease cleavage sequence, a first mobile linker, and a second mobile linker are introduced at any position in the sequence from amino acid 108 (EU numbering) (Kabat numbering 108) to amino acid 131 (EU numbering) (Kabat numbering 131) of the antibody light chain constant region. (B15) The ligand-binding molecule according to (B11), wherein the protease cleavage sequence, or the protease cleavage sequence and a first mobile linker, or the protease cleavage sequence, a first mobile linker, and a second mobile linker are located within the antibody VH or within the antibody VL. (B16) The ligand-binding molecule described in (B15), wherein the protease cleavage sequence, or the protease cleavage sequence and a first mobile linker, or the protease cleavage sequence, a first mobile linker, and a second mobile linker are introduced at any position in a sequence selected from the group consisting of amino acids VH7 (Kabat numbering) to 16 (Kabat numbering), amino acids 40 (Kabat numbering) to 47 (Kabat numbering), amino acids 55 (Kabat numbering) to 69 (Kabat numbering), amino acids 73 (Kabat numbering) to 79 (Kabat numbering), amino acids 83 (Kabat numbering) to 89 (Kabat numbering), amino acids 95 (Kabat numbering) to 99 (Kabat numbering), and amino acids 101 (Kabat numbering) to 113 (Kabat numbering). (B17) The ligand-binding molecule described in (B15), wherein the protease cleavage sequence, or the protease cleavage sequence and a first mobile linker, or the protease cleavage sequence, a first mobile linker, and a second mobile linker are introduced at any position in a sequence selected from the group consisting of amino acids VL7 (Kabat numbering) to 19 (Kabat numbering), amino acids 39 (Kabat numbering) to 46 (Kabat numbering), amino acids 49 (Kabat numbering) to 62 (Kabat numbering), and amino acids 96 (Kabat numbering) to 107 (Kabat numbering). (B18) The ligand-binding molecule according to (B11), wherein the protease cleavage sequence, or the protease cleavage sequence and a first mobile linker, or the protease cleavage sequence, a first mobile linker, and a second mobile linker are located near the boundary between the antibody constant region and the antibody VH, or / and near the boundary between the antibody constant region and the antibody VL. (B19) The ligand-binding molecule described in (B18), wherein the protease cleavage sequence, or the protease cleavage sequence and a first mobile linker, or the protease cleavage sequence, a first mobile linker, and a second mobile linker are introduced at any position in the sequence from amino acid VH109 (Kabat numbering) to amino acid 122 (EU numbering) of the antibody heavy chain constant region. (B20) The ligand-binding molecule described in (B18), wherein the protease cleavage sequence, or the protease cleavage sequence and a first mobile linker, or the protease cleavage sequence, a first mobile linker, and a second mobile linker are introduced at any position in the sequence from amino acid VL104 (Kabat numbering) to amino acid 113 (EU numbering) (Kabat numbering position 113) of the constant region of the antibody light chain. (B21) The ligand-binding molecule according to any one of (B11) to (B20), wherein the antibody VL and the antibody VH in the ligand-binding molecule are associated, and this association is resolved when the protease cleavage sequence is cleaved by the protease. (B22) The ligand is a molecule having biological activity, and the ligand-binding molecule inhibits the biological activity of the ligand by binding to it, the ligand-binding molecule according to any one of (B1) to (B21). (B23) The ligand-binding molecule according to any one of (B1) to (B22), wherein the ligand is a cytokine or chemokine. (B24) The ligand-binding molecule according to any one of (B1) to (B22), wherein the ligand is selected from interleukins, interferons, hematopoietic factors, the TNF superfamily, chemokines, cell growth factors, and the TGF-β family. (B25) The ligand-binding molecule according to any one of (B1) to (B22), wherein the ligand is CXCL10, IL-12, PD-1, IL-6R, or IL-1Ra. (B26) The ligand is CXCL10, the ligand-binding molecule comprises antibody VH and antibody VL, and the ligand-binding molecule is: (a) Having antibody VH containing H-CDR1 (sequence number: 374), H-CDR2 (sequence number: 375), and H-CDR3 (sequence number: 376); antibody VL containing L-CDR1 (sequence number: 377), L-CDR2 (sequence number: 378), and L-CDR3 (sequence number: 379); or (b) Having antibody VH containing H-CDR1 (sequence number: 380), H-CDR2 (sequence number: 381), and H-CDR3 (sequence number: 382); antibody VL containing L-CDR1 (sequence number: 383), L-CDR2 (sequence number: 384), and L-CDR3 (sequence number: 385); or (c) Having antibody VH and antibody VL that compete with (a) or (b); or (d) The ligand-binding molecule according to (B25), having antibody VH and antibody VL that bind to the same epitope as (a) or (b). (B27) The ligand-binding molecule according to (B26), wherein the ligand-binding molecule is an antibody comprising an antibody heavy chain selected from the sequences shown in SEQ ID NOs: 4-14, 23-27, 33, 59, 60, and 346-367, or an antibody light chain selected from the sequences shown in SEQ ID NOs: 15-22, 1146-1160, 1282-1380, and 1386-1389. (B28) The ligand is IL-12, the ligand-binding molecule comprises antibody VH and antibody VL, and the ligand-binding molecule is: (a) Having antibody VH containing H-CDR1 (sequence number: 386), H-CDR2 (sequence number: 387), and H-CDR3 (sequence number: 388); antibody VL containing L-CDR1 (sequence number: 389), L-CDR2 (sequence number: 390), and L-CDR3 (sequence number: 391); or (b) Having antibody VH and antibody VL that compete with (a); or (c) A ligand-binding molecule according to (B25), having antibody VH and antibody VL that bind to the same epitope as in (a). (B29) The ligand-binding molecule is the ligand-binding molecule described in (B28), wherein the ligand-binding molecule is an antibody containing the antibody heavy chain shown in Sequence ID No. 146. (B30) The ligand is PD-1, and the ligand-binding molecule comprises antibody VH and antibody VL, and the ligand-binding molecule is: (a) Having antibody VH containing H-CDR1 (sequence number: 392), H-CDR2 (sequence number: 393), and H-CDR3 (sequence number: 394); antibody VL containing L-CDR1 (sequence number: 395), L-CDR2 (sequence number: 396), and L-CDR3 (sequence number: 397); or (b) Having antibody VH and antibody VL that compete with (a); or (c) A ligand-binding molecule according to (B25), having antibody VH and antibody VL that bind to the same epitope as in (a). (B31) The ligand-binding molecule according to (B30), wherein the ligand-binding molecule is an antibody comprising an antibody heavy chain selected from the sequences shown in SEQ ID NOs: 304 and 305, or an antibody light chain selected from the sequences shown in SEQ ID NOs: 306-315 and 322. (B32) The ligand is IL-6R (IL-6 receptor), the ligand-binding molecule comprises antibody VH and antibody VL, and the ligand-binding molecule is: (a) Having antibody VH containing H-CDR1 with SEQ ID NO: 398, H-CDR2 with SEQ ID NO: 399, and H-CDR3 with SEQ ID NO: 400; antibody VL containing L-CDR1 with SEQ ID NO: 401, L-CDR2 with SEQ ID NO: 402, and L-CDR3 with SEQ ID NO: 403; or (b) Having antibody VH and antibody VL that compete with (a); or (c) A ligand-binding molecule according to (B25), having antibody VH and antibody VL that bind to the same epitope as in (a). (B33) The ligand-binding molecule according to (B32), wherein the ligand-binding molecule is an antibody comprising an antibody heavy chain selected from sequences shown in SEQ ID NOs: 153-156, 157-159, and 404-470, or an antibody light chain selected from sequences shown in SEQ ID NOs: 471-535. (B34) The ligand-binding molecule is an IgG antibody, according to any one of (B1) to (B33). (B35) A ligand-binding molecule according to any one of (B1) to (B34) that is bound to the ligand. (B36) A ligand-binding molecule according to any one of (B1) to (B34) which is fused with the ligand. (B37) The ligand-binding molecule described in (B36), wherein the ligand-binding molecule does not bind to another ligand when it is fused with a ligand. (B38) The ligand-binding molecule according to (B36) or (B37), wherein the ligand-binding molecule is fused with the ligand via a linker. (B39) The ligand-binding molecule according to (B38), wherein the linker does not contain a protease cleavage sequence. (B40) The ligand-binding molecule according to any one of (B36) to (B39), wherein the ligand is CXCL10, the ligand-binding molecule contains an antibody light chain and an antibody heavy chain, and the antibody light chain or the antibody heavy chain is fused with the ligand. (B41) The ligand-binding molecule described in (B40), wherein the protease cleavage sequence is included in the antibody light chain or the antibody heavy chain. (B42) The ligand is CXCL10, and the antibody light chain contained in the ligand-binding molecule is fused with the ligand, and the ligand-binding molecule is: (a) Having an antibody heavy chain containing H-CDR1 with SEQ ID NO: 374, H-CDR2 with SEQ ID NO: 375, and H-CDR3 with SEQ ID NO: 376, and an antibody light chain containing L-CDR1 with SEQ ID NO: 377, L-CDR2 with SEQ ID NO: 378, and L-CDR3 with SEQ ID NO: 379; or (b) Having an antibody heavy chain containing H-CDR1 with SEQ ID NO: 380, H-CDR2 with SEQ ID NO: 381, and H-CDR3 with SEQ ID NO: 382, ​​and an antibody light chain containing L-CDR1 with SEQ ID NO: 383, L-CDR2 with SEQ ID NO: 384, and L-CDR3 with SEQ ID NO: 385, A ligand-binding molecule as described in (B40) or (B41). (B43) The ligand is a modified CXCL10 represented by Sequence ID No. 370, the ligand-binding molecule described in any one of (B40) to (B42). (B44) The ligand-binding molecule according to any one of (B40) to (B43), wherein the antibody light chain contained in the ligand-binding molecule is fused with the ligand, and the series of polypeptides in which CXCL10 and the antibody light chain are fused contains the sequence shown in SEQ ID NO: 372. (B45) The ligand-binding molecule according to any one of (B36) to (B39), wherein the ligand is PD-1, the ligand-binding molecule contains an antibody light chain and an antibody heavy chain, and the antibody light chain or the antibody heavy chain is fused with the ligand. (B46) The ligand-binding molecule described in (B45), wherein the protease cleavage sequence is included in the antibody light chain or the antibody heavy chain. (B47) The ligand-binding molecule according to (B45) or (B46), wherein the ligand is PD-1, the antibody light chain comprises L-CDR1 corresponding to SEQ ID NO: 395, L-CDR2 corresponding to SEQ ID NO: 396, and L-CDR3 corresponding to SEQ ID NO: 397, and the antibody heavy chain comprises H-CDR1 corresponding to SEQ ID NO: 392, H-CDR2 corresponding to SEQ ID NO: 393, and H-CDR3 corresponding to SEQ ID NO: 394. (B48) The ligand is PD-1, represented by Sequence ID No. 320, and is a ligand-binding molecule as described in any one of (B45) to (B47). (B49) The ligand-binding molecule according to any one of (B45) to (B48), wherein the ligand is PD-1, and the antibody heavy chain contained in the ligand-binding molecule is fused with the ligand, and the series of polypeptides in which PD-1 and the antibody heavy chain are fused include a sequence selected from the sequences shown in SEQ ID NOs: 323 and 324. (B50) The ligand-binding molecule according to any one of (B45) to (B48), wherein the ligand is PD-1, and the antibody light chain contained in the ligand-binding molecule is fused with the ligand, and the series of polypeptides in which PD-1 and the antibody light chain are fused include a sequence selected from the sequences shown in SEQ ID NOs: 325 to 334. (B51) The ligand-binding molecule according to any one of (B36) to (B39), wherein the ligand is IL-12, the ligand-binding molecule includes an antibody light chain and an antibody heavy chain, and the antibody light chain or the antibody heavy chain is fused with the ligand. (B52) The protease cleavage sequence is a ligand-binding molecule as described in (B51), which is included in the antibody light chain or the antibody heavy chain. (B53) The ligand-binding molecule according to (B51) or (B52), wherein the ligand is IL-12, the antibody light chain comprises L-CDR1 (sequence number: 389), L-CDR2 (sequence number: 390), and L-CDR3 (sequence number: 391), and the antibody heavy chain comprises H-CDR1 (sequence number: 386), H-CDR2 (sequence number: 387), and H-CDR3 (sequence number: 388). (B54) The ligand-binding molecule according to any one of (B36) to (B39), wherein the ligand is IL-6R, the ligand-binding molecule contains an antibody light chain and an antibody heavy chain, and the antibody light chain or the antibody heavy chain is fused with the ligand. (B55) The protease cleavage sequence is a ligand-binding molecule as described in (B54), which is included in the antibody light chain or the antibody heavy chain. (B56) The ligand-binding molecule according to (B54) or (B55), wherein the ligand is IL-6R, the antibody light chain comprises L-CDR1 corresponding to SEQ ID NO: 401, L-CDR2 corresponding to SEQ ID NO: 402, and L-CDR3 corresponding to SEQ ID NO: 403, and the antibody heavy chain comprises H-CDR1 corresponding to SEQ ID NO: 398, H-CDR2 corresponding to SEQ ID NO: 399, and H-CDR3 corresponding to SEQ ID NO: 400. (B57) A complex formed of the ligand and any one of the ligand-binding molecules described in (B1) to (B34) that is bound to the ligand. (B58) A fusion protein in which the ligand is fused with any one of the ligand-binding molecules described in (B1) to (B34). (B59) The fusion protein described in (B58), wherein the ligand-binding molecule does not bind to any other ligand when it is fused with a ligand. (B60) The fusion protein according to (B58) or (B59), wherein the ligand-binding molecule is fused with the ligand via a linker. (B61) The fusion protein according to (B60), wherein the linker does not contain a protease cleavage sequence. (B62) The fusion protein according to (B60) or (B61), wherein the linker is a linker made of a glycine-serine polymer. (B63) The fusion protein according to any one of (B58) to (B62), wherein the ligand is CXCL10, the ligand-binding molecule contains an antibody light chain and an antibody heavy chain, and the antibody light chain or the antibody heavy chain is fused with the ligand. (B64) The fusion protein according to (B63), wherein the protease cleavage sequence is included in the antibody light chain or antibody heavy chain of the ligand-binding molecule. (B65) The ligand is CXCL10, and the antibody light chain contained in the ligand-binding molecule is fused with the ligand, and the ligand-binding molecule is: (a) Having an antibody heavy chain containing H-CDR1 with SEQ ID NO: 374, H-CDR2 with SEQ ID NO: 375, and H-CDR3 with SEQ ID NO: 376, and an antibody light chain containing L-CDR1 with SEQ ID NO: 377, L-CDR2 with SEQ ID NO: 378, and L-CDR3 with SEQ ID NO: 379; or (b) Having an antibody heavy chain containing H-CDR1 with SEQ ID NO: 380, H-CDR2 with SEQ ID NO: 381, and H-CDR3 with SEQ ID NO: 382, ​​and an antibody light chain containing L-CDR1 with SEQ ID NO: 383, L-CDR2 with SEQ ID NO: 384, and L-CDR3 with SEQ ID NO: 385, The fusion protein described in (B63) or (B64). (B66) The ligand is a CXCL10 variant represented by Sequence ID No. 370, the fusion protein according to any one of (B63) to (B65). (B67) The fusion protein according to any one of (B63) to (B66), wherein the antibody light chain contained in the ligand-binding molecule is fused with the ligand, and the series of polypeptides fused with CXCL10 and the antibody light chain contain the sequence shown in SEQ ID NO: 372. (B68) The fusion protein according to any one of (B58) to (B62), wherein the ligand is PD-1, the ligand-binding molecule contains an antibody light chain and an antibody heavy chain, and the antibody light chain or the antibody heavy chain is fused with the ligand. (B69) The ligand-binding molecule described in (B68), wherein the protease cleavage sequence is included in the antibody light chain or the antibody heavy chain. (B70) The ligand is PD-1, the antibody light chain comprises L-CDR1 of SEQ ID NO: 395, L-CDR2 of SEQ ID NO: 396, and L-CDR3 of SEQ ID NO: 397, and the antibody heavy chain comprises H-CDR1 of SEQ ID NO: 392, H-CDR2 of SEQ ID NO: 393, and H-CDR3 of SEQ ID NO: 394, as described in (B68) or (B69). (B71) The ligand is PD-1, represented by Sequence ID No. 320, and the fusion protein is one of the descriptions in (B68) to (B70). (B72) The fusion protein according to any one of (B68) to (B71), wherein the ligand is PD-1, and an antibody heavy chain contained in the ligand-binding molecule is fused with the ligand, and the series of polypeptides fused with PD-1 and the antibody heavy chain include a sequence selected from the sequences shown in SEQ ID NOs: 323 and 324. (B73) The ligand is PD-1, and an antibody light chain contained in the ligand-binding molecule is fused with the ligand, and the series of polypeptides fused with PD-1 and the antibody light chain include a sequence selected from the sequences shown in SEQ ID NOs: 325 to 334, the fusion protein according to any one of (B68) to (B71). (B74) The fusion protein according to any one of (B58) to (B62), wherein the ligand is IL-12, the ligand-binding molecule contains an antibody light chain and an antibody heavy chain, and the antibody light chain or the antibody heavy chain is fused with the ligand. (B75) The fusion protein according to (B74), wherein the protease cleavage sequence is included in the antibody light chain or the antibody heavy chain. (B76) The ligand is IL-12, the antibody light chain comprises L-CDR1 of SEQ ID NO: 389, L-CDR2 of SEQ ID NO: 390, and L-CDR3 of SEQ ID NO: 391, and the antibody heavy chain comprises H-CDR1 of SEQ ID NO: 386, H-CDR2 of SEQ ID NO: 387, and H-CDR3 of SEQ ID NO: 388, as described in (B74) or (B75). (B78) The ligand is IL-6R, the ligand-binding molecule comprises an antibody light chain and an antibody heavy chain, and the antibody light chain or the antibody heavy chain is fused with the ligand, the fusion protein according to any one of (B58) to (B62). (B78) The fusion protein according to (B77), wherein the protease cleavage sequence is included in the antibody light chain or the antibody heavy chain. (B79) The ligand is IL-6R, the antibody light chain comprises L-CDR1 of SEQ ID NO: 401, L-CDR2 of SEQ ID NO: 402, and L-CDR3 of SEQ ID NO: 403, and the antibody heavy chain comprises H-CDR1 of SEQ ID NO: 398, H-CDR2 of SEQ ID NO: 399, and H-CDR3 of SEQ ID NO: 400, as described in (B77) or (B78). A pharmaceutical composition comprising a ligand-binding molecule described in any one of (B80)(B1) to (B56). A pharmaceutical composition comprising a ligand-binding molecule and a ligand as described in any one of (B81)(B1) to (B35). A pharmaceutical composition comprising the complex described in (B82)(B57). A pharmaceutical composition comprising a fusion protein described in any one of (B83), (B58), to (B79). A method for producing a ligand-binding molecule described in any one of (B84)(B1) to (B56). (B85) The method of production according to (B84), comprising introducing a protease cleavage sequence into a molecule that can bind to a ligand. (B86) A method for producing a fusion protein according to any one of (B58) to (B79), comprising fusing a ligand-binding molecule having a protease cleavage sequence with its ligand. A polynucleotide encoding a ligand-binding molecule as described in any one of (B87)(B1) through (B56). A vector containing polynucleotides as described in (B88)(B87). A host cell containing the polynucleotides described in (B89)(B87) or the vector described in (B88). A method for producing a ligand-binding molecule according to any one of (B1) to (B56), comprising the step of culturing the host cells described in (B90) and (B89). A polynucleotide encoding a fusion protein as described in any one of (B92)(B58) to (B79). A vector containing the polynucleotides described in (B92)(B91). A host cell containing the polynucleotide described in (B93)(B91) or the vector described in (B92). A method for producing a fusion protein according to any one of (B58) to (B79), comprising the step of culturing the host cells described in (B94) or (B93). [Brief explanation of the drawing]

[0012] [Figure 1] This figure shows a fusion protein of an IgG antibody and a ligand, containing a VH molecule of a ligand-linker-antiligand antibody that is specifically released in target tissue, and one mode of its activation. The ligand and anti-ligand antibody are linked by a linker. [Figure 2] This figure shows an IgG antibody that specifically releases a ligand in target tissue, and one mode of its activation. The anti-ligand antibody, in which a protease cleavage sequence is inserted near the VH-CH1 boundary, and the ligand are mixed and administered to the individual. [Figure 3] This figure shows an IgG antibody that specifically releases a ligand in target tissue, and one mode of its activation. An anti-ligand antibody, in which a protease cleavage sequence is inserted near the VH-CH1 boundary, is administered to an individual. The administered antibody binds to a ligand already present in the body, and the subsequent activation mode is the same as shown in Figure 2. [Figure 4] This figure shows the results of evaluating the interaction between MabCXCL10 and human CXCL10 using Biacore. [Figure 5A] This figure shows a model of an antibody molecule created by inserting a protease cleavage sequence near the boundary between the antibody variable region and the constant region of MabCXCL10. [Figure 5B]This diagram shows the names of each heavy chain variant created, the insertion locations of the protease cleavage sequences, and the inserted amino acid sequences. The insertion sites are indicated by "[insert]". [Figure 5C] This diagram shows the name of each light chain variant created, the insertion site of the protease cleavage sequence, and the inserted amino acid sequence. The insertion site is indicated by [insert]. [Figure 6A] This figure shows the results of evaluating the interaction between an antibody molecule created by inserting a protease cleavage sequence near the boundary between the variable and constant regions of the heavy chain of MabCXCL10 and human CXCL10 using Biacore. [Figure 6B] This figure shows the results of evaluating the interaction between an antibody molecule created by inserting a protease cleavage sequence near the boundary between the variable and constant regions of the MabCXCL10 light chain and human CXCL10 using Biacore. [Figure 7-1] (A) This figure shows the results of evaluating the degree of cleavage by electrophoresis using reduced SDS-PAGE and detection with Coomassie Brilliant Blue (CBB) after treating an antibody molecule created by inserting a protease cleavage sequence near the boundary between the variable and constant regions of the heavy chain of MabCXCL10 with protease (MT-SP1). Of the two new bands created by the protease treatment, the band that appeared around 15 kDa is the band originating from the VH region, and the band that appeared at the 25-50 kDa position is the band originating from the constant region. [Figure 7-2] (A) is a continuation of (A), and (B) is a figure showing the results of evaluating the degree of cleavage by reduced SDS-PAGE after protease (MT-SP1) treatment of antibody molecules created by inserting protease cleavage sequences into the variable and constant regions of the light chain of MabCXCL10. Two new bands originating from the cleaved light chain have been created by the protease treatment. [Figure 7-3] This is a continuation of (B). [Figure 8]This figure shows the names of each heavy chain variant created by inserting a protease cleavage sequence and a mobile linker sequence near the boundary between the variable and constant regions of MabCXCL10, the insertion locations of the protease cleavage sequence and mobile linker sequence, and the inserted amino acid sequences. The insertion site is indicated by [insert]. [Figure 9] This figure shows the results of evaluating the interaction between an antibody molecule created by inserting a protease cleavage sequence and a movable linker sequence near the boundary between the variable and constant regions of the heavy chain of MabCXCL10 and human CXCL10 using Biacore. [Figure 10A] This figure shows the results of evaluating the degree of cleavage by electrophoresis using reduced SDS-PAGE and detection by CBB after treating an antibody molecule created by inserting a protease cleavage sequence and a linker sequence near the boundary between the variable and constant regions of the heavy chain of MabCXCL10 with protease (uPA, MT-SP1). Of the two new bands created by the protease treatment, the band that appeared around 15 kDa is the band originating from the VH region, and the band that appeared at the 25-50 kDa position is the band originating from the constant region. [Figure 10B] This figure is a continuation of Figure 10A. [Figure 11A] This figure shows the results of evaluating whether CXCL10 is released by treating the MabCXCL10a and CXCL10 complex with a protease (MT-SP1). [Figure 11B] This figure shows the results of evaluating whether CXCL10 is released by treating the EEIVHC006a / EEIVL and CXCL10 complex with a protease (MT-SP1). [Figure 12]This figure shows the names of each heavy chain created by substituting a portion of the amino acid sequence near the boundary between the variable and constant regions of MabCXCL10 with a protease cleavage sequence and a movable linker sequence, the sites where amino acids were inserted and modified, the inserted sequences, and the amino acid sequences after insertion and modification. Insertion sites are indicated with [insert]. In the "Insertion and Modification Sites" column, amino acid residues shown with a strikethrough indicate that the inserted sequence was removed during insertion, i.e., substituted with the C-terminal amino acid of the inserted sequence. [Figure 13] This figure shows the results of evaluating the degree of cleavage by electrophoresis using reduced SDS-PAGE and detection by CBB after treating an antibody molecule created by substituting a portion of the amino acid sequence near the boundary between the variable and constant regions of MabCXCL10 with a protease cleavage sequence and a movable linker with protease (uPA, MT-SP1). Of the two new bands created by the protease treatment, the band that appeared around 15 kDa is the band originating from the VH region, and the band that appeared at the 25-50 kDa position is the band originating from the constant region. [Figure 14] This diagram shows the luciferase activity (luminescence value). [Figure 15] These are the SDS-PAGE results before and after cleavage of the CXCL10-anti-CXCL10 antibody fusion protein with protease. [Figure 16] This diagram shows the luciferase activity (luminescence value). [Figure 17] This figure shows the results of reduced SDS-PAGE evaluating the protease cleavage of an anti-IL-12 neutralizing antibody into which a protease cleavage sequence and a mobile linker sequence were introduced. [Figure 18] This figure shows the production of interferon-gamma when IL-12 and antibody are added. NoAb is a sample in which only IL-12 was added without antibody, and NoIL-12 is a sample in which neither IL-12 nor antibody was added. [Figure 19A] This figure shows the cleavage of an antibody by a protease. [Figure 19B] This figure shows the cleavage of an antibody by a protease. [Figure 20A]This figure shows the results of cleavage using various proteases. [Figure 20B] This figure shows the results of cleavage using various proteases. [Figure 21] This figure shows the results of cleavage using various proteases. [Figure 22A] This figure shows the results of cleaving the MRA modified organism with protease. [Figure 22B] This figure shows the results of cleaving the MRA modified organism with protease. [Figure 22C] This figure shows the results of cleaving the MRA modified organism with protease. [Figure 22D] This figure shows the results of cleaving the MRA modified organism with protease. [Figure 22E] This figure shows the results of cleaving the MRA modified organism with protease. [Figure 22F] This figure shows the results of cleaving the MRA modified organism with protease. [Figure 22G] This figure shows the results of cleaving the MRA modified organism with protease. [Figure 22H] This figure shows the results of cleaving the MRA modified organism with protease. [Figure 22I] This figure shows the results of cleaving the MRA modified organism with protease. [Figure 23A] This figure shows the results of cleaving the MRA modified organism with protease. [Figure 23B] This figure shows the results of cleaving the MRA modified organism with protease. [Figure 23C] This figure shows the results of cleaving the MRA modified organism with protease. [Figure 24A] This figure shows the results of cleaving the MRA modified organism with protease. [Figure 24B] This figure shows the results of cleaving the MRA modified organism with protease. [Figure 24C] This figure shows the results of cleaving the MRA modified organism with protease. [Figure 24D] This figure shows the results of cleaving the MRA modified organism with protease. [Figure 24E] This figure shows the results of cleaving the MRA modified organism with protease. [Figure 25A] This figure shows the results of cleaving the MRA modified organism with protease. [Figure 25B] This figure shows the results of cleaving the MRA modified organism with protease. [Figure 26] This graph compares real-time graphs showing the binding of PD-1 to 5C4-bio in a binding evaluation sample containing protease-treated antibody and untreated antibody. The thick black line represents the binding evaluation sample containing protease-treated antibody, and the thin gray line represents the binding evaluation sample containing untreated antibody. The X-axis represents the measurement time (seconds), with the start of measurement set to 0 seconds. The Y-axis shows the binding. The name of each graph indicates the antibody contained in the evaluation sample; the None (antigen only) graph uses only the antigen as the evaluation sample, without mixing in antibodies. [Figure 27] This shows the electrophoresis results of protease-treated antibodies and untreated antibodies. The Protease(+) lane represents protease-treated antibodies, and the Protease(-) lane represents untreated antibodies. [Figure 28] This graph compares real-time graphs showing the binding of protease-treated antibodies and untreated antibodies to PD-1. The thick black line represents the protease-treated antibody, and the thin gray line represents the untreated antibody. The X-axis represents the measurement time (seconds), with the start of measurement set to 0 seconds. The Y-axis shows the binding time. The name of each graph indicates the antibody used; the "None" graph uses only PBS buffer and no antibody. [Figure 29]This figure compares real-time graphs showing the binding of free PD-1 to 5C4-bio in samples treated with protease in the presence of PD-1 and samples not treated with protease in the presence of PD-1. The thick black line represents the protease-treated sample, and the thin gray line represents the untreated sample. The X-axis represents the measurement time (seconds), with the start of measurement set to 0 seconds. The Y-axis shows the binding. The names of each graph indicate the antibodies contained in the sample; the "Antigen and Protease" graph contains only PD-1 in the sample and no antibodies. [Figure 30] This figure compares real-time graphs showing the binding of free PD-1 to 5C4-bio in solutions of protease-treated fusion protein and untreated protein. The thick black line represents the protease-treated sample, and the thin gray line represents the untreated sample. The X-axis represents the measurement time (seconds), with the start of measurement set to 0 seconds. The Y-axis shows the binding. The name of each graph indicates the antibody in the fusion protein. In the None (antigen only) graph, only the antigen PD-1 was used as the evaluation sample without the fusion protein. In the graph labeled 5C4H-G1T4 / 5C4L-KT0, only the 5C4H-G1T4 / 5C4L-KT0 antibody was used without the fusion protein. [Figure 31] This is the electrophoresis result of protease-treated antibody-PD-1 fusion proteins. The Protease(+) lane represents protease-treated fusion proteins, and the Protease(-) lane represents untreated fusion proteins. [Figure 32] This figure shows the in vivo cleavage efficiency of an antibody molecule containing a protease cleavage sequence, administered to mice. [Modes for carrying out the invention]

[0013] In the present invention, polypeptides typically refer to peptides and proteins having a length of about 4 amino acids or more. While polypeptides in the present invention are typically polypeptides consisting of artificially designed sequences, they are not particularly limited and may, for example, be polypeptides of biological origin. They may also be natural polypeptides, synthetic polypeptides, recombinant polypeptides, etc. Furthermore, fragments of the above-mentioned polypeptides are also included in the polypeptides of the present invention.

[0014] In this specification, amino acids are represented by one-letter codes, three-letter codes, or both, for example, Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, Val / V.

[0015] For modifying amino acids in the amino acid sequence of polypeptides, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately employed. In addition, several known methods can be used for modifying amino acids by substituting them with amino acids other than natural ones (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing tRNA in which a non-natural amino acid is bound to the complementary amber suppressor tRNA of the UAG codon (amber codon), one of the stop codons, is also suitably used.

[0016] In this specification, the term "and / or" used to describe amino acid modification sites includes any combination of "and" and "or" as appropriate. Specifically, for example, "amino acids 37, 45, and / or 47 are substituted" includes the following variations of amino acid modification: (a) No. 37, (b) No. 45, (c) No. 47, (d) No. 37 and No. 45, (e) No. 37 and No. 47, (f) No. 45 and No. 47, (g) No. 37, No. 45 and No. 47.

[0017] In this specification, expressions that include the one-letter or three-letter codes of the original and modified amino acids before and after a number representing a specific position may be used as appropriate to indicate amino acid modifications. For example, the modification F37V or Phe37Val, used when making amino acid substitutions in the antibody variable region, represents the substitution of Phe at position 37, as represented by Kabat numbering, to Val. That is, the number represents the position of the amino acid as represented by Kabat numbering, the one-letter or three-letter code of the amino acid listed before it represents the original amino acid, and the one-letter or three-letter code of the amino acid listed after it represents the substituted amino acid. Similarly, the modification P238A or Pro238Ala, used when making amino acid substitutions in the Fc region included in the antibody constant region, represents the substitution of Pro at position 238, as represented by EU numbering, to Ala. That is, the number represents the position of the amino acid as represented by EU numbering, the one-letter or three-letter code of the amino acid listed before it represents the original amino acid, and the one-letter or three-letter code of the amino acid listed after it represents the substituted amino acid.

[0018] This invention relates to a ligand-binding molecule having a cleavage site, wherein the binding to the ligand is weakened when the cleavage site is cleaved. The ligand-binding molecule of this invention is a polypeptide and refers to a molecule capable of binding to a ligand.

[0019] The ligand-binding molecule of the present invention is a molecule capable of binding to a ligand, particularly a molecule capable of binding to a ligand in an uncleaved state. Here, "binding" usually refers to binding by interactions mainly involving non-covalent bonds such as electrostatic forces, van der Waals forces, and hydrogen bonds. Preferred examples of ligand-binding modes of the ligand-binding molecule of the present invention, but not limited to these, include antigen-antibody reactions in which an antigen-binding region, antigen-binding molecule, antibody, and antibody fragment bind to an antigen.

[0020] Furthermore, "ligand-binding capability" means that the ligand-binding molecule can bind to the ligand even if the ligand and the ligand are separate molecules, and does not mean that the ligand-binding molecule and the ligand are linked by a covalent bond. For example, the fact that the ligand and the ligand-binding molecule are covalently bonded via a linker does not mean that it is ligand-binding capability. Also, "weakening of ligand binding" means that the ability to bind is weakened. For example, if the ligand and the ligand-binding molecule are covalently bonded via a linker, the cleavage of that linker does not mean that the binding to the ligand is weakened. In this invention, as long as the ligand-binding molecule is ligand-binding capability, the ligand-binding molecule may be linked to the ligand via a linker or the like.

[0021] The ligand-binding molecule of the present invention is limited only to the fact that it binds to the ligand in an uncleaved state; any molecule with any structure can be used as long as it can bind to the target ligand in an uncleaved state. Examples of ligand-binding molecules, though not limited to these, include, for example, the heavy chain variable region (VH) and light chain variable region (VL) of antibodies, single-domain antibodies (sdAb), a module called the A domain of about 35 amino acids contained in Avimer, a cell membrane protein present in living organisms (International Publication WO2004 / 044011, WO2005 / 040229), Adnectin containing the 10Fn3 domain, a protein-binding domain in fibronectin, a glycoprotein expressed on the cell membrane (International Publication WO2002 / 032925), Affibody (International Publication WO1995 / 001937), which uses an IgG-binding domain as a scaffold to form a bundle of three helices consisting of 58 amino acids of Protein A, and ankyrin repeats, which have a structure in which a turn containing 33 amino acid residues and two antiparallel helical and loop subunits are repeatedly stacked. Examples include DARPins (Designed Ankyrin Repeat proteins) (International Publication WO2002 / 020565), which are regions exposed on the molecular surface of repeat (AR); Anticalin, etc. (International Publication WO2003 / 029462), which are four loop regions in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) that support one side of a barrel structure twisted towards the center by eight highly conserved antiparallel strands; and recessed regions of parallel sheet structures within a horseshoe-shaped structure in which leucine-rich repeat (LRR) modules, which do not possess the structure of immunoglobulins, are repeatedly stacked, as part of the acquired immune system of jawless fish such as lampreys and hagfish (International Publication WO2008 / 016854).

[0022] In this specification, the term “antibody” is used in its broadest sense and encompasses a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0023] Methods for producing antibodies with desired binding activity are known to those skilled in the art. The following is an example of a method for producing antibodies that bind to IL-6R (anti-IL-6R antibodies). Antibodies that bind to antigens other than IL-6R can also be produced as appropriate in accordance with the following examples.

[0024] Anti-IL-6R antibodies can be obtained as polyclonal or monoclonal antibodies using known methods. Mammalian-derived monoclonal antibodies are preferably produced as anti-IL-6R antibodies. Mammalian-derived monoclonal antibodies include those produced by hybridomas and those produced by host cells transformed with expression vectors containing antibody genes using genetic engineering techniques. Antibodies referred to in this application include "humanized antibodies" and "chimeric antibodies."

[0025] Monoclonal antibody-producing hybridomas can be produced using known techniques, for example, as follows: Mammals are immunized according to a standard immunization method using the IL-6R protein as a sensitizing antigen. The resulting immune cells are fused with known parent cells by a standard cell fusion method. Next, hybridomas that produce anti-IL-6R antibodies can be selected by screening monoclonal antibody-producing cells using a standard screening method.

[0026] Specifically, the production of monoclonal antibodies is carried out as follows: First, the IL-6R protein, which can be used as a sensitizing antigen for antibody acquisition, can be obtained by expressing the IL-6R gene. That is, suitable host cells are transformed by inserting the gene sequence encoding IL-6R into a known expression vector. The desired human IL-6R protein is purified from the host cells or culture supernatant by a known method. To obtain soluble IL-6R from the culture supernatant, soluble IL-6R is expressed, for example, as described by Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968). Alternatively, purified native IL-6R protein can also be used as a sensitizing antigen.

[0027] The purified IL-6R protein can be used as a sensitizing antigen for immunization against mammals. A partial peptide of IL-6R can also be used as a sensitizing antigen. In this case, the partial peptide can be obtained by chemical synthesis from the amino acid sequence of human IL-6R. It can also be obtained by incorporating a part of the IL-6R gene into an expression vector and expressing it. Furthermore, it can be obtained by degrading the IL-6R protein using a protease, but the region and size of the IL-6R peptide used as a partial peptide are not particularly limited to any special form. Preferably, the number of amino acids constituting the peptide to be used as a sensitizing antigen is at least 5, for example, 6 or more, or 7 or more. More specifically, a peptide of 8 to 50 residues, preferably 10 to 30 residues, can be used as a sensitizing antigen.

[0028] Furthermore, fusion proteins obtained by fusing a desired partial polypeptide or peptide of the IL-6R protein with a different polypeptide can be used as sensitization antigens. For example, antibody Fc fragments or peptide tags can be suitably used to produce fusion proteins used as sensitization antigens. A vector expressing a fusion protein can be produced by fusing genes encoding two or more desired polypeptide fragments in-frame, and inserting the fusion gene into an expression vector as described above. The method for producing fusion proteins is described in Molecular Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58 (1989) Cold Spring Harbor Lab. press). Methods for obtaining IL-6R used as a sensitization antigen and immunization methods using it are also specifically described in WO2003 / 000883, WO2004 / 022754, WO2006 / 006693, etc.

[0029] While the mammals immunized with the sensitizing antigen are not limited to specific animals, it is preferable to select them considering their compatibility with the parent cells used for cell fusion. Generally, rodents such as mice, rats, hamsters, rabbits, and monkeys are preferred.

[0030] The animals described above are immunized with the sensitizing antigen according to known methods. For example, a common method is to administer the sensitizing antigen to mammals intraperitoneally or subcutaneously. Specifically, the sensitizing antigen, diluted to an appropriate dilution ratio with PBS (Phosphate-Buffered Saline) or physiological saline, is mixed with a conventional adjuvant, such as Freund's complete adjuvant, if desired, and emulsified. After emulsification, the sensitizing antigen is administered to mammals several times every 4 to 21 days. A suitable carrier may also be used during immunization with the sensitizing antigen. In particular, when a partial peptide with a small molecular weight is used as the sensitizing antigen, it may be desirable to immunize with the sensitizing antigen peptide bound to a carrier protein such as albumin or keyhole limpet hemocyanin.

[0031] Furthermore, hybridomas that produce the desired antibody can also be produced using DNA immunization as follows. DNA immunization is an immunization method in which a vector DNA constructed in such a manner that a gene encoding an antigen protein can be expressed in the immunized animal is administered, and the sensitized antigen is expressed in the immunized animal, thereby providing immune stimulation. Compared to general immunization methods in which protein antigens are administered to immunized animals, DNA immunization is expected to have the following advantages. - It is possible to maintain the structure of membrane proteins such as IL-6R and deliver immunostimulation. - There is no need to purify immune antigens.

[0032] To obtain the monoclonal antibody of the present invention by DNA immunization, first, DNA expressing the IL-6R protein is administered to an immunized animal. The DNA encoding IL-6R can be synthesized by known methods such as PCR. The obtained DNA is inserted into a suitable expression vector and administered to an immunized animal. Commercial expression vectors such as pcDNA3.1 can be suitably used as the expression vector. Commonly used methods can be used to administer the vector into a living organism. For example, DNA immunization is performed by introducing gold particles to which the expression vector is adsorbed into the cells of an immunized animal using a gene gun. Furthermore, antibodies that recognize IL-6R can also be produced using the method described in International Publication WO 2003 / 104453.

[0033] After the mammal is immunized in this manner and an increase in antibody titers binding to IL-6R in the serum is confirmed, immune cells are collected from the mammal and used for cell fusion. Splenocytes, in particular, may be used as preferred immune cells.

[0034] Mammalian myeloma cells are used as the cells fused with the aforementioned immune cells. It is preferable that the myeloma cells possess appropriate selection markers for screening. A selection marker refers to a trait that allows (or prevents) survival under specific culture conditions. Known selection markers include hypoxanthine-guanine-phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) or thymidine kinase deficiency (hereinafter abbreviated as TK deficiency). Cells lacking HGPRT or TK are hypoxanthine-aminopterin-thymidine sensitive (hereinafter abbreviated as HAT sensitive). HAT-sensitive cells cannot synthesize DNA in HAT-selective medium and die, but when fused with normal cells, they can continue DNA synthesis using the normal cell's salvage pathway and thus proliferate even in HAT-selective medium.

[0035] HGPRT-deficient and TK-deficient cells can be selected in media containing 6-thioguanine, 8-azaguanine (hereinafter abbreviated as 8AG), or 5'-bromodeoxyuridine, respectively. Normal cells that incorporate these pyrimidine analogs into their DNA will die. On the other hand, cells lacking these enzymes and unable to incorporate these pyrimidine analogs can survive in the selective medium. Another selection marker, known as G418 resistance, confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) via the neomycin resistance gene. Various myeloma cells suitable for cell fusion are known.

[0036] Examples of such myeloma cells include P3 (P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (C. Eur. J. Immunol. (1976) 6 (7), 511-519), MPC-11 (Cell (1976) 8 (3), 405-415), SP2 / 0 (Nature (1978) 276 (5685), 269-270), FO (J. Immunol. Methods (1980) 35 (1-2), 1-21), S194 / 5.XX0.BU.1 (J. Exp. Med.(1978)148 (1), 313-323), R210 (Nature(1979)277 (5692), 131-133), etc., can be suitably used.

[0037] Cell fusion between the immune cells and myeloma cells is basically performed according to known methods, such as the method of Köhler and Myrstein et al. (Methods Enzymol. (1981) 73, 3-46). More specifically, the cell fusion can be carried out, for example, in a normal nutrient culture medium in the presence of a cell fusion promoter. Examples of fusion promoters include polyethylene glycol (PEG) and Sendai virus (HVJ), and additional adjuvants such as dimethyl sulfoxide may be added as desired to further enhance fusion efficiency.

[0038] The ratio of immune cells to myeloma cells can be set arbitrarily. For example, it is preferable to use 1 to 10 times more immune cells than myeloma cells. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium, MEM culture medium, or other common culture mediums used for this type of cell culture can be used, and serum supplements such as fetal bovine serum (FCS) may be suitably added.

[0039] Cell fusion is performed by thoroughly mixing predetermined amounts of the immune cells and myeloma cells in the culture medium, and then adding a PEG solution (for example, with an average molecular weight of about 1000 to 6000) that has been preheated to about 37°C, usually at a concentration of 30 to 60% (w / v). The desired fused cells (hybridomas) are formed by the gradual mixing of the mixture. Subsequently, the appropriate culture medium mentioned above is added sequentially, and the process of centrifugation and removal of the supernatant is repeated, thereby removing cell fusion agents and other substances unfavorable to hybridoma growth.

[0040] The hybridomas obtained in this manner can be selected by culturing them in a standard selective culture medium, such as HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Culturing with the HAT culture medium can be continued for a sufficient time (usually several days to several weeks) to kill cells other than the desired hybridoma (non-fusion cells). Subsequently, screening and single cloning of hybridomas that produce the desired antibody is performed using a standard limiting dilution method.

[0041] The hybridomas obtained in this way can be selected by using a selective culture medium corresponding to the selection markers present in the myeloma used for cell fusion. For example, cells lacking HGPRT or TK can be selected by culturing them in HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells that successfully fuse with normal cells can be selectively proliferated in the HAT culture medium. Culturing with the HAT culture medium is continued for a sufficient amount of time for cells other than the desired hybridoma (non-fused cells) to die. Specifically, generally, the desired hybridoma can be selected by culturing for several days to several weeks. Subsequently, screening and single cloning of hybridomas that produce the desired antibody can be performed using the usual limiting dilution method.

[0042] Screening and single cloning of desired antibodies can be suitably carried out by known antigen-antibody reaction-based screening methods. For example, a monoclonal antibody that binds to IL-6R can bind to IL-6R expressed on the cell surface. Such monoclonal antibodies can be screened, for example, by FACS (fluorescence activated cell sorting). FACS is a system that allows for the measurement of antibody binding to the cell surface by analyzing cells contacted with a fluorescent antibody using laser light and measuring the fluorescence emitted by individual cells.

[0043] To screen hybridomas that produce the monoclonal antibody of the present invention by FACS, cells expressing IL-6R are first prepared. Preferred cells for screening are mammalian cells that overexpress IL-6R. By using untransformed mammalian cells as the host cell as a control, the antibody binding activity to IL-6R on the cell surface can be selectively detected. That is, by selecting hybridomas that produce antibodies that do not bind to host cells but bind to IL-6R-overexpressing cells, hybridomas that produce IL-6R monoclonal antibodies can be obtained.

[0044] Alternatively, the binding activity of antibodies against immobilized IL-6R-expressing cells can be evaluated based on the principles of ELISA. For example, IL-6R-expressing cells are immobilized in the wells of an ELISA plate. The culture supernatant of hybridomas is brought into contact with the immobilized cells in the wells, and antibodies that bind to the immobilized cells are detected. If the monoclonal antibody is derived from a mouse, the antibody bound to the cells can be detected by an anti-mouse immunoglobulin antibody. Hybridomas that produce the desired antibody with antigen-binding ability, selected through these screenings, can be cloned by methods such as limiting dilution.

[0045] The hybridomas producing monoclonal antibodies thus created can be subcultured in a normal culture medium. Furthermore, these hybridomas can be stored for extended periods in liquid nitrogen.

[0046] The hybridoma can be cultured according to conventional methods, and the desired monoclonal antibody can be obtained from the culture supernatant. Alternatively, the hybridoma can be administered to a compatible mammal to proliferate, and the monoclonal antibody can be obtained from its ascites fluid. The former method is suitable for obtaining high-purity antibodies.

[0047] Antibodies encoded by antibody genes cloned from antibody-producing cells such as hybridomas can also be suitably utilized. By incorporating the cloned antibody gene into a suitable vector and introducing it into a host, the antibody encoded by the gene is expressed. Methods for isolating antibody genes, introducing them into vectors, and transforming host cells have already been established, for example, by Vandamme et al. (Eur.J. Biochem.(1990)192 (3), 767-775). Methods for producing recombinant antibodies are also known, as described below.

[0048] For example, cDNA encoding the variable region (V region) of the anti-IL-6R antibody can be obtained from hybridoma cells that produce anti-IL-6R antibodies. To do this, total RNA is usually extracted from the hybridoma first. Methods such as the following can be used to extract mRNA from cells. - Guanidine ultracentrifugation (Biochemistry (1979) 18 (24), 5294-5299) - AGPC method (Anal. Biochem. (1987) 162 (1), 156-159)

[0049] The extracted mRNA can be purified using an mRNA Purification Kit (GE Healthcare Biosciences), etc. Alternatively, kits for directly extracting total mRNA from cells are commercially available, such as the QuickPrep mRNA Purification Kit (GE Healthcare Biosciences). mRNA can be obtained from hybridomas using such kits. From the obtained mRNA, cDNA encoding the antibody V region can be synthesized using reverse transcriptase. cDNA can be synthesized using an AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation), etc. Furthermore, for cDNA synthesis and amplification, the SMART RACE cDNA amplification kit (Clontech) and the 5'-RACE method using PCR (Proc. Natl. Acad. Sci. USA (1988) 85 (23), 8998-9002, Nucleic Acids Res. (1989) 17 (8), 2919-2932) may be used as appropriate. Furthermore, during the process of synthesizing cDNA, appropriate restriction enzyme sites, as described later, can be introduced at both ends of the cDNA.

[0050] The target cDNA fragment is purified from the obtained PCR product and then ligated to vector DNA. A recombinant vector is thus prepared, introduced into E. coli or other organisms, and after colony selection, the desired recombinant vector can be prepared from the E. coli that formed the colonies. Then, whether or not the recombinant vector possesses the target cDNA base sequence is confirmed by known methods, such as dideoxynucleotide chain intermination.

[0051] To obtain genes encoding variable regions, the 5'-RACE method using primers for variable region gene amplification is a convenient approach. First, cDNA is synthesized using RNA extracted from hybridoma cells as a template, yielding a 5'-RACE cDNA library. Commercially available kits, such as the SMART RACE cDNA amplification kit, can be used as appropriate for synthesizing the 5'-RACE cDNA library.

[0052] The obtained 5'-RACE cDNA library is used as a template to amplify the antibody gene by PCR. Primers for mouse antibody gene amplification can be designed based on known antibody gene sequences. These primers have different nucleotide sequences for each immunoglobulin subclass. Therefore, it is desirable to determine the subclass in advance using a commercially available kit such as the Iso Strip mouse monoclonal antibody isotyping kit (Roche Diagnostics).

[0053] Specifically, for example, when the goal is to obtain genes encoding mouse IgG, primers capable of amplifying genes encoding γ1, γ2a, γ2b, and γ3 as heavy chains, and κ and λ chains as light chains, can be used. To amplify the variable region genes of IgG, a primer that anneals to the constant region close to the variable region is generally used for the 3' side. On the other hand, for the 5' side primer, the primers included with the 5' RACE cDNA library preparation kit are used.

[0054] Using the PCR product thus amplified, an immunoglobulin consisting of a combination of heavy and light chains can be reconstituted. The binding activity of the reconstituted immunoglobulin to IL-6R can be used as an indicator to screen for the desired antibody. For example, when the goal is to obtain an antibody against IL-6R, it is even more preferable that the antibody's binding to IL-6R is specific. Antibodies that bind to IL-6R can be screened, for example, as follows: (1) A step of contacting IL-6R expressing cells with an antibody containing a V region encoded by cDNA obtained from a hybridoma, (2) A step to detect the binding of IL-6R-expressing cells to an antibody, and (3) A step of selecting an antibody that binds to IL-6R expressing cells.

[0055] Methods for detecting the binding of antibodies to IL-6R-expressing cells are known. Specifically, the binding of antibodies to IL-6R-expressing cells can be detected by methods such as FACS, as mentioned earlier. Fixed specimens of IL-6R-expressing cells can be used as appropriate to evaluate the binding activity of antibodies.

[0056] As a screening method for antibodies using binding activity as an indicator, the panning method using phage vectors is also suitably employed. When antibody genes are obtained from a polyclonal antibody-expressing cell population as a library of heavy chain and light chain subclasses, the screening method using phage vectors is advantageous. Genes encoding the variable regions of the heavy chain and light chain can be linked with a suitable linker sequence to form a single-chain Fv (scFv). By inserting the gene encoding scFv into a phage vector, a phage expressing scFv on its surface can be obtained. After contact between this phage and the desired antigen, the phage bound to the antigen can be recovered, thereby recovering the DNA encoding scFv with the desired binding activity. By repeating this operation as needed, scFv with the desired binding activity can be enriched.

[0057] After obtaining the cDNA encoding the V region of the target anti-IL-6R antibody, the cDNA is digested by restriction enzymes that recognize restriction enzyme sites inserted at both ends of the cDNA. Preferred restriction enzymes recognize and digest base sequences that appear infrequently in the base sequence constituting the antibody gene. Furthermore, to insert one copy of the digested fragment into the vector in the correct orientation, insertion of a restriction enzyme that provides an adhesive end is preferable. By inserting the cDNA encoding the V region of the anti-IL-6R antibody, digested as described above, into a suitable expression vector, an antibody expression vector can be obtained. At this time, if the gene encoding the antibody constant region (C region) and the gene encoding the V region are fused in-frame, a chimeric antibody is obtained. Here, a chimeric antibody means that the constant region and the variable region originate from different sources. Therefore, in addition to heterologous chimeric antibodies such as mouse-human, human-human allologous chimeric antibodies are also included in the chimeric antibodies of this invention. A chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector that already has a constant region. Specifically, for example, a restriction enzyme recognition sequence for a restriction enzyme that digests the V region gene can be appropriately placed on the 5' end of an expression vector containing DNA encoding the desired antibody constant region (C region). A chimeric antibody expression vector is constructed by in-frame fusion of the two, which have been digested with the same combination of restriction enzymes.

[0058] To produce an anti-IL-6R monoclonal antibody, the antibody gene is incorporated into an expression vector so that it is expressed under the control of an expression regulatory region. This regulatory region for antibody expression includes, for example, enhancers and promoters. Furthermore, an appropriate signal sequence may be added to the amino terminus so that the expressed antibody is secreted extracellularly. For example, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 536) may be used as the signal sequence, but other suitable signal sequences may also be added. The expressed polypeptide is cleaved at the carboxyl terminus of the above sequence, and the cleaved polypeptide can be secreted extracellularly as a mature polypeptide. Subsequently, recombinant cells expressing DNA encoding the anti-IL-6R antibody can be obtained by transforming a suitable host cell with this expression vector.

[0059] An "antibody fragment" refers to a molecule other than the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments are not limited to these, but include Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0060] The terms "full-length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a native antibody, or having a heavy chain containing an Fc region as defined herein.

[0061] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of an antibody (VH and VL, respectively) typically have a similar structure, with each domain containing four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0062] As used herein, the terms “complementarity-determining region” or “CDR” refer to the regions of the variable domain of an antibody that are hypervariable in sequence and / or form structurally defined loops (“hypervariable loops”) and / or antigen contact residues (“antigen contacts”). Typically, an antibody contains six CDRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Illustrative CDRs as used herein include: (a) Hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contact occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and, (d) A combination of (a), (b), and / or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Unless otherwise indicated, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein in accordance with Kabat et al.

[0063] The "framework" or "FR" refers to variable domain residues other than complementarity-determining region (CDR) residues. The variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the sequences of the CDR and FR usually appear in the VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0064] In this specification, the term “constant region” or “constant domain” refers to the portion of an antibody other than the variable region. For example, an IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons, composed of two identical disulfide-linked light chains and two identical heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by a heavy chain constant region (CH) containing the CH1 domain, hinge region, CH2 domain, and CH3 domain. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chains of native antibodies may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0065] The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0066] In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes the Fc region of the native sequence and mutant Fc regions. In one embodiment, in the case of human IgG1, the heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain, provided that the lysine (Lys447) or glycine-lysine (Gly446-Lys447) at the C-terminus of the Fc region is present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0067] The ligand-binding molecule of the present invention is a polypeptide containing a cleavage site. The cleavage site can be cleaved, for example, by an enzyme, reduced by a reducing agent, or photodegraded. The cleavage site may be located at any position in the polypeptide, as long as cleavage weakens the binding of the ligand-binding molecule to the ligand. Furthermore, the polypeptide may contain one or more cleavage sites.

[0068] Furthermore, the ligand-binding molecule of the present invention exhibits weaker ligand binding (i.e., reduced) in the cleaved state compared to the uncleaved state. In embodiments where the binding of the ligand-binding molecule to the ligand is an antigen-antibody reaction, the reduction in ligand binding can be evaluated by the ligand-binding activity of the ligand-binding molecule.

[0069] The binding activity of ligand-binding molecules and ligands can be evaluated using well-known methods such as FACS, ELISA format, ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), BIACORE method utilizing surface plasmon resonance (SPR), and BLI (Bio-Layer Interferometry) method (Octet) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010). The ALPHA screen is performed using ALPHA technology, which employs two beads, a donor and an acceptor, based on the following principle: Molecules bound to the donor bead interact with molecules bound to the acceptor bead, and an emission signal is detected only when the two beads are in close proximity. A photosensitiver within the donor bead, excited by a laser, converts surrounding oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and, upon reaching the nearby acceptor bead, triggers a chemiluminescent reaction within the bead, ultimately emitting light. When the molecules bound to the donor bead and the molecules bound to the acceptor bead do not interact, the singlet oxygen produced by the donor bead does not reach the acceptor bead, and therefore no chemiluminescent reaction occurs.

[0070] For example, a biotin-labeled ligand-binding molecule is bound to a donor bead, and a ligand tagged with glutathione S-transferase (GST) is bound to an acceptor bead. In the absence of competing untagged ligand-binding molecules, the ligand-binding molecule and the ligand interact, producing a signal in the 520-620 nm range. Untagged ligand-binding molecules compete with the interaction between the tagged ligand-binding molecule and the ligand. The relative binding affinity can be determined by quantifying the decrease in fluorescence that results from this competition. Biotinylation of ligand-binding molecules such as antibodies using sulfo-NHS-biotin is a well-known method. As a method for tagging ligands with GST, a GST-fusion ligand can be expressed in cells containing a vector capable of expressing a fusion gene in which a polynucleotide encoding the ligand and a polynucleotide encoding GST are fused in frame, and then purified using a glutathione column. The obtained signals can be suitably analyzed by fitting them to a one-site competition model that utilizes nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

[0071] When one of the substances whose interaction is to be observed (ligand) is fixed onto a gold thin film on a sensor chip, and light is shone from the back of the sensor chip so as to cause total internal reflection at the interface between the gold thin film and glass, a region of reduced reflectance intensity (SPR signal) is formed in a part of the reflected light. When the other substance whose interaction is to be observed (analyte) is flowed onto the surface of the sensor chip and the ligand and analyte bind, the mass of the immobilized ligand molecule increases, and the refractive index of the solvent on the surface of the sensor chip changes. This change in refractive index causes the position of the SPR signal to shift (conversely, when the bond dissociates, the signal position returns to its original position). The Biacore system plots the amount of the above shift, i.e., the change in mass on the sensor chip surface, on the vertical axis and displays the change in mass over time as measurement data (sensorgram). From the sensorgram curve, kinetics: the binding rate constant (ka) and the dissociation rate constant (kd) can be determined, and the dissociation constant (KD) can be determined from the ratio of these constants. Inhibition measurement methods and equilibrium value analysis methods are also suitably used in the BIACORE method. Examples of inhibition assays are described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010, and examples of equilibrium value analysis methods are described in Methods Enzymol. 2000;323:325-40.

[0072] A reduced ability of a ligand-binding molecule to bind to a ligand means, for example, that, based on the measurement method described above, the amount of ligand bound per test ligand-binding molecule is 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, 15% or less, particularly preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less compared to the control ligand-binding molecule. Any desired index may be used as an indicator of binding activity, for example, the dissociation constant (KD) may be used. When using the dissociation constant (KD) as an evaluation index for binding activity, a larger dissociation constant (KD) of the test ligand-binding molecule with respect to the ligand compared to the dissociation constant (KD) of the control ligand-binding molecule with respect to the ligand indicates that the binding activity of the test ligand-binding molecule with respect to the ligand is weaker than that of the control ligand-binding molecule. A weakened ability to bind to a ligand means, for example, that the keying factor (KD) of the test ligand-binding molecule relative to the ligand is 2 times or more, preferably 5 times or more, 10 times or more, and particularly preferably 100 times or more, compared to the keying factor (KD) of the control ligand-binding molecule relative to the ligand. Examples of control ligand-binding molecules include uncleaved ligand-binding molecules.

[0073] In one embodiment of the present invention, the ligand is released from the ligand-binding molecule by cleavage at a cleavage site. Here, if the ligand is bound to a portion of the ligand-binding molecule via a linker and there is no cleavage site on the linker, the ligand will be released while still connected to that portion of the ligand-binding molecule via the linker (see, for example, Figure 1). In this way, even if the ligand is released together with a portion of the ligand-binding molecule, as long as it is released from the majority of the ligand-binding molecule, it can be said that the ligand has been released from the ligand-binding molecule.

[0074] One method for detecting the release of a ligand from a ligand-binding molecule by cleavage at a cleavage site is to detect the ligand using a ligand-detection antibody that recognizes the ligand. When the ligand-binding molecule is an antibody fragment, it is preferable that the ligand-detection antibody binds to an epitope similar to that of the ligand-binding molecule. Ligand detection using ligand-detection antibodies can be confirmed by well-known methods such as FACS, ELISA format, ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), BIACORE method utilizing surface plasmon resonance (SPR), and BLI (Bio-Layer Interferometry) method (Octet) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010). For example, when detecting ligand release using Octet, ligand release can be detected by biotinylating a ligand-recognizing antibody, contacting it with a biosensor, and then measuring its binding to the ligand in the sample. Specifically, ligand release can be detected by measuring the amount of ligand using a ligand-detection antibody in a sample containing ligand-binding molecules and ligand before or after protease treatment, and comparing the amount of ligand detected in the sample before and after protease treatment. Furthermore, ligand release can be detected by measuring the amount of ligand using a ligand-detection antibody in a sample containing protease, ligand-binding molecules, and ligand, and in a sample containing ligand-binding molecules and ligand without protease, and comparing the amount of ligand detected in the sample with and without protease. More specifically, ligand release can be detected by the method in the embodiment of this application. When a ligand-binding molecule is fused with a ligand to form a fusion protein, ligand release can be detected by measuring the amount of ligand using a ligand-detection antibody in a sample containing the fusion protein before or after protease treatment, and comparing the amount of ligand detected in the sample before and after protease treatment. Furthermore, ligand release can be detected by measuring the amount of ligand using a ligand detection antibody in samples containing a protease and a fusion protein, and in samples containing a fusion protein but without a protease, and comparing the amount of ligand detected in the samples with and without protease. More specifically, ligand release can be detected by the method described in the present invention.

[0075] Furthermore, in embodiments where the biological activity of a ligand is inhibited upon binding to a ligand-binding molecule, the release of the ligand from the ligand-binding molecule can be detected by measuring the biological activity of the ligand in the sample. Specifically, ligand release can be detected by measuring and comparing the biological activity of the ligand in a sample containing a ligand-binding molecule and a ligand before or after protease treatment. Ligand release can also be detected by measuring and comparing the biological activity of the ligand in a sample containing a protease, a ligand-binding molecule, and a ligand, and in a sample containing a ligand-binding molecule and a ligand without a protease. When a ligand-binding molecule is fused with a ligand to form a fusion protein, ligand release can be detected by measuring and comparing the biological activity of the ligand in a sample containing the fusion protein before or after protease treatment. Ligand release can also be detected by measuring and comparing the biological activity of the ligand in a sample containing a protease and a fusion protein, and in a sample containing a fusion protein without a protease.

[0076] In one embodiment of the present invention, the cleavage site includes a protease cleavage sequence and is cleaved by a protease.

[0077] In this specification, the term "protease" refers to an enzyme such as an endopeptidase or exopeptidase that hydrolyzes peptide bonds, usually an endopeptidase. The proteases used in the present invention are limited only by their ability to cleave protease cleavage sequences, and their type is not particularly limited. In some embodiments, target tissue-specific proteases are used. Target tissue-specific proteases are, for example, (1) Proteases that are expressed at higher levels in target tissue than in normal tissue, (2) Proteases that have higher activity in target tissue than in normal tissue, (3) Proteases expressed at higher levels in target cells than in normal cells, (4) Proteases that have higher activity in target cells than in normal cells, This can refer to either of the above. In a more specific embodiment, cancer tissue-specific proteases or inflammatory tissue-specific proteases are used.

[0078] In this specification, the term “target tissue” means tissue containing at least one target cell. In some embodiments of the present invention, the target tissue is cancerous tissue. In some embodiments of the present invention, the target tissue is inflammatory tissue.

[0079] The term "cancer tissue" means tissue containing at least one cancer cell. Therefore, it refers to all cell types that contribute to the formation of a tumor mass, including cancer cells and endothelial cells, such as cancer tissue containing cancer cells and blood vessels. In this specification, "tumor" means a foci of tumor tissue. The term "tumor" is generally used to mean either a benign or malignant neoplasm.

[0080] In this specification, "inflammatory tissue" includes, for example, the following: Joints in rheumatoid arthritis and osteoarthritis • Lungs (alveoli) in bronchial asthma and COPD • Digestive organs in inflammatory bowel disease, Crohn's disease, and ulcerative colitis • Fibrotic tissue in fibrosis of the liver, kidneys, and lungs • Tissues that are being rejected in organ transplants • Blood vessels and heart (myocardium) in arteriosclerosis and heart failure • Visceral fat in metabolic syndrome • Skin tissue in atopic dermatitis and other skin inflammations • Spinal nerves in herniated discs and chronic lower back pain

[0081] In some types of target tissues, proteases that are specifically expressed or specifically activated, or proteases that are thought to be associated with the disease state of the target tissue (target tissue-specific proteases), are known. For example, proteases that are specifically expressed in cancer tissue are disclosed in international publications WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846, among others. Furthermore, proteases thought to be associated with inflammation have been disclosed in J Inflamm (Lond). 2010; 7: 45., Nat Rev Immunol. 2006 Jul;6(7):541-50., Nat Rev Drug Discov. 2014 Dec;13(12):904-27., Respir Res. 2016 Mar 4;17:23., Dis Model Mech. 2014 Feb;7(2):193-203., and Biochim Biophys Acta. 2012 Jan;1824(1):133-45.

[0082] In addition to proteases that are specifically expressed in target tissues, there are also proteases that are specifically activated in target tissues. For example, proteases may be expressed in an inactive form and then become active, and in many tissues, substances that inhibit the active protease exist, and the activity is controlled by the activation process and the presence of inhibitors (Nat Rev Cancer. 2003 Jul;3(7):489-501.). In target tissues, the active protease may escape inhibition and be specifically activated. Active proteases can be measured using methods that employ antibodies that recognize active proteases (PNAS 2013 Jan 2; 110(1): 93-98.) or by fluorescently labeling the peptide recognized by the protease, quenching it before cleavage, and then emitting light after cleavage (Nat Rev Drug Discov. 2010 Sep;9(9):690-701. doi: 10.1038 / nrd3053.).

[0083] From one perspective, the term "target tissue-specific protease" is, (i) Proteases expressed at higher levels in target tissue than in normal tissue, (ii) Proteases that have higher activity in target tissue than in normal tissue, (iii) Proteases expressed at higher levels in target cells than in normal cells, (iv) Proteases that have higher activity in target cells than in normal cells, It can refer to any of the following:

[0084] While not meant to be interpreted restrictively, specific proteases include cysteine ​​proteases (including cathepsin family B, L, S, etc.), aspartyl proteases (cathepsin D, E, K, O, etc.), serine proteases (matryptase (including MT-SP1), cathepsin A and G), thrombin, plasmin, urokinase (uPA), tissue plasminogen activator (tPA), elastase, proteinase 3, thrombin, kallikrein, trip Metalloproteinases (including tase and chymase), metalloproteinases (including both membrane-bound (MMP14-17 and MMP24-25) and secreted (MMP1-13, MMP18-23 and MMP26-28) metalloproteinases (MMP1-28), protease A disintegrin and metalloproteinase (ADAM), metalloproteinases with A disintegrin or thrombospongin motifs (ADAMTS), meprin (meprin α(meprin alpha, meprin beta, CD10 (CALLA), prostate-specific antigen (PSA), regmine, TMPRSS3, TMPRSS4, neutrophil elastase (HNE), beta-secretase (BACE), fibroblast-activating protein alpha (FAP), granzyme B, guanidinobenzoate (GB), hepsin, neprilysin, NS3 / 4A, HCV-NS3 / 4, calpain, ADAMDEC1, renin, cathepsin C, cathepsin V / L2, cathepsin X / Z / P, Kurjipain, Otsubine 2, Kallikrein-related peptidases (KLKs (KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14)), Bone morphogenetic protein 1 (BMP-1), Activated protein C, Blood coagulation-related proteases (Factor VIIa, Factor IXa, Factor Xa, Factor XIa, Factor XIIa), HtrA1, lactoferrin, malapsin, PACE4, DESC1, dipeptidyl peptidase 4 (DPP-4), TMPRSS2, cathepsin F, cathepsin H, cathepsin L2, cathepsin O, cathepsin S, granzyme A, gepsin calpain 2, glutamate carboxypeptidase 2, AMSH-LikeExamples include proteases, AMSH, gamma secretase, anti-plasmin cleavage enzyme (APCE), Decysin 1, N-Acetylated Alpha-Linked Acidic Dipeptidase-Like 1 (NAALADL1), and furin.

[0085] From another perspective, target tissue-specific proteases can refer to cancer tissue-specific proteases or inflammatory tissue-specific proteases.

[0086] Examples of cancer tissue-specific proteases include those disclosed in international publications WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846, which are specifically expressed in cancer tissue.

[0087] The type of cancer tissue-specific protease that exhibits high specificity in the target cancer tissue yields a greater reduction in side effects. It is preferable that the concentration of the cancer tissue-specific protease in cancer tissue is at least five times higher than that in normal tissue, more preferably at least ten times higher, even more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. Furthermore, it is preferable that the activity of the cancer tissue-specific protease in cancer tissue is at least twice as high as that in normal tissue, more preferably at least three times higher, four times higher, five times higher, ten times higher, even more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. Furthermore, cancer tissue-specific proteases may be bound to the cell membrane of cancer cells, or they may not be bound to the cell membrane and may be secreted extracellularly. If cancer tissue-specific proteases are not bound to the cell membrane of cancer cells, it is preferable that the cancer tissue-specific protease is located inside or near the cancer tissue in order for the cytotoxicity by immune cells to be specific to cancer cells. In this specification, "near the cancer tissue" means within the range in which the cancer tissue-specific protease cleavage sequence is cleaved and exerts a ligand-binding activity reduction effect. However, it is preferable that this range does not damage normal cells as much as possible. From another perspective, cancer tissue-specific proteases are, (i) Proteases expressed at higher levels in cancer tissue than in normal tissue, (ii) Proteases that have higher activity in cancer tissue than in normal tissue, (iii) Proteases expressed at higher levels in cancer cells than in normal cells, (iv) Proteases that have higher activity in cancer cells than in normal cells, It is one of the following: Cancer tissue-specific proteases may be used individually or in combination of two or more. The number of types of cancer tissue-specific proteases can be appropriately determined by a person skilled in the art, taking into consideration the type of cancer being treated.

[0088] From the above viewpoint, among the proteases exemplified above, serine proteases and metalloproteases are preferred as cancer tissue-specific proteases, matryptase (including MT-SP1), urokinase (uPA), and metalloproteases are more preferred, and MT-SP1, uPA, MMP-2, and MMP-9 are even more preferred.

[0089] The type of inflammation-specific protease that exhibits high specificity in the inflammatory tissue being treated is preferable to achieve a reduction in side effects. It is preferable that the concentration of the inflammation-specific protease in inflammatory tissue is at least five times higher than that in normal tissue, more preferably at least ten times higher, even more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. Furthermore, it is preferable that the activity of the inflammation-specific protease in inflammatory tissue is at least twice as high as that in normal tissue, more preferably at least three times, four times, five times, ten times, even more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. Furthermore, the inflammation tissue-specific protease may be bound to the cell membrane of inflammatory cells, or it may not be bound to the cell membrane and may be secreted extracellularly. If the inflammation tissue-specific protease is not bound to the cell membrane of inflammatory cells, it is preferable that the inflammation tissue-specific protease is located inside or near the inflammatory tissue in order for the cytotoxicity by immune cells to be specific to inflammatory cells. In this specification, "near the inflammatory tissue" means within the range in which the inflammation tissue-specific protease cleavage sequence is cleaved and exerts a ligand-binding activity reduction effect. However, it is preferable that this range does not damage normal cells as much as possible. From another perspective, inflammation-specific proteases are, (i) Proteases expressed at higher levels in inflammatory tissue than in normal tissue, (ii) Proteases that have higher activity in inflammatory tissue than in normal tissue, (iii) Proteases expressed at higher levels in inflammatory cells than in normal cells, (iv) Proteases that have higher activity in inflammatory cells than in normal cells, It is one of the following: Inflammatory tissue-specific proteases may be used individually or in combination of two or more types. The number of types of inflammatory tissue-specific proteases can be appropriately determined by those skilled in the art, taking into consideration the disease condition being treated.

[0090] From the above perspective, among the proteases exemplified above, metalloproteases are preferred as inflammatory tissue-specific proteases, and among metalloproteases, ADAMTS5, MMP-2, MMP-7, MMP-9, and MMP-13 are more preferred.

[0091] A protease cleavage sequence is a specific amino acid sequence that is specifically recognized by a target tissue-specific protease when a polypeptide is hydrolyzed by that protease in an aqueous solution. From the standpoint of reducing side effects, the protease cleavage sequence is preferably an amino acid sequence that is hydrolyzed with high specificity by a target tissue-specific protease that is more specifically expressed in or more specifically activated in the target tissue / cells being treated. Specific examples of protease cleavage sequences include, for example, target sequences that are specifically hydrolyzed by proteases specifically expressed in cancer tissue, inflammatory tissue-specific proteases, etc., as disclosed in International Publications WO2013 / 128194, WO2010 / 081173, WO2009 / 025846, etc. Artificially modified sequences, such as those with amino acid mutations introduced into known target sequences that are specifically hydrolyzed by proteases, can also be used. Furthermore, protease cleavage sequences identified by methods known to those skilled in the art, as described in Nature Biotechnology 19, 661 - 667 (2001), may also be used. Furthermore, naturally occurring protease cleavage sequences may also be used. For example, sequences that undergo protease cleavage in proteins whose molecular shape changes upon protease cleavage can be used, such as TGFβ changing to its latent form upon protease cleavage.

[0092] Examples of protease cleavage sequences, but not limited to these, include International Publications WO2015 / 116933, WO2015 / 048329, WO2016 / 118629, WO2016 / 179257, WO2016 / 179285, WO2016 / 179335, WO2016 / 179003, WO2016 / 046778, WO2016 / 014974, U.S. Patent Publication US2016 / 0289324, U.S. Patent Publication US2016 / 0311903, PNAS (2000) 97: 7754-7759, Biochemical Journal (2010) 426: The sequences shown in pp. 219-228 and Beilstein J Nanotechnol. (2016) 7: 364-373 can be used. As described above, the protease cleavage sequence is more preferably an amino acid sequence that is specifically hydrolyzed by a suitable target tissue-specific protease. Among amino acid sequences that are specifically hydrolyzed by target tissue-specific proteases, the following amino acid sequences are preferred. LSGRSDNH (Sequence ID: 3, MT-SP1, can be cut by uPA) PLGLAG (Sequence ID: 34, can be cut by MMP-2 and MMP-9) VPLSLTMG (Sequence number: 35, can be cut by MMP-7) The following sequences can also be used as protease cleavage sequences. TSTSGRSANPRG (Sequence number: 66, MT-SP1, can be cut by uPA) ISSGLLSGRSDNH (Sequence number: 67, MT-SP1, can be cut by uPA) AVGLLAPPGGLSGRSDNH (Sequence number: 68, MT-SP1, can be cut by uPA) GAGVPMSMRGGAG (Sequence ID: 69, can be cut by MMP-1) GAGIPVSLRSGAG ​​(Sequence number: 70, can be cut by MMP-2) GPLGIAGQ (Sequence ID: 71, can be cut by MMP-2) GGPLGMLSQS (Sequence ID: 72, can be cut by MMP-2) PLGLWA (Sequence ID: 73, can be cut by MMP-2) GAGRPFSMIMGAG (Sequence ID: 74, can be cut by MMP-3) GAGVPLSLTMGAG (Sequence number: 75, can be cut by MMP-7) GAGVPLSLYSGAG (Sequence number: 76, can be cut by MMP-9) AANLRN (Sequence ID: 77, can be cleaved by MMP-11) AQAYVK (Sequence ID: 78, can be disconnected by MMP-11) AANYMR (Sequence ID: 79, can be cleaved by MMP-11) AAALTR (Sequence ID: 80, can be cut by MMP-11) AQNLMR (Sequence ID: 81, can be cleaved by MMP-11) AANYTK (Sequence ID: 82, can be cut by MMP-11) GAGPQGLAGQRGIVAG (Sequence ID: 83, can be cleaved by MMP-13) PRFKIIGG (Sequence ID: 84, cleavable by pro-urokinase) PRFRIIGG (SEQ ID NO: 85, cleavable by pro-urokinase) GAGSGRSAG (Sequence ID: 86, can be cut by uPA) SGRSA (Sequence ID: 87, can be cleaved by uPA) GSGRSA (Sequence ID: 88, can be cleaved by uPA) SGKSA (Sequence ID: 89, can be cut by uPA) SGRSS (Sequence ID: 90, can be cut by uPA) SGRRA (Sequence ID: 91, can be cut by uPA) SGRNA (Sequence ID: 92, cleavable by uPA) SGRKA (Sequence ID: 93, can be cut by uPA) QRGRSA (Sequence ID: 94, can be cleaved by tPA) GAGSLLKSRMVPNFNAG (Sequence ID: 95, cleavable by cathepsin B) TQGAAA (Sequence ID: 96, cleavable by cathepsin B) GAAAAA (Sequence ID: 97, cleavable by cathepsin B) GAGAAG (Sequence ID: 98, cleavable by cathepsin B) AAAAAG (Sequence ID: 99, cleavable by cathepsin B) LCGAAI (Sequence ID: 100, cleavable by cathepsin B) FAQALG (Sequence ID: 101, cleavable by cathepsin B) LLQANP (Sequence ID: 102, cleavable by cathepsin B) LAAANP (Sequence ID: 103, cleavable by cathepsin B) LYGAQF (Sequence ID: 104, cleavable by cathepsin B) LSQAQG (Sequence ID: 105, cleavable by cathepsin B) ASAASG (Sequence ID: 106, cleavable by cathepsin B) FLGASL (Sequence ID: 107, cleavable by cathepsin B) AYGATG (Sequence ID: 108, cleavable by cathepsin B) LAQATG (Sequence ID: 109, cleavable by cathepsin B) GAGSGVVIATVIVITAG (Sequence ID: 110, cleavable by cathepsin L) APMAEGGG (Sequence ID: 111, cleavable by meprin α and meprin β) EAQGDKII (Sequence ID: 112, cleavable by meprin α and meprin β) LAFSDAGP (Sequence ID: 113, cleavable by meprin α and meprin β) YVADAPK (Sequence ID: 114, cleavable by meprin α and meprin β) RRRRR (Sequence ID: 115, can be cleaved by Fuhrin) RRRRRR (Sequence ID: 116, cleavable by Fuhrin) GQSSRHRRAL (Sequence ID: 117, cleavable by Fuhrin) SSRHRRALD (Sequence ID: 118) RKSSIIIRMRDVVL (Sequence ID: 119, cleavable by plasminogen) SSSFDKGKYKKGDDA (Sequence ID: 120, cleavable by staphylokinase) SSSFDKGKYKRGDDA (Sequence ID: 121, cleavable by staphylokinase) IEGR (Sequence ID: 122, can be cut by FactorIXa) IDGR (Sequence ID: 123, can be cut by FactorIXa) GGSIDGR (Sequence ID: 124, can be cut by FactorIXa) GPQGIAGQ (Sequence ID: 125, cleavable by collagenase) GPQGLLGA (Sequence ID: 126, can be cleaved by collagenase) GIAGQ (Sequence ID: 127, cleavable by collagenase) GPLGIAG (Sequence ID: 128, cleavable by collagenase) GPEGLRVG (Sequence ID: 129, can be cleaved by collagenase) YGAGLGVV (Sequence ID: 130, cleavable by collagenase) AGLGVVER (Sequence ID: 131, cleavable by collagenase) AGLGISST (Sequence ID: 132, cleavable by collagenase) EPQALAMS (Sequence ID: 133, cleavable by collagenase) QALAMSAI (Sequence ID: 134, can be cleaved by collagenase) AAYHLVSQ (Sequence ID: 135, can be cleaved by collagenase) MDAFLESS (Sequence ID: 136, can be cleaved by collagenase) ESLPVVAV (Sequence ID: 137, can be cleaved by collagenase) SAPAVESE (Sequence ID: 138, can be cleaved by collagenase) DVAQFVLT (Sequence ID: 139, cleavable by collagenase) VAQFVLTE (Sequence ID: 140, can be disconnected by Collagenase) AQFVLTEG (Sequence ID: 141, can be cleaved by collagenase) PVQPIGPQ (Sequence ID: 142, cleavable by collagenase) LVPRGS (Sequence ID: 143, can be cut by Thrombin) TSGSGRSANARG (Sequence ID: 335) TSQSGRSANQRG (Sequence ID: 336) TSPSGRSAYPRG (Sequence ID: 337) TSGSGRSATPRG (Sequence ID: 338) TSQSGRSATPRG (Sequence ID: 339) TSASGRSATPRG (Sequence ID: 340) TSYSGRSAVPRG(Sequence ID: 341) TSYSGRSANFRG(Sequence ID: 342) TSSSGRSATPRG (Sequence ID: 343) TSTTGRSASPRG(Sequence ID: 344) TSTSGRSANPRG (Sequence ID: 345)

[0093] The sequences shown in Table 1 can also be used as protease cleavage sequences.

[0094] [Table 1] TIFF2026108884000003.tif245150TIFF2026108884000004.tif245150TIFF2026108884 000005.tif245150TIFF2026108884000006.tif245150TIFF2026108884000007.tif24515 0TIFF2026108884000008.tif245150TIFF2026108884000009.tif245150TIFF2026108884 000010.tif231150TIFF2026108884000011.tif229153TIFF2026108884000012.tif55153

[0095] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1161) In this case, X1 to X8 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0096] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1162) In this context, X1 to X8 each represent a single amino acid, where X1 is an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is A, X8 is an amino acid selected from D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0097] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1163) In this case, X1 to X8 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, F, L, M, P, Q, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is A, D, E, X8 is an amino acid selected from F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0098] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1164) In this context, X1 to X8 each represent a single amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is A, X8 is an amino acid selected from D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0099] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1165) In this context, X1 to X8 each represent a single amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, G, H, I, K, L, M, N, Q, R, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is A, X8 is an amino acid selected from D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0100] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1166) In this case, X1 to X8 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from E, F, K, M, N, P, Q, R, S, and W; X7 is A, D, E, F, G, X8 is an amino acid selected from H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0101] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1167) In this case, X1 to X8 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and X7 is an amino acid selected from Y; X8 is an amino acid selected from A, D, F, G, L, M, P, Q, V, and W; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0102] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1168) In this case, X1 to X8 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and X7 is an amino acid selected from Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, I, K, N, T and W.

[0103] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1169) In this sequence, X1 through X8 each represent a single amino acid. X1 is an amino acid selected from A, G, I, P, Q, S, and Y; X2 is an amino acid selected from K or T; X3 is G; X4 is R; X5 is S; X6 is A; X7 is an amino acid selected from H, I, and V; and X8 is an amino acid selected from H, V, and Y.

[0104] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1170) In this sequence, X1 through X8 each represent a single amino acid: X1 is Y; X2 is an amino acid selected from S and T; X3 is G; X4 is R; X5 is S; X6 is an amino acid selected from A and E; X7 is an amino acid selected from N and V; and X8 is an amino acid selected from H, P, V, and Y.

[0105] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1171) In this case, X1 to X9 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and X7 is an amino acid selected from Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0106] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1172) In this case, X1 to X9 each represent one amino acid, where X1 is an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is A, X8 is an amino acid selected from D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0107] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1173) In this case, X1 to X9 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, F, L, M, P, Q, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is A, D, E, X8 is an amino acid selected from F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0108] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1174) Among them, X1 to X9 each represent an amino acid. X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0109] The following can also be used as the protease cleavage sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO: 1175) Among them, X1 to X9 each represent an amino acid. X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, G, H, I, K, L, M, N, Q, R, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0110] The following can also be used as a protease cleavage sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO: 1176) In this case, X1 to X9 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from E, F, K, M, N, P, Q, R, S, and W; X7 is A, D, E, F, G, X8 is an amino acid selected from H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0111] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1177) In this case, X1 to X9 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and X7 is an amino acid selected from Y; X8 is an amino acid selected from A, D, F, G, L, M, P, Q, V, and W; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0112] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1178) In this case, X1 to X9 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and X7 is an amino acid selected from Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, I, K, N, T, and W; X9 is an amino acid selected from A, G, H, I, L, and R.

[0113] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1179) In this sequence, X1 through X9 each represent a single amino acid. X1 is an amino acid selected from A, G, I, P, Q, S, and Y; X2 is an amino acid selected from K or T; X3 is G; X4 is R; X5 is S; X6 is A; X7 is an amino acid selected from H, I, and V; X8 is an amino acid selected from H, V, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0114] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1180) In this sequence, X1 through X9 each represent a single amino acid: X1 is Y; X2 is an amino acid selected from S and T; X3 is G; X4 is R; X5 is S; X6 is an amino acid selected from A and E; X7 is an amino acid selected from N and V; X8 is an amino acid selected from H, P, V and Y; and X9 is an amino acid selected from A, G, H, I, L and R.

[0115] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1392) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, X7 is an amino acid selected from M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0116] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1393) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, M, N, P, Q, X7 is an amino acid selected from R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0117] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1394) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, F, L, M, P, Q, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, M, N, P, Q, R, X7 is an amino acid selected from S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0118] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1395) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, M, N, P, X7 is an amino acid selected from Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0119] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1396) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, G, H, I, K, L, M, N, Q, R, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, M, N, P, X7 is an amino acid selected from Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0120] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1397) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being E, F, K, M, N, P, Q, R, X7 is an amino acid selected from S and W; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0121] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1398) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, X7 is an amino acid selected from M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, F, G, L, M, P, Q, V, and W; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0122] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1399) Among them, X1 to X11 each represent one amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, I, K, N, T and W.

[0123] The following can also be used as the protease cleavage sequence: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 1400) Among them, X1 to X11 each represent one amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, G, I, P, Q, S and Y; X2 is an amino acid selected from K or T; X3 is G; X4 is R; X5 is S; X6 is A; X7 is an amino acid selected from H, I and V; X8 is an amino acid selected from H, V and Y.

[0124] The following can also be used as the protease cleavage sequence: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1401) In this sequence, X1 to X11 each represent a single amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being Y; X2 being an amino acid selected from S and T; X3 being G; X4 being R; X5 being S; X6 being an amino acid selected from A and E; X7 being an amino acid selected from N and V; and X8 being an amino acid selected from H, P, V, and Y.

[0125] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 1402) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, X7 is an amino acid selected from M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0126] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1403) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, M, N, P, X7 is an amino acid selected from Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0127] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 1404) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, F, L, M, P, Q, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, M, N, P, Q, R, X7 is an amino acid selected from S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0128] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 1405) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, M, N, P, X7 is an amino acid selected from Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0129] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 1406) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, G, H, I, K, L, M, N, Q, R, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, M, N, P, X7 is an amino acid selected from Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0130] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1407) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being E, F, K, M, N, P, Q, R, X7 is an amino acid selected from S and W; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0131] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1408) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, X7 is an amino acid selected from M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, F, G, L, M, P, Q, V, and W; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0132] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1409) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, X7 is an amino acid selected from M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, I, K, N, T, and W; X9 is an amino acid selected from A, G, H, I, L, and R.

[0133] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 1410) In this sequence, X1 to X11 each represent a single amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, G, I, P, Q, S, and Y; X2 being an amino acid selected from K or T; X3 being G; X4 being R; X5 being S; X6 being A; X7 being an amino acid selected from H, I, and V; X8 being an amino acid selected from H, V, and Y; and X9 being an amino acid selected from A, G, H, I, L, and R.

[0134] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1411) In this sequence, X1 to X11 each represent a single amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being Y; X2 being an amino acid selected from S and T; X3 being G; X4 being R; X5 being S; X6 being an amino acid selected from A and E; X7 being an amino acid selected from N and V; X8 being an amino acid selected from H, P, V, and Y; and X9 being an amino acid selected from A, G, H, I, L, and R.

[0135] In addition to using the above-mentioned protease cleavage sequences, new protease cleavage sequences may be obtained through screening. For example, new protease cleavage sequences can be searched for by changing the interaction between the cleavage sequence and the enzyme's active and recognized residues based on the results of crystal structure analysis of known protease cleavage sequences. Furthermore, new protease cleavage sequences can be searched for by modifying the amino acids in known protease cleavage sequences and confirming their interaction with the protease. As another example, sequences cleaved by proteases can be searched for by displaying a peptide library using in vitro display methods such as phage display or ribosome display, or by confirming the interaction with the protease using a peptide array immobilized on a chip or beads. The interaction between the protease cleavage sequence and the protease can be confirmed by observing protease cleavage in vitro or in vivo.

[0136] By quantifying the amount of cleavage fragments after protease treatment, separated by electrophoresis methods such as SDS-PAGE, it is possible to evaluate the protease cleavage sequence, protease activity, and the cleavage rate of molecules into which the protease cleavage sequence has been introduced. The following is one non-limiting embodiment of a method for evaluating the cleavage rate of molecules into which the protease cleavage sequence has been introduced. For example, when evaluating the cleavage rate of an antibody variant into which a protease cleavage sequence has been introduced using recombinant human u-Plasminogen Activator / Urokinase (human uPA, huPA) (R&D Systems; 1310-SE-010) or recombinant human Matriptase / ST14 Catalytic Domain (human MT-SP1, hMT-SP1) (R&D Systems; 3946-SE-010), the mixture is reacted for 1 hour under conditions of huPA 40 nM or hMT-SP1 3 nM, antibody variant 100 μg / mL, PBS, and 37°C, and then subjected to a capillary electrophoresis immunoassay. Wes (Protein Simple) can be used for the capillary electrophoresis immunoassay, but is not limited to this method. Alternatively, the sample may be separated by SDS-PAGE or similar methods and then detected by Western blotting. For detecting light chains before and after cleavage, an anti-human lambda chain HRP-labeled antibody (abcam; ab9007) can be used, but any antibody capable of detecting cleavage fragments can be used. By outputting the area of ​​each peak obtained after protease treatment using Wes-specific software (Compass for SW; Protein Simple), the cleavage rate (%) of the antibody-modified molecule can be calculated using the formula: (cleavage light chain peak area) * 100 / (cleavage light chain peak area + uncleaved light chain peak area). The cleavage rate can be calculated as long as protein fragments before and after protease treatment can be detected, and the cleavage rate can be calculated for various proteins, not just antibody-modified molecules, that have been introduced with a protease cleavage sequence.

[0137] After administering a molecule containing a protease cleavage sequence to an animal, the in vivo cleavage rate can be calculated by detecting the administered molecule in a blood sample. For example, after administering an antibody variant containing a protease cleavage sequence to a mouse, plasma is collected from the blood sample, and the antibody is purified using Dynabeads Protein A (Thermo; 10001D) by a method known to those skilled in the art. The protease cleavage rate of the antibody variant can then be evaluated by subjecting it to a capillary electrophoresis immunoassay. While Wes (Protein Simple) can be used for the capillary electrophoresis immunoassay, the method is not limited to this; alternative methods such as separation by SDS-PAGE followed by detection by Western Blotting are also possible, and the method is not limited to these. For detecting the light chain of the antibody variant recovered from the mouse, an anti-human lambda chain HRP-labeled antibody (abcam; ab9007) can be used, but any antibody capable of detecting cleavage fragments can be used. By outputting the area of ​​each peak obtained by capillary electrophoresis immunoassay using Wes-specific software (Compass for SW; Protein Simple), and calculating the light chain retention ratio as (light chain peak area) / (heavy chain peak area), it is possible to calculate the proportion of the full-length light chain that remained uncleaved in the mouse body. Calculating the cleavage efficiency in vivo is possible if protein fragments recovered from the body can be detected, and the cleavage rate can be calculated for various proteins, not just antibody modifiers, that have been introduced with protease cleavage sequences. By calculating the cleavage rate using the method described above, it is possible to compare, for example, the in vivo cleavage rates of antibody modifiers with different cleavage sequences, and also to compare the cleavage rates of the same antibody modifier across different animal models, such as normal mouse models and tumor-transplanted mouse models.

[0138] For example, the protease cleavage sequences exemplified in Table 1 were all newly discovered by the present inventors. Polypeptides containing these protease cleavage sequences are all useful as protease substrates that are hydrolyzed by the action of proteases. That is, the present invention provides protease substrates containing sequences shown in SEQ ID NOs: 1161-1180, 1392-1411, and sequences selected from the sequences listed in Table 1. The protease substrates of the present invention can be used, for example, as a library for selecting those with properties appropriate for the purpose when incorporating them into ligand-binding molecules. Specifically, their protease sensitivity can be evaluated in order to selectively cleave ligand-binding molecules by proteases localized in lesions. Ligand-binding molecules bound to ligands may reach lesions after being administered to a living organism and coming into contact with various proteases. Therefore, it is desirable to have sensitivity to proteases localized in lesions while having as high a tolerance as possible to other proteases. To select a desirable protease cleavage sequence according to the purpose, protease resistance can be determined by comprehensively analyzing the sensitivity of each protease substrate to various proteases beforehand. Based on the obtained protease resistance spectra, a protease cleavage sequence with the required sensitivity and resistance can be found. Alternatively, ligand-binding molecules incorporating protease cleavage sequences reach the lesion not only through enzymatic action by proteases, but also through various environmental stresses such as pH changes, temperature, and redox stress. Even in response to such external factors, it is possible to select a protease cleavage sequence with desirable properties for the purpose based on information comparing the resistance of various protease substrates.

[0139] In one embodiment of the present invention, a movable linker is further added to either one or both ends of the protease cleavage sequence. The movable linker at one end of the protease cleavage sequence may be referred to as the first movable linker, and the movable linker at the other end may be referred to as the second movable linker. In a particular embodiment, the protease cleavage sequence and the movable linker include one of the following formulas. (Protease cleavage sequence) (First movable linker)-(Protease cleavage sequence) (Protease cleavage sequence)-(Second movable linker) (First movable linker) - (Protease cleavage sequence) - (Second movable linker) In this embodiment, the movable linker is preferably a peptide linker. The first movable linker and the second movable linker are independently and optionally present, and are identical or different movable linkers containing at least one flexible amino acid (such as Gly). For example, the protease cleavage sequence contains a sufficient number of residues to obtain the desired protease accessibility (amino acids optionally selected from Arg, Ile, Gln, Glu, Cys, Tyr, Trp, Thr, Val, His, Phe, Pro, Met, Lys, Gly, Ser, Asp, Asn, Ala, etc., particularly Gly, Ser, Asp, Asn, Ala, especially Gly and Ser, especially Gly, etc.).

[0140] A movable linker suitable for use at both ends of a protease cleavage sequence typically improves protease access to the protease cleavage sequence, thereby increasing the efficiency of protease cleavage. Suitable movable linkers are readily selectable, and suitable options can be chosen from various lengths, including 3 to 12 amino acids, as well as 1 to 20 amino acids (e.g., Gly), 2 to 15 amino acids, 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids. In some embodiments of the present invention, the movable linker is a peptide linker with 1 to 7 amino acids.

[0141] Examples of movable linkers include, but are not limited to, glycine polymers (G)n, glycine-serine polymers (e.g., including (GS)n, (GSGGS:SEQ ID NO:45)n and (GGGS:SEQ ID NO:36)n, where n is at least an integer of 1), glycine-alanine polymers, alanine-serine polymers, and other movable linkers known in the prior art. Among these, glycine and glycine-serine polymers are attracting attention because these amino acids are relatively unstructured and easily function as neutral tethers between components. Examples of movable linkers made of glycine-serine polymers include, but are not limited to, the following: Ser Gly·Ser(GS) Ser·Gly(SG) Gly·Gly·Ser(GGS) Gly·Ser·Gly (GSG) Ser·Gly·Gly (SGG) Gly·Ser·Ser (GSS) Ser·Ser·Gly (SSG) Ser·Gly·Ser(SGS) Gly·Gly·Gly·Ser(GGGS, Sequence ID: 36) Gly·Gly·Ser·Gly (GGSG, Sequence ID: 37) Gly·Ser·Gly·Gly (GSGG, Sequence ID: 38) Ser·Gly·Gly·Gly (SGGG, Sequence ID: 39) Gly·Ser·Ser·Gly(GSSG, Sequence ID: 40) Gly·Gly·Gly·Gly·Ser(GGGGS, Sequence ID: 41) Gly·Gly·Gly·Ser·Gly (GGGSG, Sequence ID: 42) Gly·Gly·Ser·Gly·Gly(GGSGG, Sequence ID: 43) Gly·Ser·Gly·Gly·Gly(GSGGG, Sequence ID: 44) Gly·Ser·Gly·Gly·Ser(GSGGS, Sequence ID: 45) Ser·Gly·Gly·Gly·Gly (SGGGG, Sequence ID: 46) Gly·Ser·Ser·Gly·Gly (GSSGG, Sequence ID: 47) Gly·Ser·Gly·Ser·Gly(GSGSG, Sequence ID: 48) Ser·Gly·Gly·Ser·Gly (SGGSG, Sequence ID: 49) Gly·Ser·Ser·Ser·Gly(GSSSG, Sequence ID: 50) Gly·Gly·Gly·Gly·Gly·Ser(GGGGGS, Sequence ID: 51) Ser·Gly·Gly·Gly·Gly·Gly (SGGGGG, Sequence ID: 52) Gly·Gly·Gly·Gly·Gly·Gly·Ser(GGGGGGS, Sequence ID: 53) Ser·Gly·Gly·Gly·Gly·Gly·Gly(SGGGGGG, Sequence ID: 54) (Gly·Gly·Gly·Gly·Ser(GGGGS, Sequence ID: 41))n (Ser·Gly·Gly·Gly·Gly(SGGGG, Sequence ID: 46))n Examples include [n is an integer greater than or equal to 1]. However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0142] In some embodiments of the present invention, the ligand-binding molecule comprises antibody VH and antibody VL. Examples of ligand-binding molecules comprising VH and VL include, but are not limited to, Fv, scFv, Fab, Fab', Fab'-SH, F(ab')2, complete antibodies, and the like.

[0143] In some embodiments of the present invention, the ligand-binding molecule includes an Fc region. When using the Fc region of an IgG antibody, the type is not limited, and it is possible to use Fc regions such as IgG1, IgG2, IgG3, and IgG4. For example, it is possible to use an Fc region containing one sequence selected from the amino acid sequences shown in SEQ ID NOs: 55, 56, 57, and 58, or Fc region variants modified from these Fc regions. Furthermore, in some embodiments of the present invention, the ligand-binding molecule includes the antibody constant region.

[0144] In some more specific embodiments of the present invention, the ligand-binding molecule is an antibody. When an antibody is used as the ligand-binding molecule, binding to the ligand is achieved by a variable region. In some even more specific embodiments, the ligand-binding molecule is an IgG antibody. When an IgG antibody is used as the ligand-binding molecule, the type is not limited, and IgG1, IgG2, IgG3, IgG4, etc., can be used. Even when an IgG antibody is used as the ligand-binding molecule, binding to the ligand is achieved by a variable region, and binding to the ligand can be achieved by one or both of the two variable regions of the IgG antibody.

[0145] In some embodiments of the present invention, cleavage of the cleavage site / protease cleavage sequence in the ligand-binding molecule cleaves the ligand-binding domain in the ligand-binding molecule, thereby weakening its binding to the ligand. For example, in an embodiment where an IgG antibody is used as the ligand-binding molecule, a cleavage site / protease cleavage sequence is provided in the antibody variable region, so that in the cleaved state, the antibody variable region cannot be fully formed, and thus weakening its binding to the ligand.

[0146] In this specification, "association" can be rephrased as referring to a state in which, for example, two or more polypeptide regions interact. Generally, hydrophobic bonds, hydrogen bonds, ionic bonds, etc., are formed between the target polypeptide regions to create an aggregate. One common example of association is in antibodies, such as natural antibodies, where the heavy chain variable region (VH) and the light chain variable region (VL) are known to maintain a paired structure through non-covalent bonds between them.

[0147] In some embodiments of the present invention, VH and VL contained in a ligand-binding molecule associate. Furthermore, the association between antibody VH and antibody VL can be resolved, for example, by cleavage of a cleavage site / protease cleavage sequence. Resolution of association can be rephrased as, for example, the resolution of all or part of the interaction state of two or more polypeptide regions. Resolution of association between VH and VL may mean that the interaction between VH and VL is resolved entirely, or that some of the interaction between VH and VL is resolved. The ligand-binding molecules of the present invention include ligand-binding molecules in which the association between antibody VL or a part thereof and antibody VH or a part thereof is resolved by cleavage of a cleavage site or by cleavage of a protease cleavage sequence by a protease.

[0148] In some embodiments of the present invention, the ligand-binding molecule contains antibody VH and antibody VL, and when the cleavage site / protease cleavage sequence of the ligand-binding molecule is not cleaved, antibody VH and antibody VL in the ligand-binding molecule are associated, and cleavage of the cleavage site / protease cleavage sequence dissociates the association of antibody VH and antibody VL in the ligand-binding molecule. The cleavage site / protease cleavage sequence in the ligand-binding molecule may be located at any position in the ligand-binding molecule as long as cleavage weakens the binding of the ligand-binding molecule to the ligand.

[0149] In some further embodiments of the present invention, the ligand-binding molecule comprises antibody VH, antibody VL, and antibody constant region. The VH and VL, and CH and CL domains of antibodies are known to interact with each other via many amino acid side chains, as described by Rothlisberger et al. (J Mol Biol. 2005 Apr 8;347(4):773-89.). While VH-CH1 and VL-CL are known to be able to form stable structures as Fab domains, as reported, the amino acid side chains between VH and VL are generally 10 -5 M to 10 -8The interaction occurs with a dissociation constant in the M range, and it is thought that the proportion of associated states formed when only the VH domain and VL domain are present is small.

[0150] In some embodiments of the present invention, a ligand-binding molecule containing antibody VH and antibody VL is provided with a cleavage site / protease cleavage sequence. While all heavy-chain-light-chain interactions exist between the two peptides in the Fab structure before cleavage, when the cleavage site / protease cleavage sequence is cleaved, the interaction between the peptide containing VH (or a portion of VH) and the peptide containing VL (or a portion of VL) is weakened, and the association of VH and VL is resolved.

[0151] In one embodiment of the present invention, the cleavage site / protease cleavage sequence is located within the antibody constant region. In a more specific embodiment, the cleavage site / protease cleavage sequence is located on the variable region side of amino acid 140 (EU numbering) in the antibody heavy chain constant region, preferably on the variable region side of amino acid 122 (EU numbering) in the antibody heavy chain constant region. In some specific embodiments, the cleavage site / protease cleavage sequence is introduced at any position in the sequence from amino acid 118 (EU numbering) to amino acid 140 (EU numbering) in the antibody heavy chain constant region. In another, more specific embodiment, the cleavage site / protease cleavage sequence is located on the variable region side of amino acid 130 (EU numbering) (Kabat numbering 130) in the constant region of the antibody light chain, preferably on the variable region side of amino acid 113 (EU numbering) (Kabat numbering 113) in the constant region of the antibody light chain, and on the variable region side of amino acid 112 (EU numbering) (Kabat numbering 112) in the constant region of the antibody light chain. In some specific embodiments, the cleavage site / protease cleavage sequence is introduced at any position in the sequence from amino acid 108 (EU numbering) (Kabat numbering 108) to amino acid 131 (EU numbering) (Kabat numbering 131) in the constant region of the antibody light chain.

[0152] In one embodiment of the present invention, the cleavage site / protease cleavage sequence is located within antibody VH or antibody VL. In a more specific embodiment, the cleavage site / protease cleavage sequence is located on the antibody constant region side of the 7th (Kabat numbered) amino acid of antibody VH, preferably on the antibody constant region side of the 40th (Kabat numbered) amino acid of antibody VH, more preferably on the antibody constant region side of the 101st (Kabat numbered) amino acid of antibody VH, even more preferably on the antibody constant region side of the 109th (Kabat numbered) amino acid of antibody VH, and on the antibody constant region side of the 111th (Kabat numbered) amino acid of antibody VH. In a more specific embodiment, the cleavage site / protease cleavage sequence is located closer to the antibody constant region than the 7th (Kabat numbered) amino acid of antibody VL, preferably closer to the antibody constant region than the 39th (Kabat numbered) amino acid of antibody VL, more preferably closer to the antibody constant region than the 96th (Kabat numbered) amino acid of antibody VL, and even more preferably closer to the antibody constant region than the 104th (Kabat numbered) amino acid of antibody VL and closer to the antibody constant region than the 105th (Kabat numbered) amino acid of antibody VL. In some more specific embodiments, the cleavage site / protease cleavage sequence is introduced at the positions of residues that form loop structures and residues close to loop structures in antibody VH or antibody VL. Loop structures in antibody VH or antibody VL refer to the parts of antibody VH or antibody VL that do not form secondary structures such as α-helices and β-sheets. Specifically, the positions of residues that form loop structures and residues close to loop structures are: from amino acid 7 (Kabat numbering) to amino acid 16 (Kabat numbering) in antibody VH, from amino acid 40 (Kabat numbering) to amino acid 47 (Kabat numbering), from amino acid 55 (Kabat numbering) to amino acid 69 (Kabat numbering), from amino acid 73 (Kabat numbering) to amino acid 79 (Kabat numbering), from amino acid 83 (Kabat numbering) to amino acid 89 (Kabat numbering), and from amino acid 95 (Kabat numbering) This can refer to the range from t-numbering to amino acid 99 (Kabat numbering), from amino acid 101 (Kabat numbering) to amino acid 113 (Kabat numbering), from antibody VL7 (Kabat numbering) to amino acid 19 (Kabat numbering), from amino acid 39 (Kabat numbering) to amino acid 46 (Kabat numbering), from amino acid 49 (Kabat numbering) to amino acid 62 (Kabat numbering), and from amino acid 96 (Kabat numbering) to amino acid 107 (Kabat numbering). In some more specific embodiments, the cleavage site / protease cleavage sequence is introduced at any position in the sequence from amino acid 7 (Kabat numbering) to amino acid 16 (Kabat numbering) of antibody VH, from amino acid 40 (Kabat numbering) to amino acid 47 (Kabat numbering), from amino acid 55 (Kabat numbering) to amino acid 69 (Kabat numbering), from amino acid 73 (Kabat numbering) to amino acid 79 (Kabat numbering), from amino acid 83 (Kabat numbering) to amino acid 89 (Kabat numbering), from amino acid 95 (Kabat numbering) to amino acid 99 (Kabat numbering), and from amino acid 101 (Kabat numbering) to amino acid 113 (Kabat numbering). In some more specific embodiments, the cleavage site / protease cleavage sequence is introduced at any position in the sequence from amino acid VL7 (Kabat numbering) to amino acid 19 (Kabat numbering), from amino acid 39 (Kabat numbering) to amino acid 46 (Kabat numbering), from amino acid 49 (Kabat numbering) to amino acid 62 (Kabat numbering), and from amino acid 96 (Kabat numbering) to amino acid 107 (Kabat numbering).

[0153] In one embodiment of the present invention, the cleavage site / protease cleavage sequence is located near the boundary between antibody VH and the antibody constant region. The boundary between antibody VH and the antibody heavy chain constant region can refer to the area between amino acid 101 (Kabat numbering) of antibody VH and amino acid 140 (EU numbering) of the antibody heavy chain constant region, preferably between amino acid 109 (Kabat numbering) of antibody VH and amino acid 122 (EU numbering) of the antibody heavy chain constant region, or between amino acid 111 (Kabat numbering) of antibody VH and amino acid 122 (EU numbering) of the antibody heavy chain constant region. Furthermore, when antibody VH and the antibody light chain constant region are linked, the area near the boundary between antibody VH and the antibody light chain constant region can refer to the area between amino acid 101 (Kabat numbering) of antibody VH and amino acid 130 (EU numbering (Kabat numbering 130)) of the antibody light chain constant region, preferably between amino acid 109 (Kabat numbering) of antibody VH and amino acid 113 (EU numbering) (Kabat numbering 113) of the antibody light chain constant region, or between amino acid 111 (Kabat numbering) of antibody VH and amino acid 112 (EU numbering) (Kabat numbering 112) of the antibody light chain constant region.

[0154] In one embodiment, the cleavage site / protease cleavage sequence is located near the boundary between the antibody VL and the antibody constant region. The area near the boundary between the antibody VL and the antibody light chain constant region can refer to the area between amino acid 96 (Kabat numbering) of the antibody VL and amino acid 130 (EU numbering) (Kabat numbering 130) of the antibody light chain constant region, preferably between amino acid 104 (Kabat numbering) of the antibody VL and amino acid 113 (EU numbering) (Kabat numbering 113) of the antibody light chain constant region, or between amino acid 105 (Kabat numbering) of the antibody VL and amino acid 112 (EU numbering) (Kabat numbering 112) of the antibody light chain constant region. When the antibody VL and the antibody heavy chain constant region are linked, the area near the boundary between the antibody VL and the antibody heavy chain constant region can refer to the area between amino acid 96 (Kabat numbering) of the antibody VL and amino acid 140 (EU numbering) of the antibody heavy chain constant region, preferably between amino acid 104 (Kabat numbering) of the antibody VL and amino acid 122 (EU numbering) of the antibody heavy chain constant region, or between amino acid 105 (Kabat numbering) of the antibody VL and amino acid 122 (EU numbering) of the antibody heavy chain constant region.

[0155] Multiple cleavage sites / protease cleavage sequences can be provided within the ligand-binding molecule, for example, at multiple locations selected from within the antibody constant region, within antibody VH, within antibody VL, near the boundary between antibody VH and the antibody constant region, and near the boundary between antibody VL and the antibody constant region. Furthermore, a person skilled in the art familiar with the present invention can change the shape of the molecule containing antibody VH, antibody VL, and the antibody constant region, such as by swapping antibody VH and antibody VL, and such molecular shape does not deviate from the scope of the present invention.

[0156] In this specification, the term “ligand” refers to a bioactive molecule. Bioactive molecules typically function by interacting with receptors on the cell surface, thereby biologically stimulating, inhibiting, or otherwise modulating them, and these are usually thought to be involved in intracellular signaling pathways that harbor such receptors.

[0157] In this specification, the term "ligand" encompasses any desired molecule that exerts biological activity by interacting with a biomolecule. For example, a ligand not only means a molecule that interacts with a receptor, but also a molecule that exerts biological activity by interacting with that molecule. For instance, receptors that interact with such molecules, or their binding fragments, are also included in the definition of ligand. For example, the ligand-binding site of a protein known as a receptor, or a protein containing a site on which such a receptor interacts with other molecules, are included in the definition of ligand in this invention. Specifically, soluble receptors, soluble fragments of receptors, extracellular domains of transmembrane receptors, and polypeptides containing these are included in the definition of ligand in this invention.

[0158] The ligands of the present invention can typically exert desired biological activity by binding to one or more binding partners. The binding partners of the ligand can be extracellular, intracellular, or transmembrane proteins. In one embodiment, the binding partner of the ligand is an extracellular protein, such as a soluble receptor. In another embodiment, the binding partner of the ligand is a membrane-bound receptor. The ligands of the present invention can specifically bind to their binding partners with dissociation constants (KD) of 10 μM, 1 μM, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 500 pM, 400 pM, 350 pM, 300 pM, 250 pM, 200 pM, 150 pM, 100 pM, 50 pM, 25 pM, 10 pM, 5 pM, 1 pM, 0.5 pM, or 0.1 pM or less.

[0159] Examples of biologically active molecules include, but are not limited to, cytokines, chemokines, polypeptide hormones, growth factors, apoptosis-inducing factors, PAMPs, DAMPs, nucleic acids, or fragments thereof. In detailed embodiments, interleukins, interferons, hematopoietic factors, the TNF superfamily, chemokines, cell growth factors, the TGF-β family, myokines, adipokines, or neurotrophic factors may be used as ligands. In more detailed embodiments, ligands may include CXCL10, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFN-α, IFN-β, IFN-g, MIG, I-TAC, RANTES, MIP-1a, MIP-1b, IL-1R1 (Interleukin-1 receptor, type I), IL-1R2 (Interleukin-1 receptor, type II), IL-1RAcP (Interleukin-1 receptor accessory protein), or IL-1Ra (protein accession No. NP_776214, mRNA accession No. NM_173842.2).

[0160] Chemokines are a family of homogeneous serum proteins between 7 and 16 kDa, originally characterized by their ability to induce leukocyte migration. Most chemokines possess four characteristic cysteine ​​(Cys) and are classified into the chemokine classes CXC, or alpha; CC, or beta; C, or gamma; and CX3C, or delta, based on the motif indicated by the first two cysteines. Two disulfide bonds are formed between the first and third cysteines, and between the second and fourth cysteines. Generally, disulfide crosslinking is considered necessary, and Clark-Lewis and collaborators reported that disulfide bonds are decisive to chemokine activity, at least for CXCL10 (Clark-Lewis et al., J. Biol. Chem. 269:16075-16081, 1994). The only exception to the rule of having four cysteine ​​residues is lymphotactin, which has only two cysteine ​​residues. Therefore, lymphotactin manages to maintain its functional structure with only one disulfide bond. Furthermore, the CXC or alpha subfamilies are classified into two groups based on the presence of an ELR motif (Glu-Leu-Arg) preceding the first cysteine: ELR-CXC chemokines and non-ELR-CXC chemokines (see, for example, Clark-Lewis, above, and Belperio et al., "CXC Chemokines in Angiogenesis," J. Leukoc. Biol. 68:1-8, 2000).

[0161] Interferon-inducible protein 10 (IP-10 or CXCL10) is induced by interferon-γ and TNF-α and produced by keratinocytes, endothelial cells, fibroblasts, and monocytes. IP-10 is thought to play a role in the recruitment of activated T cells to sites of tissue inflammation (Dufour, et al., "IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking," J Immunol., 168:3195-204, 2002). Furthermore, IP-10 may play a role in hypersensitivity reactions. Furthermore, it may also play a role in the development of inflammatory demyelinating neuropathies (Kieseier, et al., "Chemokines and chemokine receptors in inflammatory demyelinating neuropathies: a central role for IP-10," Brain 125:823-34, 2002).

[0162] Studies have shown that IP-10 may be useful for stem cell engraftment following transplantation (Nagasawa, T., Int. J. Hematol. 72:408-11, 2000), stem cell recruitment (Gazitt, Y., J. Hematother Stem Cell Res 10:229-36, 2001; Hattori et al., Blood 97:3354-59, 2001), and enhancement of antitumor immunity (Nomura et al., Int. J. Cancer 91:597-606, 2001; Mach and Dranoff, Curr. Opin. Immunol. 12:571-75, 2000). For example, the biological activity of chemokines has been discussed in reports known to those skilled in the art (Bruce, L. et al., "Radiolabeled Chemokine binding assays," Methods in Molecular Biology (2000) vol. 138, pp129-134; Raphaele, B. et al., "Calcium Mobilization," Methods in Molecular Biology (2000) vol. 138, pp143-148; Paul D. Ponath et al., "Transwell Chemotaxis," Methods in Molecular Biology (2000) vol. 138, pp113-120 Humana Press. Totowa, New Jersey).

[0163] Examples of CXCL10's biological activities include binding to the CXCL10 receptor (CXCR3), CXCL10-induced calcium facilitation, CXCL10-induced cell chemotaxis, binding of CXCL10 to glycosaminoglycans, and CXCL10 oligomerization. Methods for measuring the physiological activity of CXCL10 include measuring the cell migration activity of CXCL10, the Reporter assay using CXCR3 stable expression cell lines (see PLoS One. 2010 Sep 13;5(9):e12700.), and PathHunter, which utilizes B-Arrestin recruitment induced in the early stages of GPCR signaling. TM Examples include the β-Arrestin recruitment assay.

[0164] Interleukin-12 (IL-12) is a heterodimer cytokine consisting of disulfide-linked glycosyl polypeptide chains of 30 and 40 kD. Cytokines are synthesized and secreted by antigen-presenting cells, including dendritic cells, monocytes, macrophages, B cells, Langerhans cells, and keratinocytes, as well as natural killer (NK) cells. IL-12 mediates various biological processes and has been referred to as an NK cell stimulating factor (NKSF), a T cell stimulating factor, a cytotoxic T lymphocyte maturation factor, and an EBV-transformed B cell lineage factor.

[0165] Interleukin-12 can bind to IL-12 receptors expressed on the cytoplasmic membrane of cells (e.g., T cells, NK cells), thereby altering (e.g., initiating, inhibiting) biological processes. For example, the binding of IL-12 to the IL-12 receptor stimulates the proliferation of pre-activated T and NK cells, enhances the cytolytic activity of cytotoxic T cells (CTLs), NK cells, and LAK (lymphokine-activated killer) cells, induces the production of γ interferon (IFNγ) by T and NK cells, and induces the differentiation of naive Th0 cells into Th1 cells that produce IFNγ and IL-2. In particular, IL-12 is absolutely essential for the generation of cytolytic cells (e.g., NK, CTLs) and for establishing cellular immune responses (e.g., Th1 cell-mediated immune responses). Thus, IL-12 is absolutely crucial in the generation and regulation of both prophylactic immunity (e.g., eradication of infectious diseases) and pathological immune responses (e.g., autoimmunity).

[0166] Methods for measuring the physiological activity of IL-12 include measuring the cell proliferation activity of IL-12, STAT4 reporter assays, IL-12-induced cell activation (cell surface marker expression, cytokine production, etc.), and IL-12-induced promotion of cell differentiation.

[0167] The protein Programmed Death 1 (PD-1) is an inhibitory member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells (Okazaki et al. (2002) Curr. Opin. Immunol. 14:391779-82, Bennett et al. (2003) J Immunol 170:711-8). CD28 and ICOS, the first members of this family, were discovered through their functional effects on increased T cell proliferation after the addition of monoclonal antibodies (Hutloff et al. (1999) Nature 397:263-266, Hansen et al. (1980) Immunogenics 10:247-260). PD-1 was discovered by screening for different expression levels in apoptotic cells (Ishida et al. (1992) EMBO J. 11:3887-95). Other members of the family, CTLA-4 and BTLA, were discovered by screening for different expression levels in cytotoxic T lymphocytes and TH1 cells, respectively. CD28, ICOS, and CTLA-4 all have unpaired cysteine ​​residues, which enable homodimerization. In contrast, PD-1 is thought to exist as a monomer and does not have the unpaired cysteine ​​characteristic of other CD28 family members.

[0168] The PD-1 gene is a 55 kDa type I transmembrane protein that is part of the Ig gene superfamily. PD-1 contains a membrane-proximal immunoreceptor tyrosine inhibitor motif (ITIM) and a membrane-distal tyrosine-based switch motif (ITSM). PD-1 is structurally similar to CTLA-4 but lacks the MYPPPY motif (SEQ ID NO: 537), which is crucial for B7-1 and B7-2 binding. Two ligands for PD-1, PD-L1 and PD-L2, have been identified and have been shown to negatively regulate T cell activation upon binding to PD-1 (Freeman et al. (2000) J Exp Med 192:1027-34, Latchman et al. (2001) Nat Immunol 2:261-8, Carter et al. (2002) Eur J Immunol 32:634-43). Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1 but not to other CD28 family members. PD-L1, one ligand for PD-1, is abundant in various human cancers (Dong et al. (2002) Nat. Med. 8:787-9). The interaction between PD-1 and PD-L1 results in a decrease in tumor-infiltrating lymphocytes, reduced T cell receptor-mediated proliferation, and immune evasion by cancer cells (Dong et al. (2003) J. Mol. Med. 81:281-7, Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314, Konishi et al. (2004) Clin. Cancer Res. 10:5094-100). Immunosuppression can be reversed by inhibiting the local interaction between PD-L1 and PD-1, and the effect is additive when the interaction between PD-L2 and PD-2 is similarly inhibited (Iwai et al. (2002) Proc. Nat' l. Acad. Sci. USA 99:12293-7, Brown et al. (2003) J. Immunol. 170:1257-66).

[0169] PD-1 is an inhibitory member of the CD28 family expressed on activated B cells, T cells, and myeloid cells. PD-1-deficient animals develop a variety of autoimmune phenotypes, including autoimmune cardiomyopathy and lupus-like syndrome with arthritis and nephritis (Nishimura et al. (1999) Immunity 11:141-51, Nishimura et al. (2001) Science 291:319-22). Furthermore, PD-1 has been shown to play an important role in autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease (GVHD), type 1 diabetes mellitus, and rheumatoid arthritis (Salama et al. (2003) J Exp Med 198:71-78, Prokunia and Alarcon-Riquelme (2004) Hum Mol Genet 13:R143, Nielsen et al. (2004) Lupus 13:510). In mouse B-cell tumor lines, PD-1 ITSM is BCR-mediated Ca 2+ It has been revealed that this process is essential for inhibiting tyrosine phosphorylation of downstream effector molecules (Okazaki et al. (2001) PNAS 98:13866-71).

[0170] In some embodiments of the present invention, the ligand is a cytokine. Cytokines are a family of secreted cell signaling proteins involved in immunomodulatory and inflammatory processes, secreted by glial cells in the nervous system and numerous cells in the immune system. Cytokines can be classified as proteins, peptides, or glycoproteins and encompass a large and diverse family of regulators. Cytokines bind to cell surface receptors and induce intracellular signaling, which can lead to the regulation of enzyme activity, upregulation or downregulation of several genes and their transcription factors, or feedback inhibition. In some embodiments, the cytokines of the present invention include immunomodulatory factors such as interleukins (ILs) and interferons (IFNs). Suitable cytokines may include proteins derived from one or more of the following types: the four α-helix bundle families (including the IL-2 subfamily, the IFN subfamily, and the IL-10 subfamily); the IL-1 family (including IL-1 and IL-8); and the IL-17 family. Cytokines may also include those classified as type 1 cytokines that enhance cellular immune responses (e.g., IFN-γ, TGF-β, etc.) or type 2 cytokines that favor antibody responses (e.g., IL-4, IL-10, IL-13, etc.).

[0171] In some embodiments of the present invention, the ligand is a chemokine. Chemokines generally act as chemotaxis, recruiting immune effector cells to chemokine expression sites. This is considered useful for expressing specific chemokine genes, for example, together with cytokine genes, for the purpose of recruiting other immune system components to the treatment site. Such chemokines include CXCL10, RANTES, MCAF, MIP1-α, and MIP1-β. Those skilled in the art will recognize that certain cytokines also have chemotaxis and can be classified under the term chemokines.

[0172] Furthermore, in some embodiments of the present invention, modified cytokines, chemokines, etc. (e.g., Annu Rev Immunol. 2015;33:139-67.) or fusion proteins containing them (e.g., Stem Cells Transl Med. 2015 Jan; 4(1): 66-73.) can be used as ligands.

[0173] In some embodiments of the present invention, the ligand is selected from CXCL10, PD-1, IL-12, IL-6R, IL-1R1, IL-1R2, IL-1RAcP, and IL-1Ra. The CXCL10, PD-1, IL-12, IL-6R, IL-1R1, IL-1R2, IL-1RAcP, and IL-1Ra may have the same sequence as the naturally occurring CXCL10, PD-1, IL-12, IL-6R, IL-1R1, IL-1R2, IL-1RAcP, and IL-1Ra, or they may be modified compounds that have a different sequence from the naturally occurring CXCL10, PD-1, IL-12, IL-6R, IL-1R1, IL-1R2, IL-1RAcP, and IL-1Ra but retain physiological activity while having the corresponding naturally occurring ligand. To obtain ligand variants, the ligand sequence may be artificially modified for various purposes, preferably by making modifications that prevent protease cleavage (protease-resistant).

[0174] In some embodiments of the present invention, the biological activity of a ligand is inhibited by binding to an uncleaved ligand-binding molecule. The embodiments in which the biological activity of a ligand is inhibited are not limited, but examples include embodiments in which the binding of an uncleaved ligand-binding molecule to a ligand substantially or significantly interferes with or competes with the binding of the ligand to its binding partner. When an antibody or fragment thereof having ligand-neutralizing activity is used as the ligand-binding molecule, the biological activity of the ligand can be inhibited by the ligand-binding molecule exerting its neutralizing activity upon binding to the ligand.

[0175] In one embodiment of the present invention, it is preferable that the uncleaved ligand-binding molecule can sufficiently neutralize the biological activity of the ligand by binding to it. That is, it is preferable that the biological activity of the ligand bound to the uncleaved ligand-binding molecule is lower than the biological activity of the ligand not bound to the uncleaved ligand-binding molecule. Although not limited thereto, for example, the biological activity of the ligand bound to the uncleaved ligand-binding molecule may be 90% or less, preferably 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, particularly preferably 20% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less compared to the biological activity of the ligand not bound to the uncleaved ligand-binding molecule. By sufficiently neutralizing the biological activity of the ligand, it is expected that when the ligand-binding molecule is administered, it will be possible to prevent the ligand from exerting its biological activity before reaching the target tissue. Alternatively, the present invention provides a method for neutralizing the biological activity of a ligand. The method of the present invention includes the step of contacting a ligand whose biological activity is to be neutralized with the ligand-binding molecule of the present invention and recovering the binding product of the two. By cleaving the ligand-binding molecule of the recovered binding product, the biological activity of the ligand that was thereby neutralized can be restored. In other words, the method for neutralizing the biological activity of a ligand of the present invention may further include the step of cleaving the ligand-binding molecule of the binding product consisting of a ligand and a ligand-ligand-binding molecule to restore the biological activity of the ligand (i.e., release the neutralizing effect of the ligand-binding molecule).

[0176] In one embodiment of the present invention, the binding activity of the cleaved ligand-binding molecule to the ligand is preferably lower than the binding activity of the ligand's in vivo natural binding partner (e.g., a natural receptor for the ligand). However, the binding activity of the cleaved ligand-binding molecule to the ligand is preferably 90% or less, preferably 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, particularly preferably 20% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less compared to the amount of ligand binding between the in vivo natural binding partner (per unit binding partner). The indicator of binding activity may be any desired indicator as appropriate, for example, the dissociation constant (KD) may be used. When using the key-to-dissociation (KD) as an indicator for evaluating binding activity, a larger KD of the cleaved ligand-binding molecule relative to the ligand compared to the KD of the natural binding partner in vivo indicates that the binding activity of the cleaved ligand-binding molecule relative to the ligand is weaker than that of the natural binding partner in vivo. The KD of the cleaved ligand-binding molecule relative to the ligand is, for example, 1.1 times or more, preferably 1.5 times or more, 2 times or more, 5 times or more, 10 times or more, and particularly preferably 100 times or more, compared to the KD of the natural binding partner in vivo. By having low binding activity, or almost no binding activity, after cleavage, it is expected that the ligand-binding molecule will release the ligand and prevent it from rebinding to another ligand molecule.

[0177] It is desirable that the suppressed biological activity of the ligand be restored after the ligand-binding molecule is cleaved. It is also desirable that the ligand-binding molecule's inhibitory function on ligand activity be weakened by the reduced binding of the cleaved ligand-binding molecule to the ligand. Those skilled in the art can confirm the biological activity of a ligand by known methods, such as detecting the binding of the ligand to its binding partner.

[0178] In some embodiments of the present invention, an uncleaved ligand-binding molecule forms a complex with a ligand by antigen-antibody binding. In more specific embodiments, the complex of the ligand-binding molecule and the ligand is formed by non-covalent bonding between the ligand-binding molecule and the ligand, for example, antigen-antibody binding.

[0179] In some embodiments of the present invention, an uncleaved ligand-binding molecule fuses with a ligand to form a fusion protein, and the ligand-binding molecule portion and the ligand portion within the fusion protein further interact via antigen-antibody binding. The ligand-binding molecule and the ligand may fuse via or without a linker. Even when the ligand-binding molecule and the ligand in the fusion protein are fused via or without a linker, a non-covalent bond between the ligand-binding molecule portion and the ligand portion still exists. In other words, even in embodiments where the ligand-binding molecule is fused with the ligand, the non-covalent bond between the ligand-binding molecule portion and the ligand portion is similar to that in embodiments where the ligand-binding molecule and the ligand are not fused. When the ligand-binding molecule is cleaved, its non-covalent bond is weakened. That is, the binding between the ligand-binding molecule and the ligand is weakened. In a preferred embodiment of the present invention, a ligand-binding molecule and a ligand are fused via a linker. Any peptide linker that can be introduced by genetic engineering, or a synthetic compound linker (see, for example, Protein Engineering, 9 (3), 299-305, 1996) can be used as the linker for fusing the ligand-binding molecule and the ligand; however, a peptide linker is preferred in this embodiment. The length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art depending on the purpose. For example, but not limited to, in the case of a peptide linker: Ser Gly·Ser(GS) Ser·Gly(SG) Gly·Gly·Ser(GGS) Gly·Ser·Gly (GSG) Ser·Gly·Gly (SGG) Gly·Ser·Ser (GSS) Ser·Ser·Gly (SSG) Ser·Gly·Ser(SGS) Gly·Gly·Gly·Ser(GGGS, Sequence ID: 36) Gly·Gly·Ser·Gly (GGSG, Sequence ID: 37) Gly·Ser·Gly·Gly (GSGG, Sequence ID: 38) Ser·Gly·Gly·Gly (SGGG, Sequence ID: 39) Gly·Ser·Ser·Gly(GSSG, Sequence ID: 40) Gly·Gly·Gly·Gly·Ser(GGGGS, Sequence ID: 41) Gly·Gly·Gly·Ser·Gly (GGGSG, Sequence ID: 42) Gly·Gly·Ser·Gly·Gly(GGSGG, Sequence ID: 43) Gly·Ser·Gly·Gly·Gly(GSGGG, Sequence ID: 44) Gly·Ser·Gly·Gly·Ser(GSGGS, Sequence ID: 45) Ser·Gly·Gly·Gly·Gly (SGGGG, Sequence ID: 46) Gly·Ser·Ser·Gly·Gly (GSSGG, Sequence ID: 47) Gly·Ser·Gly·Ser·Gly(GSGSG, Sequence ID: 48) Ser·Gly·Gly·Ser·Gly (SGGSG, Sequence ID: 49) Gly·Ser·Ser·Ser·Gly(GSSSG, Sequence ID: 50) Gly·Gly·Gly·Gly·Gly·Ser(GGGGGS, Sequence ID: 51) Ser·Gly·Gly·Gly·Gly·Gly (SGGGGG, Sequence ID: 52) Gly·Gly·Gly·Gly·Gly·Gly·Ser(GGGGGGS, Sequence ID: 53) Ser·Gly·Gly·Gly·Gly·Gly·Gly(SGGGGGG, Sequence ID: 54) (Gly·Gly·Gly·Gly·Ser(GGGGS, Sequence ID: 41))n (Ser·Gly·Gly·Gly·Gly(SGGGG, Sequence ID: 46))n Examples include [n is an integer greater than or equal to 1]. However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0180] Synthetic compound linkers (chemical crosslinking agents) are crosslinking agents commonly used for crosslinking peptides, such as N-hydroxysuccinimide (NHS), disuccinimidylsverate (DSS), bis(sulfosuccinimidyl)sverate (BS3), dithiobis(succinimidylpropionate) (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), ethylene glycol bis(succinimidylsuccinate) (EGS), ethylene glycol bis(sulfosuccinimidylsuccinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimideoxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimideoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES), and these crosslinking agents are commercially available.

[0181] The present invention also relates to pharmaceutical compositions (drugs) comprising the ligand-binding molecule of the present invention and a pharmaceutically acceptable carrier, pharmaceutical compositions (drugs) comprising the ligand-binding molecule of the present invention, a ligand, and a pharmaceutically acceptable carrier, and pharmaceutical compositions (drugs) comprising a fusion protein in which the ligand-binding molecule of the present invention and a ligand are fused, and a pharmaceutically acceptable carrier.

[0182] As used herein, “treatment” (and its grammatical derivatives, e.g., “to treat,” “to treat,” etc.) means a clinical intervention intended to modify the natural course of the individual being treated, and may be carried out for preventive purposes or during the course of a clinical condition. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, reduction of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction of the rate of disease progression, recovery or mitigation of the disease state, and remission or an improved prognosis. In some embodiments, the ligand-binding molecules of the present invention can control the biological activity of a ligand and are used to delay the onset of disease or slow the progression of disease.

[0183] In this invention, a pharmaceutical composition generally refers to a drug used for the treatment or prevention of a disease, or for examination and diagnosis. Furthermore, in the present invention, the term "pharmaceutical composition containing a ligand-binding molecule" can be rephrased as "a method for treating a disease, comprising administering a ligand-binding molecule to a target for treatment," or as "the use of a ligand-binding molecule in the manufacture of a pharmaceutical for treating a disease." The term "pharmaceutical composition containing a ligand-binding molecule" can also be rephrased as "the use of a ligand-binding molecule for treating a disease." The term "pharmaceutical composition comprising a ligand-binding molecule and a ligand" can also be rephrased as "a method for treating a disease comprising administering a ligand-binding molecule and a ligand to a target for treatment," or as "the use of ligand-binding molecules and ligands in the manufacture of pharmaceuticals for treating diseases." The term "pharmaceutical composition comprising a ligand-binding molecule and a ligand" can also be rephrased as "the use of ligand-binding molecules and ligands for treating diseases." The term "pharmaceutical composition containing a fusion protein" can also be rephrased as "a method for treating a disease, comprising administering a fusion protein to a target," or as "the use of a fusion protein in the manufacture of a pharmaceutical for treating a disease." The term "pharmaceutical composition containing a fusion protein" can also be rephrased as "the use of a fusion protein for treating a disease."

[0184] In some embodiments of the present invention, a composition containing a ligand-binding molecule can be administered to an individual. The ligand-binding molecule administered to the individual binds to a ligand already present in the individual, for example, in the blood or tissue, and is further transported within the body while bound to the ligand. The ligand-binding molecule transported to the target tissue is cleaved in the target tissue, weakening its binding to the ligand and releasing the bound ligand in the target tissue. The released ligand can exert biological activity in the target tissue and treat diseases originating from the target tissue. In embodiments in which the ligand-binding molecule suppresses the biological activity of the ligand while it is bound to the ligand, and the ligand-binding molecule is cleaved specifically in the target tissue, the biological activity of the ligand can be exerted only after cleavage in the target tissue without the ligand exhibiting biological activity during transport, thereby treating diseases and minimizing systemic side effects.

[0185] In some embodiments of the present invention, a composition containing a ligand-binding molecule and a composition containing a ligand can be administered to an individual separately or simultaneously. Alternatively, a composition containing both a ligand-binding molecule and a ligand can be administered to an individual. When a composition containing both a ligand-binding molecule and a ligand is administered to an individual, the ligand-binding molecule and the ligand in the composition may form a complex. When both a ligand-binding molecule and a ligand are administered to an individual, the ligand-binding molecule binds to the administered ligand and is transported within the body while bound to the ligand. Upon delivery to a target tissue, the ligand-binding molecule is cleaved in the target tissue, reducing its binding to the ligand and releasing the bound ligand in the target tissue. The released ligand can exert biological activity in the target tissue, treating diseases originating from the target tissue. In embodiments where the ligand-binding molecule suppresses the biological activity of the ligand while bound, and the ligand-binding molecule is cleaved specifically in the target tissue, the ligand's biological activity can be exerted only after cleavage in the target tissue, without the ligand exhibiting biological activity during transport, thus treating diseases and minimizing systemic side effects. Ligand-binding molecules administered to an organism can bind not only to the administered ligand but also to ligands naturally present within the organism, allowing for the transport of both the naturally present ligand and the administered ligand within the organism while bound. In other words, the present invention also provides a method for producing a ligand complex, comprising the steps of contacting a ligand-binding molecule with a ligand and recovering a complex consisting of the ligand-binding molecule and the ligand. The complex of the present invention can be incorporated into a pharmaceutical composition, for example, by compounding it with a pharmaceutically acceptable carrier.

[0186] In some embodiments of the present invention, a fusion protein, which is a fusion of a ligand-binding molecule and a ligand, can be administered to an individual. In these embodiments, the ligand-binding molecule and ligand in the fusion protein form the fusion protein with or without the linker, but a non-covalent bond between the ligand-binding molecule portion and the ligand portion still exists. When a fusion protein, which is a fusion of a ligand-binding molecule and a ligand, is administered to an individual, the fusion protein is transported in the body, and the ligand-binding molecule portion in the fusion protein is cleaved in the target tissue, thereby weakening the non-covalent bond between the ligand-binding molecule portion and the ligand, and releasing the ligand and a portion of the ligand-binding molecule from the fusion protein. The released ligand and a portion of the ligand-binding molecule exert the biological activity of the ligand in the target tissue, and can treat diseases originating from the target tissue. In embodiments in which the biological activity of the ligand is suppressed when the ligand-binding molecule is bound to the ligand, and the ligand-binding molecule is cleaved specifically in the target tissue, the biological activity of the ligand can be exerted only after cleavage in the target tissue, without the biological activity of the ligand in the fusion protein being exerted during transport, thereby treating diseases and minimizing systemic side effects. Therefore, based on the present invention, a method for administering a ligand to a subject requiring ligand administration is provided, comprising the following steps: [1] A step of contacting a ligand with the ligand conjugate of the present invention to obtain a conjugated product comprising both; and [2] A step of administering the binding product of [1] to a subject that requires ligand administration.

[0187] The pharmaceutical compositions of the present invention can be formulated using methods known to those skilled in the art. For example, they can be administered parenterally in the form of sterile solutions with water or other pharmaceutically acceptable liquids, or as injectable suspensions. For example, they can be formulated by mixing them with pharmacokinetically acceptable carriers or media, specifically sterile water or saline solution, vegetable oil, emulsifiers, suspensions, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, etc., in a unit dose form generally required for pharmaceutical practice. The amount of active ingredient in these formulations is set to obtain an appropriate volume within the indicated range.

[0188] Sterile compositions for injection can be formulated in accordance with standard formulation procedures using a vehicle such as distilled water for injection. Examples of aqueous solutions for injection include physiological saline, glucose, and isotonic solutions containing other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). Appropriate solubilizers, such as alcohols (ethanol, etc.), polyalcohols (propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (polysorbate 80™, HCO-50, etc.), may be used in combination.

[0189] Examples of oily solutions include sesame oil and soybean oil, and benzyl benzoate and / or benzyl alcohol may also be used as solubilizers. Furthermore, buffers (e.g., phosphate buffer and sodium acetate buffer), analgesics (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants may be added. The prepared injection solution is usually filled into appropriate ampoules.

[0190] The pharmaceutical composition of the present invention is preferably administered by parenteral administration. For example, compositions in the form of injection, nasal administration, pulmonary administration, or transdermal administration may be administered. For example, it may be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.

[0191] The method of administration may be appropriately selected depending on the patient's age and symptoms. The dosage of the pharmaceutical composition containing the ligand-binding molecule may be set, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight per dose. Alternatively, for example, a dosage of 0.001 to 100,000 mg per patient may be set, but the present invention is not necessarily limited to these values. The dosage and method of administration will vary depending on the patient's weight, age, symptoms, etc., but a person skilled in the art can set an appropriate dosage and method of administration considering these conditions.

[0192] The present invention also relates to a method for producing a ligand-binding molecule in which binding to a ligand is weakened when cleaved, or a fusion protein obtained by fusing such ligand-binding molecule with a ligand. In one embodiment of the present invention, a method for producing a ligand-binding molecule or a fusion protein is provided, which includes introducing a protease cleavage sequence into a molecule that can bind to a ligand.

[0193] Examples of methods for introducing protease cleavage sequences into ligand-binding molecules include inserting a protease cleavage sequence into the amino acid sequence of a ligand-binding polypeptide, or replacing a portion of the amino acid sequence of a ligand-binding polypeptide with a protease cleavage sequence.

[0194] Inserting amino acid sequence A into amino acid sequence B means dividing amino acid sequence B into two parts without deleting any, and connecting the two parts with amino acid sequence A (i.e., creating a new amino acid sequence such as "first half of amino acid sequence B - amino acid sequence A - second half of amino acid sequence B"). Introducing amino acid sequence A into amino acid sequence B means dividing amino acid sequence B into two parts and connecting the two parts with amino acid sequence A. In addition to inserting amino acid sequence A into amino acid sequence B, it is also possible to delete one or more amino acid residues, including amino acid residues in amino acid sequence B adjacent to amino acid sequence A, and then connect the two parts with amino acid sequence A (i.e., replacing a part of amino acid sequence B with amino acid sequence A).

[0195] One example of a method for obtaining a molecule capable of binding to a ligand is to obtain a ligand-binding region that has the ability to bind to a ligand. The ligand-binding region can be obtained, for example, by a method using a known antibody production method. The antibody obtained by this method may be used as is in the ligand-binding region, or only the Fv region of the obtained antibody may be used, and if the Fv region is single-stranded (also referred to as "sc") and capable of recognizing the antigen, only the single-stranded portion may be used. Alternatively, the Fab region including the Fv region may be used.

[0196] While specific antibody production methods are well known to those skilled in the art, monoclonal antibodies, for example, may be produced by hybridoma (Kohler and Milstein, Nature 256:495 (1975)) or recombinant methods (U.S. Patent No. 4,816,567). They may also be isolated from phage antibody libraries (Clackson et al., Nature 352:624-628 (1991); Marks et al., J.Mol.Biol. 222:581-597 (1991)). They may also be isolated from a single B cell clone (N. Biotechnol. 28(5): 253-457 (2011)).

[0197] Humanized antibodies are also called reshaped human antibodies. Specifically, known examples include humanized antibodies obtained by transplanting the CDR of an antibody from a non-human animal, such as a mouse antibody, into a human antibody. Common genetic recombination techniques for obtaining humanized antibodies are also known. Specifically, Overlap Extension PCR is a known method for transplanting the CDR of a mouse antibody into the FR of a human antibody.

[0198] A vector for humanized antibody expression can be created by inserting into an expression vector, in a way that fuses in-frame, DNA encoding an antibody variable region consisting of three CDRs and four FRs with DNA encoding a human antibody constant region. After introducing the embedded vector into a host to establish recombinant cells, the recombinant cells are cultured and the DNA encoding the humanized antibody is expressed, thereby producing the humanized antibody in the cultured cell culture (see European Patent Application Publication No. 239400, International Publication No. 1996 / 002576).

[0199] If necessary, amino acid residues in the FR can be substituted so that the reconstituted human antibody CDR forms an appropriate antigen-binding site. For example, amino acid sequence mutations can be introduced into the FR by applying the PCR method used to transplant mouse CDRs into human FRs.

[0200] Transgenic animals possessing the entire repertoire of human antibody genes (see International Publications 1993 / 012227, 1992 / 003918, 1994 / 002602, 1994 / 025585, 1996 / 034096, and 1996 / 033735) can be used as immunized animals, and desired human antibodies can be obtained by DNA immunization.

[0201] Furthermore, a technique for obtaining human antibodies by panning using a human antibody library is also known. For example, the Fv region of a human antibody is expressed on the surface of a phage as a single-chain antibody (also called "scFv") by phage display. A phage expressing an antigen-binding scFv can be selected. By analyzing the genes of the selected phage, the DNA sequence encoding the Fv region of the antigen-binding human antibody can be determined. After determining the DNA sequence of the antigen-binding scFv, an expression vector can be created by fusing the Fv region sequence in-frame with the sequence of the desired human antibody C region and then inserting it into a suitable expression vector. The human antibody can be obtained by introducing the expression vector into suitable expression cells as described above and expressing the gene encoding the human antibody. These methods are already publicly known (see International Publications 1992 / 001047, 1992 / 020791, 1993 / 006213, 1993 / 011236, 1993 / 019172, 1995 / 001438, and 1995 / 015388).

[0202] A molecule into which a protease cleavage sequence has been introduced to a ligand-binding molecule becomes the ligand-binding molecule of the present invention. The ligand-binding molecule can be optionally cleaved by treatment with a protease corresponding to the protease cleavage sequence. For example, by contacting a molecule into which a protease cleavage sequence has been introduced to a ligand-binding molecule with a protease and examining the molecular weight of the product after protease treatment using electrophoresis such as SDS-PAGE, it is possible to confirm whether or not the protease cleavage sequence has been cleaved.

[0203] Furthermore, by quantifying the amount of cleavage fragments after protease treatment separated by electrophoresis methods such as SDS-PAGE, it is possible to evaluate protease activity and the cleavage rate of molecules into which protease cleavage sequences have been introduced. The following is one non-limiting embodiment of a method for evaluating the cleavage rate of molecules into which protease cleavage sequences have been introduced. For example, when evaluating the cleavage rate of an antibody variant into which a protease cleavage sequence has been introduced using recombinant human u-Plasminogen Activator / Urokinase (human uPA, huPA) (R&D Systems; 1310-SE-010) or recombinant human Matriptase / ST14 Catalytic Domain (human MT-SP1, hMT-SP1) (R&D Systems; 3946-SE-010), the mixture is reacted for 1 hour under conditions of huPA 40 nM or hMT-SP1 3 nM, antibody variant 100 μg / mL, PBS, and 37°C, and then subjected to a capillary electrophoresis immunoassay. Wes (Protein Simple) can be used for the capillary electrophoresis immunoassay, but is not limited to this method. Alternatively, the sample may be separated by SDS-PAGE or similar methods and then detected by Western blotting. For detecting light chains before and after cleavage, an anti-human lambda chain HRP-labeled antibody (abcam; ab9007) can be used, but any antibody capable of detecting cleavage fragments can be used. By outputting the area of ​​each peak obtained after protease treatment using Wes-specific software (Compass for SW; Protein Simple), the cleavage rate (%) of the antibody-modified molecule can be calculated using the formula: (cleavage light chain peak area) * 100 / (cleavage light chain peak area + uncleaved light chain peak area). The cleavage rate can be calculated as long as protein fragments before and after protease treatment can be detected, and the cleavage rate can be calculated for various proteins, not just antibody-modified molecules, that have been introduced with a protease cleavage sequence.

[0204] After administering a molecule containing a protease cleavage sequence to an animal, the in vivo cleavage rate can be calculated by detecting the administered molecule in a blood sample. For example, after administering an antibody variant containing a protease cleavage sequence to a mouse, plasma is collected from the blood sample, and the antibody is purified using Dynabeads Protein A (Thermo; 10001D) by a method known to those skilled in the art. The protease cleavage rate of the antibody variant can then be evaluated by subjecting it to a capillary electrophoresis immunoassay. While Wes (Protein Simple) can be used for the capillary electrophoresis immunoassay, the method is not limited to this; alternative methods such as separation by SDS-PAGE followed by detection by Western Blotting are also possible, and the method is not limited to these. For detecting the light chain of the antibody variant recovered from the mouse, an anti-human lambda chain HRP-labeled antibody (abcam; ab9007) can be used, but any antibody capable of detecting cleavage fragments can be used. By outputting the area of ​​each peak obtained by capillary electrophoresis immunoassay using Wes-specific software (Compass for SW; Protein Simple), and calculating the light chain retention ratio as (light chain peak area) / (heavy chain peak area), it is possible to calculate the proportion of the full-length light chain that remained uncleaved in the mouse body. Calculating the cleavage efficiency in vivo is possible if protein fragments recovered from the body can be detected, and the cleavage rate can be calculated for various proteins, not just antibody modifiers, that have been introduced with protease cleavage sequences. By calculating the cleavage rate using the method described above, it is possible to compare, for example, the in vivo cleavage rates of antibody modifiers with different cleavage sequences, and also to compare the cleavage rates of the same antibody modifier across different animal models, such as normal mouse models and tumor-transplanted mouse models.

[0205] The present invention also relates to polynucleotides that encode ligand-binding molecules whose binding to ligands is weakened when cleaved, or to polynucleotides that encode fusion proteins formed by fusing such ligand-binding molecules with ligands.

[0206] The polynucleotides in this invention are typically loaded (inserted) into a suitable vector and introduced into host cells. The vector is not particularly limited as long as it stably holds the inserted nucleic acid. For example, if E. coli is used as the host, the pBluescript vector (Stratagene) is preferred as a cloning vector, but various commercially available vectors can be used. When using a vector for the purpose of producing the ligand-binding molecule or fusion protein of this invention, an expression vector is particularly useful. The expression vector is not particularly limited as long as it is a vector that expresses the ligand-binding molecule in vitro, in E. coli, in cultured cells, or in living organisms. For example, the pBEST vector (Promega) is preferred for in vitro expression, the pET vector (Invitrogen) is preferred for E. coli, the pME18S-FL3 vector (GenBank Accession No. AB009864) is preferred for cultured cells, and the pME18S vector (Mol Cell Biol. 8:466-472 (1988)) is preferred for living organisms. The insertion of the DNA of the present invention into a vector can be carried out by conventional methods, for example, by a ligase reaction using restriction enzyme sites (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.4-11.11).

[0207] There are no particular restrictions on the host cells used, and various host cells can be used depending on the purpose. Examples of cells used to express ligand-binding molecules or fusion proteins include bacterial cells (e.g., Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, Bacillus subtilis), fungal cells (e.g., yeast, Aspergillus), insect cells (e.g., Drosophila S2, Spodoptera SF9), animal cells (e.g., CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells), and plant cells. Vector introduction into host cells can be performed by known methods such as calcium phosphate precipitation, electroporation (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), lipofectamine (GIBCO-BRL), and microinjection.

[0208] To secrete ligand-binding molecules or fusion proteins expressed in host cells into the lumen of the endoplasmic reticulum, the pericellular lumen, or the extracellular environment, appropriate secretion signals can be incorporated into the target ligand-binding molecule or fusion protein. These signals may be endogenous or heterologous to the target ligand-binding molecule or fusion protein.

[0209] In the above manufacturing method, if the ligand-binding molecule or fusion protein of the present invention is secreted into the culture medium, the culture medium is recovered. If the ligand-binding molecule or fusion protein of the present invention is produced inside a cell, the cell is first lysed, and then the ligand-binding molecule or fusion protein is recovered.

[0210] To recover and purify the ligand-binding molecule or fusion protein of the present invention from recombinant cell cultures, known methods can be used, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyl apatite chromatography, and lectin chromatography.

[0211] It will be understood by those skilled in the art that any combination of one or more embodiments described herein is included in the present invention, provided that it does not contradict the common technical knowledge of those skilled in the art. Furthermore, any invention that excludes any combination of one or more embodiments described herein from the present invention should be considered as an invention intended and described herein, provided that it does not contradict the common technical knowledge of those skilled in the art. [Examples]

[0212] The following are examples of the methods and compositions of the present invention. In light of the general description above, it will be understood that various other embodiments may be implemented.

[0213] Example 1: Challenges of previously reported immunocytokines and protease-activating cytokines Immunocytokines that target antigens expressed in cancer tissue have generally been created by fusing the target cytokine to the terminal of targeting IgG or scFv (Expert Opin Investig Drugs. 2009 Jul;18(7):991-1000., Curr Opin Immunol. 2016 Jun;40:96-102.). Because cytokines such as IL-2, IL-12, and TNF are highly toxic, it is hoped that delivering these cytokines to the tumor site using antibodies will reduce side effects while enhancing efficacy (Non-patent Literature 4, 5, 6). However, all of these methods have challenges, such as not showing sufficient clinical efficacy with systemic administration, having a narrow therapeutic window, and being too toxic to be administered systemically. A major reason for this is that even immunocytokines, when administered systemically, are exposed to the entire body, which can cause systemic effects and toxicity, or they can only be administered at extremely low doses to avoid toxicity. Furthermore, immunocytokines that bind to cancer antigens are internalized and eliminated by cancer cells within the tumor, making it difficult to expose the tumor site to cytokines in some cases. There are also reports that the antitumor effect was the same whether immunocytokines were antibodies that bind to cancer antigens fused with IL-2 or antibodies that do not bind to cancer antigens fused with IL-2 (Non-patent Literature 7).

[0214] As a method to reduce the systemic effects of immunocytokines, a major challenge, molecules have been reported in which cytokines and cytokine receptors are linked by a linker that is cleaved by a protease highly expressed in cancer. Cytokines are inhibited by cytokine receptors linked by the linker, but when the linker is cleaved by the protease, the cytokine receptor is released and the cytokine becomes active. Examples include molecules in which TNFalpha and TNFR are linked by a linker cleaved by uPA (Non-Patent Literature 8), and molecules in which IL-2 and IL-2R are linked by a linker cleaved by MMP-2 (Non-Patent Literature 9). However, even before linker cleavage, the cytokines in these molecules possess biological activity, and linker cleavage only increases their activity by about 10 times. Two reasons for this are that the affinity between cytokines and cytokine receptors is not strong, so cytokines retain some activity even before protease cleavage, or that cytokine receptors can still bind to cytokines even after the linker has been cleaved by the protease, thereby inhibiting the biological activity of cytokines.

[0215] A molecule has been reported in which anti-IL-2 scFv is bound to IL-2 via a linker cleaved by MMP-2 instead of IL-2R (Non-Patent Document 9). In this molecule, which links IL-2 and anti-IL-2 scFv via a protease-cleaved linker, it is natural to use anti-IL-2 scFv, which does not have a strong affinity for IL-2, considering that IL-2 is released upon linker cleavage, just as with molecules that link cytokines and cytokine receptors. Furthermore, unlike the IgG-IL-2 fusions mentioned above, these reported protease-activated cytokines lack an Fc region, and are therefore expected to have short half-lives, making it difficult to maintain high exposure levels. There is no significant difference in the pharmacokinetics of the cytokines before and after activation by protease cleavage (both have short half-lives), making it difficult to broaden the therapeutic window.

[0216] Example 2: Challenges in the application of chemokines to cancer immunotherapy Chemokines (Nature Immunology 9, 949 - 952 (2008)) are basic proteins that exert their effects via G protein-coupled receptors and belong to a group of cytokines. They act on specific leukocytes that express receptors and have the activity (chemotaxis) of causing leukocytes to migrate in the direction of the concentration gradient of the substance (Nat Cell Biol. 2016 Jan;18(1):43-53.). Chemokines are known to be produced in large quantities at the site of inflammation and to cause the migration of leukocytes from the blood vessels into the inflammatory tissue. Since controlling chemokines can control the migration of leukocytes, it is thought that this could be utilized in cancer immunotherapy. If T cells, antigen-presenting cells, and M1 macrophages can be migrated to the local site of a solid tumor, it is thought that an antitumor effect can be induced. Cytokines can exert their effects even when administered systemically, but chemokines cause cells to migrate to tissues with high concentrations through a concentration gradient, so the desired effect cannot be obtained by administering chemokines systemically. For this reason, cancer immunotherapy using systemic administration of chemokines (chemokine therapy) is not considered practical.

[0217] Example 3: Concept of a ligand-binding molecule that can release a target tissue-specific ligand by introducing a protease cleavage sequence. As shown in Examples 1 and 2, previously reported cytokine / chemokine therapies have the following problems. 1. In the case of immunocytokines, even if cytokines are targeted to solid tumors using antibodies, side effects occur because cytokines act systemically, or they can only be administered at low doses to avoid side effects, making it impossible to achieve high exposure within the tumor. 2. In cytokines activated by proteases, where cytokine receptors (or antibodies) and cytokines are linked by a linker that can be cleaved by proteases, the neutralization of cytokine activity is insufficient, and the cytokine retains some activity even before protease cleavage. 3. In the case of cytokines activated by proteases, cytokine receptors (or antibodies) can still bind to the cytokine even after the linker has been cleaved by the protease, thus inhibiting the biological activity of the cytokine. 4. For cytokines activated by proteases, the inactive form has a short half-life and short blood residence time, requiring a large dose.

[0218] To solve this problem, we believe it is important to meet the following conditions. 1. Systemically, ligands such as cytokines or chemokines are sufficiently inhibited by ligand-binding molecules (biological activity is minimized). 2. The biological activity of the ligand is restored by cleavage by protease (it becomes an active ligand). 3. Cleavage by protease causes ligand-binding molecules to lose their ligand-binding activity. 4. Compared to the ligand bound to the ligand-binding molecule before cleavage by protease, the ligand that becomes active after cleavage by protease has a shorter half-life.

[0219] As a pharmaceutical composition that satisfies the above conditions, we devised a molecule in which binding to a ligand is weakened by the cleavage of a cleavage site. First, a ligand-binding molecule can be obtained, and then a ligand-binding molecule can be created by inserting a cleavage site into the binding molecule.

[0220] Example 4: An example of an anti-ligand antibody into which a protease cleavage sequence has been introduced. Figures 1, 2, and 3 show examples of molecules using antibodies as ligand-binding molecules. In these examples, first, a neutralizing antibody against the ligand is obtained. Next, a protease cleavage sequence is introduced near the boundary between the variable region (VH or VL) and the constant region (CH1 or CL) of the anti-ligand neutralizing antibody. It is confirmed that the ligand-binding activity of the anti-ligand antibody is retained even after the introduction of the protease cleavage sequence. It is confirmed that the ligand dissociates when cleaved by the protease while bound to the anti-ligand neutralizing antibody. It is confirmed that the dissociated ligand exhibits biological activity. In Figure 1, the C-terminus of the ligand and the N-terminus of the VH molecule of the anti-ligand antibody are linked via a linker, and a protease cleavage sequence is introduced near the boundary between VH and CH1. If the affinity of the anti-ligand antibody for the ligand is sufficiently strong, the biological activity of the ligand is sufficiently inhibited. Even if this ligand-anti-ligand antibody fusion is administered systemically, the ligand is neutralized and therefore does not exert its biological activity, and the ligand-anti-ligand antibody fusion has a long half-life because it has an Fc region. When the ligand-anti-ligand antibody fusion is administered systemically, the protease cleavage sequence near the boundary between VH and CH1 is cleaved by a protease highly expressed in tumor tissue, releasing the VH molecule of the ligand-linker-anti-ligand antibody. Since VH or VL alone cannot bind to the ligand (both VH and VL are required to bind to the ligand), the neutralization of the ligand is released, and it becomes possible for it to exert its biological effect in tumor tissue. Furthermore, the VH molecule of this released ligand-linker-antiligand antibody lacks an Fc region and has a small molecular weight, resulting in a very short half-life and rapid elimination from the body, thus minimizing systemic side effects caused by the ligand. In Figure 2, the ligand and anti-ligand antibody are not linked by a linker as in Figure 1. Instead, the anti-ligand antibody, which has a protease cleavage sequence introduced near the VH-CH1 boundary, is administered mixed with the ligand. If the affinity of the anti-ligand antibody for the ligand is sufficiently strong and the amount of anti-ligand antibody is sufficient relative to the ligand concentration, the biological activity of the ligand is sufficiently inhibited. Even if this ligand-anti-ligand antibody complex is administered systemically, the ligand is neutralized and therefore does not exert its biological activity. Furthermore, the ligand-anti-ligand antibody complex has a long half-life because it possesses an Fc region. When the systemically administered ligand-anti-ligand antibody complex has its protease cleavage sequence near the VH-CH1 boundary cleaved by a protease highly expressed in tumor tissue, the VH molecule of the anti-ligand antibody is released. Since VH or VL alone cannot bind to the ligand (both VH and VL are required to bind to the ligand), the neutralization of the ligand is released, and it becomes possible for it to exert its biological effect in tumor tissue. Furthermore, because this released ligand molecule lacks an Fc region and has a small molecular weight, it has a very short half-life and is rapidly eliminated from the body, thus minimizing systemic side effects caused by the ligand. In Figure 3, an anti-ligand antibody in which a protease cleavage sequence is introduced near the boundary between VH and CH1 is administered systemically. The administered antibody binds to a ligand already present in the body, and the process thereafter is the same as described above in Figure 2. By using anti-ligand antibodies in which a protease cleavage sequence is introduced near the boundary between VH and CH1, it becomes possible to selectively release the ligand in tissues that express the protease and exert its biological effects. If the ligand is a cytokine, the cytokine can be selectively acted upon in tissues that express the protease. If the ligand is a chemokine, the chemokine will be present at high concentrations in tissues that express the protease, and its concentration will be low in the peripheral blood, allowing cells expressing chemokine receptors to migrate to tissues that express the protease.

[0221] Example 5: Preparation and evaluation of CXCL10-releasing antibody 5-1. Introduction of protease cleavage sequences into anti-CXCL10 neutralizing antibodies CXCL10 is one of the chemokines that has migratory effects on effector T cells. An expression vector for MabCXCL10 (heavy chain: EEIVH (SEQ ID NO: 1), light chain: EEIVL (SEQ ID NO: 2)), a neutralizing antibody against human CXCL10, was prepared by methods known to the art, and expression and purification were performed using FreeStyle 293 (Life Technology) by methods known to the art. The CDR sequences contained in MabCXCL10 are as follows: H-CDR1 (NNGMH, SEQ ID NO: 380), H-CDR2 (VIWFDGMNKFYVDSVKG, SEQ ID NO: 381), H-CDR3 (EGDGSGIYYYYGMDV, SEQ ID NO: 382), L-CDR1 (RASQSVSSSYLA, SEQ ID NO: 383), L-CDR2 (GASSRAT, SEQ ID NO: 384), L-CDR3 (QQYGSSPIFT, SEQ ID NO: 385). The interaction between MabCXCL10 and human CXCL10 (266-IP-010 / CF, R&D Systems) was evaluated using Biacore. Specifically, R PROTEIN A (SURE) (28-4018-60, GE Healthcare) was immobilized on a CM3 sensor chip (BR100536, GE Healthcare) using an amine coupling method with NHS·EDC. Using 20 mM ACES, 0.05% Tween20, 200 mM NaCl, pH 7.4 as the running buffer, 1.563 nM human CXCL10 was flowed as an analyte while the antibody was captured, and the binding of the antibody to the antigen at 37°C was evaluated. Figure 4 shows a sensorgram representing the time-dependent binding amount, calculated by taking the difference between the result and a blank (where only the running buffer was used as the analyte). The horizontal axis represents the start time when the analyte was started. The vertical axis represents the response (binding amount) at each time point, with the response at the time the analyte was first introduced set to 0. As shown in the sensorgram in Figure 4, binding of MabCXCL10 to human CXCL10 was confirmed. We investigated inserting protease cleavage sequences near the boundary between the variable and constant regions of the heavy or light chain of MabCXCL10. We designed the heavy and light chains shown in Figure 5 by inserting peptide sequence A (SEQ ID NO: 3), a sequence reported to be cleaved by cancer-specific urokinase (uPA) and matryptase (MT-SP1), at seven locations near the boundary between the variable and constant regions of the heavy or light chain. We also designed modified versions that do not undergo glycosylation as a result of inserting the cleavage sequences. Expression vectors encoding the heavy chain variants EEIVHA (SEQ ID NO: 4), EEIVHB (SEQ ID NO: 5), EEIVHC (SEQ ID NO: 6), EEIVHD (SEQ ID NO: 7), EEIVHE (SEQ ID NO: 8), EEIVHF (SEQ ID NO: 9), EEIVHG (SEQ ID NO: 10), EEIVHBG (SEQ ID NO: 11), EEIVHCG (SEQ ID NO: 12), EEIVHDG (SEQ ID NO: 13), EEIVHEG (SEQ ID NO: 14), and the light chain variants EEIVLA (SEQ ID NO: 15), EEIVLB (SEQ ID NO: 16), EEIVLC (SEQ ID NO: 17), EEIVLD (SEQ ID NO: 18), EEIVLE (SEQ ID NO: 19), EEIVLF (SEQ ID NO: 20), EEIVLG (SEQ ID NO: 21), and EEIVLEG (SEQ ID NO: 22) were prepared by methods known to those skilled in the art. The following IgG1 antibodies were created by combining these heavy chain variants with the natural light chain, or by combining the natural heavy chain with the light chain variant, and inserting a protease cleavage sequence near the boundary between the variable and constant regions of the heavy chain: EEIVHA / EEIVL (heavy chain SEQ ID NO: 4, light chain SEQ ID NO: 2), EEIVHB / EEIVL (heavy chain SEQ ID NO: 5, light chain SEQ ID NO: 2), EEIVHC / EEIVL (heavy chain SEQ ID NO: 6, light chain SEQ ID NO: 2), EEIVHD / EE IVL (heavy chain sequence number: 7, light chain sequence number: 2), EEIVHE / EEIVL (heavy chain sequence number: 8, light chain sequence number: 2), EEIVHF / EEIVL (heavy chain sequence number: 9, light chain sequence number: 2), EEIVHG / EEIVL (heavy chain sequence number: 10, light chain sequence number: 2), EEIVHBG / EEIVL (heavy chain sequence number: 11, light chain sequence number: 2), EEIVHCG / EEIVL (heavy chain sequence number: 12, light chain sequence number: 2), EEIVHDG / EEIVL (heavy chain SEQ ID NO: 13, light chain SEQ ID NO: 2), EEIVHEG / EEIVL (heavy chain SEQ ID NO: 14, light chain SEQ ID NO: 2), and the following IgG1 antibodies with a protease cleavage sequence inserted near the boundary between the variable and constant regions of the light chain: EEIVH / EEIVLA (heavy chain SEQ ID NO: 1, light chain SEQ ID NO: 15), EEIVH / EEIVLB (heavy chain SEQ ID NO: 1, light chain SEQ ID NO: 16), EEIVH / EEIVLC (heavy chain SEQ ID NO: 1 Light chain sequence number: 17), EEIVH / EEIVLD (heavy chain sequence number: 1, light chain sequence number: 18), EEIVH / EEIVLE (heavy chain sequence number: 1, light chain sequence number: 19), EEIVH / EEIVLF (heavy chain sequence number: 1, light chain sequence number: 20), EEIVH / EEIVLG (heavy chain sequence number: 1, light chain sequence number: 21), and EEIVH / EEIVLEG (heavy chain sequence number: 1, light chain sequence number: 22) were expressed by transient expression using FreeStyle 293 (Life Technologies) using methods known to those skilled in the art, and purified using protein A using methods known to those skilled in the art.

[0222] 5-2. Evaluation of the binding activity of anti-CXCL10 neutralizing antibodies with introduced protease cleavage sequences. Figure 6 shows the results of evaluating the interaction between the antibody prepared in 5-1 and human CXCL10 (266-IP-010 / CF, R&D Systems) using Biacore. Specifically, R PROTEIN A (SURE) (28-4018-60, GE Healthcare) was immobilized on a CM3 sensor chip (BR100536, GE Healthcare) by amine coupling using NHS·EDC. 20 mM ACES, 0.05% Tween20, 150 mM NaCl, pH 7.4 was used as the running buffer, and with the antibody captured, 3.125, 1.563, and 0.781 nM human CXCL10 were run as analytes, and the binding of the antibody to the antigen at 25°C was evaluated. Figure 6 shows the sensorgram representing the time-series binding amount, calculated by taking the difference from a blank where only the running buffer was used as the analyte. The horizontal axis represents the starting point of the analyte flow. The vertical axis represents the response (binding amount) at each time point, with the response at the start of analyte flow set to 0. As shown in the sensorgram of Figure 6, all antibodies bound to human CXCL10. In other words, it was possible to insert the protease cleavage sequence near the boundary between the antibody variable region and the constant region without losing antigen-binding activity.

[0223] 5-3. Evaluation of protease cleavage of anti-CXCL10 neutralizing antibodies with introduced protease cleavage sequences. We investigated whether the antibodies prepared in 5-1 could be cleaved by proteases. Recombinant human matryptase / ST14 catalytic domain (MT-SP1) (R&D Systems, 3946-SE-010) was used as the protease, and the mixture was reacted for 20 hours under conditions of 20 nM protease, 60 or 100 μg / mL antibody, PBS, and 37°C. The protease cleavage was then evaluated by reduced SDS-PAGE, and the results are shown in Figure 7. As a result, EEIVHA / EEIVL, EEIVHE / EEIVL, EEIVHF / EEIVL, EEIVHG / EEIVL, EEIVHEG / EEIVL, and EEIVHBG / EEIVL showed the generation of new bands between 25 kDa and 50 kDa after protease treatment. Furthermore, EEIVH / EEIVLEG, EEIVH / EEIVLF, and EEIVH / EEIVLG showed bands below 25 kDa after protease treatment. Therefore, it was confirmed that the antibodies in EEIVHA / EEIVL, EEIVHE / EEIVL, EEIVHF / EEIVL, EEIVHG / EEIVL, EEIVHEG / EEIVL, EEIVHBG / EEIVL, EEIVH / EEIVLEG, EEIVH / EEIVLF, and EEIVH / EEIVLG were cleaved by proteases.

[0224] 5-4. Introduction of a mobile linker sequence near the protease cleavage sequence of an anti-CXCL10 neutralizing antibody containing a protease cleavage sequence. In step 5-3, we investigated inserting a sequence containing a linker made of a glycine-serine polymer near the protease cleavage sequence of EEIVHC / EEIVL that was not cleaved by the recombinant human matoriptase / ST14 (MT-SP1) catalytic domain (R&D Systems, 3946-SE-010). Five types of heavy chains were designed, as shown in Figure 8. Expression vectors encoding the heavy chain variants EEIVHC002 (SEQ ID NO: 23), EEIVHC003 (SEQ ID NO: 24), EEIVHC004 (SEQ ID NO: 25), EEIVHC005 (SEQ ID NO: 26), and EEIVHC006 (SEQ ID NO: 27) were prepared using methods known to the art. These heavy chain variants were combined with the native light chain to create the following IgG1 antibodies in which a protease cleavage sequence was inserted near the boundary between the variable and constant regions of the heavy chain: EEIVHC002 / EEIVL (heavy chain SEQ ID NO: 23, light chain SEQ ID NO: 2), EEIVHC003 / EEIVL (heavy chain SEQ ID NO: 24, light chain SEQ ID NO: 2), EEIVHC004 / EEIVL (heavy chain SEQ ID NO: 25, light chain SEQ ID NO: 2), EEIVHC005 / EEIVL (heavy chain SEQ ID NO: 26, light chain SEQ ID NO: 2), and EEIVHC006 / EEIVL (heavy chain SEQ ID NO: 27, light chain SEQ ID NO: 2). These antibodies were expressed transiently using FreeStyle 293 (Life Technologies) by methods known to those skilled in the art, and purified using Protein A by methods known to those skilled in the art.

[0225] 5-5. Evaluation of the binding activity of anti-CXCL10 neutralizing antibodies incorporating protease cleavage sequences and mobile linker sequences. Figure 9 shows the results of evaluating the interaction between the antibody prepared in 5-4 and human CXCL10 (266-IP-010 / CF, R&D Systems) using Biacore. Specifically, R PROTEIN A (SURE) (28-4018-60, GE Healthcare) was immobilized on a CM3 sensor chip (BR100536, GE Healthcare) by amine coupling using NHS·EDC. 20 mM ACES, 0.05% Tween20, 300 mM NaCl, pH 7.4 was used as the running buffer, and human CXCL10 at concentrations of 6.25, 3.125, 1.563, and 0.781 nM was run as analytes with the antibody captured. The binding of the antibody to the antigen at 25°C was evaluated. Figure 9 shows the sensorgram representing the time-dependent binding amount, calculated by taking the difference from a blank where only the running buffer was used as the analyte. The horizontal axis represents the starting point of the analyte flow. The vertical axis represents the response (binding amount) at each time point, with the response at the start of analyte flow set to 0. As shown in the sensorgram of Figure 9, all antibodies bound to human CXCL10. In other words, it was possible to insert the protease cleavage sequence and the mobile linker sequence near the boundary between the antibody variable region and the constant region without losing antigen-binding activity.

[0226] 5-6. Evaluation of protease cleavage of anti-CXCL10 neutralizing antibodies incorporating protease cleavage sequences and mobile linker sequences. We investigated whether the antibodies prepared in 5-5 could be cleaved by proteases. Human urokinase (uPA) (R&D Systems, 1310-SE-010) and recombinant human matryptase / ST14 catalytic domain (MT-SP1) (R&D Systems, 3946-SE-010) were used as proteases. After reacting the antibodies with 12.5 nM protease, 133 μg / mL antibody, PBS, and 37°C for 2 and 20 hours, the protease cleavage was evaluated by reduced SDS-PAGE. The results are shown in Figure 10. As a result, EEIVHC002 / EEIVL, EEIVHC003 / EEIVL, EEIVHC004 / EEIVL, EEIVHC005 / EEIVL, and EEIVHEC006 / EEIVL showed the emergence of a new band between 25kDa and 50kDa upon protease treatment. Therefore, it was confirmed that the antibodies in EEIVHC002 / EEIVL, EEIVHC003 / EEIVL, EEIVHC004 / EEIVL, EEIVHC005 / EEIVL, and EEIVHEC006 / EEIVL were cleaved by protease. These results demonstrate that even antibodies that do not undergo protease cleavage when only the protease cleavage site is introduced near the boundary between the variable and constant regions, such as EEIVHC / EEIVL, can be made protease-cleavable by introducing a mobile linker sequence near the cleavage sequence. Therefore, it is shown that antibodies that undergo protease cleavage can be produced by arbitrarily combining a protease cleavage sequence and a mobile linker.

[0227] 5-7. Ligand activation by protease cleavage of CXCL10-anti-CXCL10 neutralizing antibody. Next, we evaluated whether human CXCL10 bound to the antibody prepared in 5-5 was released by protease treatment using Biacore. Specifically, using the antibody EEIVHC006a / EEIVL (heavy chain SEQ ID NO: 33, light chain SEQ ID NO: 2) prepared in 5-5, we prepared analytes with antigen / protease, without antigen / with protease, and without antigen / without protease. For the analytes with antigen / protease, we used those prepared by binding the antibody to human CXCL10 and then treating them with 20 nM recombinant human matryptase / ST14 catalytic domain (MT-SP1) (R&D Systems, 3946-SE-010) for 20 hours. For the antigen-free / protease-containing analytes, antibody-only preparations were used, treated with 20 nM recombinant human matryptase / ST14 catalytic domain (MT-SP1) (R&D Systems, 3946-SE-010) for 20 hours. For the antigen-containing / protease-free analytes, antibody-conjugated with human CXCL10 was used. In addition, CXCL10 was prepared as an antigen-only analyte to confirm that the response was due to CXCL10 binding. Anti-CXCL10 antibody was immobilized on a CM5 sensor chip (BR100530, GE Healthcare) using a method known to the art. Four types of analytes were run using 20 mM ACES, 0.05% Tween20, pH 7.4 as the running buffer: antigen-containing / protease-containing analytes, antigen-free / protease-containing analytes, antigen-containing / protease-free analytes, and antigen-only analytes. The binding of anti-CXCL10 antibody to human CXCL10 on the sensor chip at 25°C was evaluated. Furthermore, using MabCXCL10a (heavy chain: EEIVHa (SEQ ID NO: 65), light chain: EEIVL (SEQ ID NO: 2)), which has a Fab region similar to the antibody MabCXCL10 that does not have a protease cleavage sequence, we prepared analytes with antigen / protease, without antigen / with protease, and with antigen / without protease, similar to the antibodies EEIVHC006a / EEIVL. Similarly, anti-CXCL10 antibody was immobilized on a CM5 sensor chip (BR100530, GE Healthcare) using a method known to the art. Four types of analytes were run using 20 mM ACES, 0.05% Tween20, pH 7.4 as the running buffer: antigen-containing / protease-containing analytes, antigen-free / protease-containing analytes, antigen-containing / protease-free analytes, and antigen-only analytes (CXCL10). The binding of anti-CXCL10 antibody to human CXCL10 on the sensor chip at 25°C was evaluated. Figure 11 shows a sensorgram representing the time-dependent binding amount, calculated by taking the difference between the bound anti-CXCL10 antibody and a flow cell without immobilized anti-CXCL10 antibody. The time at which the analyte starts flowing is set as the starting point on the vertical axis. The vertical axis represents the response at each time point, with the response at the start of analyte flowing set to 100. As a result, as shown in Figure 11(A), CXCL10 is not released when MabCXCL10a, which does not have a cleavage sequence introduced, is treated with protease, but as shown in Figure 11(B), CXCL10 is released when EEIVHC006a / EEIVL is treated with protease.

[0228] 5-8. Preparation of anti-CXCL10 neutralizing antibodies in which a portion of the amino acid sequence near the boundary between the antibody variable region and constant region is replaced with a portion of the protease cleavage sequence and the mobile linker sequence, and evaluation of protease cleavage. We investigated substituting a portion of the amino acid sequence near the boundary between the variable and constant regions of the MabCXCL10 heavy chain with a protease cleavage sequence and a portion of the movable linker sequence. We designed the heavy chain shown in Figure 12 by substituting a portion of the amino acids in the heavy chain with peptide sequence A (SEQ ID NO: 3), which has been reported to be cleaved by cancer-specific urokinase (uPA) and matryptase (MT-SP1). Expression vectors encoding the heavy chain variants EESVHA009 (SEQ ID NO: 59) and EESVHA012 (SEQ ID NO: 60) were prepared by methods known to those skilled in the art. These heavy chain variants were combined with the native light chain to produce the following IgG1 antibodies: EESVHA009 / EEIVL (heavy chain SEQ ID NO: 59, light chain SEQ ID NO: 2) and EESVHA012 / EEIVL (heavy chain SEQ ID NO: 60, light chain SEQ ID NO: 2). These were expressed by transient expression using FreeStyle 293 (Life Technologies) using methods known to those skilled in the art, and then purified using protein A using methods known to those skilled in the art. We investigated whether EESVHA009 / EEIVL and EESVHA012 / EEIVL could be cleaved by proteases. Human urokinase (uPA) (R&D Systems, 1310-SE-010) and recombinant human matryptase / ST14 catalytic domain (MT-SP1) (R&D Systems, 3946-SE-010) were used as proteases. After reacting with 12.5 nM protease, 100 μg / mL antibody, PBS, and 37°C for 20 hours, protease cleavage was evaluated by reduced SDS-PAGE. The results are shown in Figure 13. As a result, EESVHA009 / EEIVL and EESVHA012 / EEIVL showed the emergence of new bands between 25 kDa and 50 kDa after protease treatment. Therefore, it was confirmed that the antibody was cleaved by the protease in EESVHA009 / EEIVL and EESVHA012 / EEIVL.

[0229] Example 6: Consideration of insertion sites for cleavage sequences that cause loss of antigen-binding ability by protease cleavage. A report has been published (International Publication WO2004 / 021861A2) concerning the creation of an antibody in which a protease cleavage sequence is inserted immediately before the aspartic acid at position 216 of the heavy chain of a human IgG1 antibody, and its functional evaluation in vitro. Although experimental data are not described, it is claimed that when this antibody is mixed with an antigen and then treated with a medium containing the corresponding protease, the antigen is released from the antigen-antibody complex. The 216th amino acid in the heavy chain of the human IgG1 antibody, which the report claimed had a protease cleavage sequence inserted, is not aspartic acid according to any of the numbering systems—Kabat numbering, EU numbering, or OU numbering—described in Kabat, E. et al. Sequences of Proteins of Immunological Interest 5th edition. On the other hand, referring to different literature, the 216th amino acid in the heavy chain of the human IgG1 antibody is thought to be aspartic acid immediately following cysteine, where a disulfide bond is formed between the heavy and light chains (Nature 344, 667 - 670 (12 April 1990), Kabat, E. et al. Sequences of Proteins of Immunological Interest 4th edition). If a protease cleavage sequence is inserted immediately before this 216th aspartic acid, it is thought that a Fab region similar to that formed when the hinge region of the antibody is cleaved by papain will be formed in the antibody that has undergone protease cleavage. Since it is generally recognized that the antigen-binding ability of an antibody is unlikely to be lost due to cleavage of the hinge region by papain, it is thought that the antigen-binding ability will not be lost even if an antibody with a protease cleavage sequence inserted immediately before the aspartic acid at position 216 is cleaved with the corresponding protease. Let's consider the case where a protease cleavage sequence is inserted immediately before the 216th amino acid (Kabat numbering) of the heavy chain of a human IgG1 antibody, as described in Kabat, E. et al. Sequences of Proteins of Immunological Interest 5th edition. This site is located several amino acids N-terminus of the 220th cysteine ​​(Kabat numbering), where a disulfide bond is formed between the heavy and light chains. Therefore, it is assumed that the effect of protease cleavage of the heavy chain at this site is similar to the effect of losing the disulfide bond formed between the heavy and light chains. Based on past literature, it is unlikely that antigen binding would be lost even in a Fab region where a disulfide bond cannot be formed between the heavy and light chains (MAbs. 2014 Jan-Feb;6(1):204-18.). Therefore, even if a protease cleavage sequence is inserted immediately before the 216th amino acid (Kabat numbering) of the heavy chain of a human IgG1 antibody as described in Kabat, E. et al. Sequences of Proteins of Immunological Interest 5th edition, it is thought that the antigen-binding ability will not be lost due to protease cleavage.

[0230] Example 7: Evaluation of the migratory activity of an anti-CXCL10 neutralizing antibody / CXCL10 complex containing a protease cleavage sequence, following protease cleavage. We evaluated whether the complex formed between the CXCL10 neutralizing antibody, which incorporated the protease cleavage sequence prepared in Example 5, and CXCL10, would release CXCL10 through protease cleavage, thereby enabling CXCL10 to exhibit cell migration activity. The cell migration activity of CXCL10 was investigated by creating Ba / F3 transfectant cells expressing mouse CXCR3 (mCXCR3) (hereinafter referred to as BaF3 / mCXCR3), and then testing these cells with HTS Transwell. TMThe evaluation was performed using -96 Permeable Support with 5.0 μm Pore Polycarbonate Membrane (Cat. 3387, Corning). Five types of analytes were prepared: CXCL10+ protease, EEIVHC006a / EEIVL+CXCL10, EEIVHC006a / EEIVL+CXCL10+ protease, EEIVHC006a / EEIVL+ protease, and MabCXCL10+CXCL10+ protease. In a 1.5 mL ProteoSave SS microtube (Cat. MS-4265M, Sumitomo Bakelite), an antibody (MabCXCL10 or EEIVHC006a / EEIVL) with a final concentration of 10 μg / mL, or hCXCL10 (Cat. 300-12, Peprotech) with a final concentration of 100 ng / mL, or both the antibody and hCXCL10, were added and left at room temperature for 30 minutes. For analytes containing protease, mouse MT-SP1 (mMT-SP1, Cat. 4735-SE-010, R&D Systems) was further added after the above reaction to a final concentration of 12.5 nM. Transfer 235 μL of each analyte to the lower chamber, and then place 2.0 × 10⁶ BaF3 / mCXCR3 cells into the upper chamber. 5 Cells were seeded at a concentration of 75 μL / well to achieve a cell / well ratio, and reacted for 6 hours. The reaction was carried out under 5% carbon dioxide and 37°C conditions. After 6 hours of reaction, 100 μL of the solution in the lower chamber was transferred to a fluorescein luminescent 96-well plate (Cat. 3912, Corning), and CellTiter-Glo was used. TM 100 μL of Luminescent Cell Viability Assay solution (Cat. G7571, Promega) was added. After reacting at room temperature for 10 minutes, the luminescence value was measured using a SpectraMax M3 multimode microplate reader (Molecular Devices) to evaluate the degree of cell migration to the lower chamber. The results are shown in Figure 14. Compared to the analyte of CXCL10+ protease, the luminescence intensity decreased when the analyte of EEIVHC006a / EEIVL+CXCL10 was added. Since luminescence intensity reflects the amount of migrating cells, it was found that EEIVHC006a / EEIVL forms a complex with CXCL10 and neutralizes the action of CXCL10. On the other hand, when the analyte of EEIVHC006a / EEIVL+CXCL10+ protease was added, the luminescence intensity recovered compared to the analyte of EEIVHC006a / EEIVL+CXCL10, and it was shown to induce cell migration similar to the case of the analyte of CXCL10+ protease. When the analyte of MabCXCL10+CXCL10+ protease using a MabCXCL10 antibody without a cleavage sequence was added, no recovery of luminescence intensity was observed. These results indicate that EEIVHC006a / EEIVL exhibits reduced neutralizing ability of CXCL10 upon antibody cleavage by protease.

[0231] Example 8: Evaluation of the migratory activity of an anti-CXCL10 neutralizing antibody-CXCL10 fusion protein incorporating a protease cleavage sequence, following protease cleavage. 8-1 Preparation of anti-CXCL10 neutralizing antibody-CXCL10 fusion protein incorporating protease cleavage sequence and evaluation of protease cleavage Using the light chain of MabCXCL10_G7 (heavy chain: G7H-G1T4 (SEQ ID NO: 368), light chain: G7L-LT0 (SEQ ID NO: 369)), a neutralizing antibody against human CXCL10, we ligated the human CXCL10 mutant hCXCL10R75A (SEQ ID NO: 370), which has been mutated to become resistant to proteases, to the N-terminus of the light chain via a linker sequence consisting of a glycine-serine polymer, thereby designing the ligand-fusion light chain hCXCL10R75A.G4SGGGG.G7L-LT0 (SEQ ID NO: 371). Furthermore, we designed a ligand-fusion light chain, hCXCL10R75A.G7L.12aa0054-LT0 (SEQ ID NO: 372), in which a sequence (SEQ ID NO: 338) cleaved by cancer-specific urokinase (uPA) and matryptase (MT-SP1) was inserted near the boundary between the antibody variable region and the antibody constant region in hCXCL10R75A.G4SGGGG.G7L-LT0. These ligand-fusion light chains were combined with the MabCXCL10_G7 heavy chain G7H-G1T4 to produce the fusion proteins: G7H-G1T4 / hCXCL10R75A.G4SGGGG.G7L-LT0 (heavy chain SEQ ID NO: 368, ligand-fusion light chain SEQ ID NO: 371) and G7H-G1T4 / hCXCL10R75A.G7L.12aa0054-LT0 (heavy chain SEQ ID NO: 368, ligand-fusion light chain SEQ ID NO: 372). These were expressed by transient expression using Expi293 (Life Technologies) using methods known to those skilled in the art, and purified using protein A using methods known to those skilled in the art. The CDR sequences for MabCXCL10_G7 are as follows: H-CDR1 (SFSIT, SEQ ID NO: 374), H-CDR2 (EITPMFGIANYAQKFQG, SEQ ID NO: 375), H-CDR3 (DGRFDVSDLLTDKPKVTINYNGMDV, SEQ ID NO: 376), L-CDR1 (SGSSSNIGSNTVN, SEQ ID NO: 377), L-CDR2 (NNDQRPS, SEQ ID NO: 378), L-CDR3 (ASWDDSLNGRV, SEQ ID NO: 379). We investigated whether these fusion proteins could be cleaved by proteases. Human urokinase (human uPA, huPA) (R&D Systems, 1310-SE-010) was used as the protease. Protease cleavage of the fusion proteins was evaluated by reduced SDS-PAGE. After reacting 0.1 mg / ml of the fusion protein with 30 nM huPA at 37°C for 1 hour, the cleavage of the fusion protein was evaluated by reduced SDS-PAGE. As a result, G7H-G1T4 / hCXCL10R75A.G4SGGGG.G7L-LT0 was not cleaved by protease treatment, whereas G7H-G1T4 / hCXCL10R75A.G7L.12aa0054-LT0, which had a protease cleavage sequence inserted, produced a new band between 15 kDa and 25 kDa after protease treatment (Figure 15), confirming cleavage by protease treatment.

[0232] 8-2 Evaluation of the migratory activity of an anti-CXCL10 neutralizing antibody-CXCL10 fusion protein with introduced protease cleavage sequence upon protease cleavage. We evaluated whether an anti-CXCL10 neutralizing antibody-CXCL10 fusion protein, which is a fusion of an anti-CXCL10 neutralizing antibody with a protease cleavage sequence and CXCL10, releases CXCL10 via protease cleavage and induces cell migration. To compare the activity of hCXCL10R75A released from the fusion protein, hCXCL10R75A-His (SEQ ID NO: 373), which exhibits the activity of hCXCL10R75A alone, was prepared and purified by the following method. A histidine tag was added to the C-terminus of the human CXCL10 mutant hCXCL10R75A (SEQ ID NO: 370), which was mutated to be resistant to protease, to prepare the histidine-tagged human CXCL10 mutant hCXCL10R75A-His (SEQ ID NO: 373). hCXCL10R75A-His (SEQ ID NO: 373) was expressed transiently using Expi293 (Life Technologies) by a method known to those skilled in the art, and purified using nickel Sepharose by a method known to those skilled in the art. Cell migration activity was evaluated using Ba / F3 transfectant cells expressing mouse CXCR3 (mCXCR3) (hereinafter BaF3 / mCXCR3) and HTS Transwell TM -96 Permeable Support with 5.0μm Pore Polycarbonate Membrane (Cat. 3387, Corning). As an analyte of uPA(+), recombinant huPA (Cat. 1310-SE, R&D systems) was added to a final concentration of 30 nM to hCXCL10R75A-His 0.15 μg / mL, fusion protein G7H-G1T4 / hCXCL10R75A.G4SGGGG.G7L-LT0 1.5 μg / mL without protease cleavage sequence, or fusion protein G7H-G1T4 / hCXCL10R75A.G7L.12aa0054-LT0 1.5 μg / mL with protease cleavage sequence in a 2.0 mL 96-well deep well plate (Cat. P-DW-20-C-S, Axygen). G7H-G1T4 / hCXCL10R75A.G4SGGGG.G7L-LT0 and G7H-G1T4 / hCXCL10R75A.G7L.12aa0054-LT0 1.5 μg / mL contain 0.15 μg / mL equivalent amount of hCXCL10R75A. For the uPA(-) analyte, hCXCL10R75A-His 0.15 μg / mL, fusion protein G7H-G1T4 / hCXCL10R75A.G4SGGGG.G7L-LT0 1.5 μg / mL without protease cleavage sequence, or fusion protein G7H-G1T4 / hCXCL10R75A.G7L.12aa0054-LT0 1.5 μg / mL with protease cleavage sequence were used. 235 μL of each solution to be analyzed was transferred to the Lower chamber, and 2.0×10 BaF3 / mCXCR3 cells were placed in the Upper chamber 5Cells were seeded at a rate of 75 μL / well to achieve a cell / well ratio, and reacted for 6 hours. The reaction was carried out under 5% carbon dioxide and 37°C conditions. After 6 hours of reaction, 100 μL of the solution in the lower chamber was transferred to OptiPlate-96 (Cat. 6005299, PerkinElmer) and CellTiter-Glo was added. TM 100 μL of Luminescent Cell Viability Assay solution (Cat. G7571, Promega) was added. After reacting at room temperature for 10 minutes, the luminescence value was measured using a SpectraMax M3 multimode microplate reader (Molecular Devices) to evaluate the degree of cell migration to the lower chamber. The results are shown in Figure 16. Compared to the case with CXCL10R75A-His added, the luminescence intensity decreased when the protease-untreated G7H-G1T4 / hCXCL10R75A.G7L.12aa0054-LT0 fusion protein was added. Since luminescence intensity reflects the amount of migrating cells, it was found that the physiological activity of CXCL10R75A in G7H-G1T4 / hCXCL10R75A.G7L.12aa0054-LT0 was neutralized. On the other hand, when protease-treated G7H-G1T4 / hCXCL10R75A.G7L.12aa0054-LT0 was added, the luminescence intensity recovered compared to when untreated G7H-G1T4 / hCXCL10R75A.G7L.12aa0054-LT0 was added, and it was shown to induce cell migration equivalent to that of CXCL10R75A-His. In G7H-G1T4 / hCXCL10R75A.G4SGGGG.G7L-LT0, which does not have a cleavage sequence, no recovery of luminescence intensity was observed even after protease treatment. From these results, it was shown that the neutralizing ability of the antibody portion in the fusion protein against CXCL10R75A decreases when G7H-G1T4 / hCXCL10R75A.G7L.12aa0054-LT0 is cleaved by protease.

[0233] Example 9: Preparation of anti-IL-12 neutralizing antibodies incorporating protease cleavage sequences and mobile linker sequences, and evaluation of IL-12 activation associated with protease cleavage. 9-1. Production of anti-IL-12 neutralizing antibodies incorporating protease cleavage sequences and mobile linker sequences. IL-12 is one of the cytokines that has an immune-activating effect. IL-12 exerts its antitumor effect by activating immune cells, but it has also been reported that systemic exposure can cause serious side effects (Nat Immunol. 2012 Jul 19;13(8):722-8.). A modified version of the Ustekinumab heavy chain, UstkH-G1T4CYTM1inP1 (SEQ ID NO: 146), was designed by inserting a sequence containing peptide sequence A (SEQ ID NO: 3), which has been reported to be cleaved by urokinase (uPA) and matryptase (MT-SP1), and a movable linker consisting of a glycine-serine polymer, near the boundary between the variable and constant regions of the heavy chain (UstkH-G1T4, heavy chain SEQ ID NO: 144) of an anti-IL-12 antibody that has the same variable region as Ustekinumab, a neutralizing antibody against human IL-12. This modified version, UstkH-G1T4CYTM1inP1 (SEQ ID NO: 146), was then combined with the Ustekinumab light chain (UstkL-kT0, SEQ ID NO: 145) to produce an expression vector encoding the modified Ustekinumab, UstkH-G1T4CYTM1inP1 / UstkL-kT0 (heavy chain SEQ ID NO: 146, light chain SEQ ID NO: 145), using methods known to the art. The Ustekinumab variant UstkH-G1T4CYTM1inP1 / UstkL-kT0 was expressed transiently using FreeStyle 293 (Life Technologies) by a method known to those skilled in the art, and purified using Protein A by a method known to those skilled in the art. The CDR sequences contained in the anti-IL-12 antibody and its variant in this example are as follows: H-CDR1 (TYWLG, SEQ ID NO: 386), H-CDR2 (IMSPVDSDIRYSPSFQG, SEQ ID NO: 387), H-CDR3 (RRPGQGYFDF, SEQ ID NO: 388), L-CDR1 (RASQGISSWLA, SEQ ID NO: 389), L-CDR2 (AASSLQS, SEQ ID NO: 390), L-CDR3 (QQYNIYPYT, SEQ ID NO: 391).

[0234] 9-2. Protease cleavage of anti-IL-12 neutralizing antibodies incorporating protease cleavage sequences and mobile linker sequences. We investigated whether the antibodies prepared in 9-1 above could be cleaved by proteases. As proteases, we used recombinant human matryptase / ST14 catalytic domain (human MT-SP1, hMT-SP1) (R&D Systems, 3946-SE-010), recombinant mouse matryptase / ST14 catalytic domain (mouse MT-SP1, mMT-SP1) (R&D Systems, 4735-SE-010), and human urokinase (human uPA, huPA) (R&D Systems, 1310-SE-010). Protease treatment was performed by adding hMT-SP1, mMT-SP1, or huPA to Ustekinumab (UstkH-G1T4 / UstkL-kT0) or a variant of Ustekinumab, UstkH-G1T4CYTM1inP1 / UstkL-kT0, to final concentrations of 10.1, 16.9, and 9.17 μM, respectively, and reacting overnight at 37°C.

[0235] 9-3. Confirmation of cleavage and evaluation of IL-12 activation by introducing cleaved protease cleavage sequences and mobile linker sequences into anti-IL-12 neutralizing antibodies. Antibody cleavage by proteases was evaluated by reduced SDS-PAGE. The results showed that UstkH-G1T4 / UstkL-kT0 was not cleaved by either protease, whereas UstkH-G1T4CYTM1inP1 / UstkL-kT0, which had a protease-cleaving sequence and a mobile linker inserted, showed new bands between 25kDa and 50kDa after protease treatment (Figure 17). Therefore, it was confirmed that anti-IL-12 neutralizing antibodies (UstkH-G1T4CYTM1inP1 / UstkL-kT0) with introduced protease-cleaving sequences and mobile linker sequences are cleaved by proteases. Next, we evaluated whether IL-12 is released from its complex with the antibody and exerts physiological activity when the antibody is cleaved by a protease. The physiological activity of IL-12 was evaluated based on the IFN-γ (interferon-gamma, also written as IFN-g) production of the NK92 human cell line. NK92 cells were cultured in a 96-well cell culture plate at a rate of 1 × 10⁶ 5Cells were seeded at a rate of cells / well. 10 ng / mL of IL-12 and protease-treated antibodies (UstkH-G1T4 / UstkL-kT0 or UstkH-G1T4CYTM1inP1 / UstkL-kT0, at concentrations of 20, 4, 0.8, 0.16, 0.032, 0.0054, and 0.0013 μg / mL, respectively) were added, and IFN-γ production after 48 hours was measured by ELISA. To evaluate the effect of antibodies on IL-12 activity, experiments were also conducted with only protease-treated IL-12 added (No Ab) without antibody. Figure 18 shows the results of measuring interferon-gamma concentrations. UstkH-G1T4 / UstkL-kT0 treated with various proteases (without protease cleavage sequences) suppressed (neutralized) the production of interferon-gamma by IL-12, reaching a level comparable to that without IL-12 (No IL-12) when the antibody concentration was 0.8 μg / mL. On the other hand, UstkH-G1T4CYTM1inP1 / UstkL-kT0 treated with various proteases (containing protease cleavage sequences) produced more interferon-gamma at all antibody concentrations compared to when UstkH-G1T4 / UstkL-kT0 without protease cleavage sequences was added. From these results, it was confirmed that UstkH-G1T4CYTM1inP1 / UstkL-kT0's neutralizing ability against IL-12 is reduced upon protease cleavage, thereby allowing IL-12 to act on cells.

[0236] Example 10 Evaluation of antibodies into which various protease cleavage sequences have been introduced into anti-human CXCL10 neutralizing antibodies. 10-1. Introduction of protease cleavage sequences into anti-human CXCL10 neutralizing antibodies Expression vectors for MabCXCL10 (heavy chain: EEIVH (SEQ ID NO: 1), light chain: EEIVL (SEQ ID NO: 2)) and MabCXCL10_G7 (heavy chain: G7H-G1T4 (SEQ ID NO: 368), light chain: G7L-LT0 (SEQ ID NO: 369)), antibodies that neutralize CXCL10, were prepared by methods known to the art, and expression and purification were performed using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) by methods known to the art. Modified MabCXCL10 heavy chains were created by inserting the cleavage sequences shown in Table 2 near the boundary between the variable and constant regions of the heavy chain of MabCXCL10 or MabCXCL10_G7, respectively. The sequences of the modified MabCXCL10 heavy chains with inserted protease cleavage sequences are shown in Table 3.

[0237] [Table 2]

[0238] [Table 3]

[0239] The modified heavy and light chains in Table 3 were combined to produce the MabCXCL10 variants and MabCXCL10_G7 variants shown in Table 4. These were expressed by transient expression using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) using methods known to those skilled in the art, and then purified using methods known to those skilled in the art with Protein A.

[0240] [Table 4]

[0241] 10-2. Evaluation of protease cleavage of anti-human CXCL10 neutralizing antibodies with multiple protease cleavage sequences introduced into the heavy chain region. We investigated whether the antibody prepared in 10-1 could be cleaved by a protease. Recombinant human matryptase / ST14 catalytic domain (human MT-SP1, hMT-SP1) (R&D Systems, 3946-SE-010) was used as the protease. The reaction was carried out for 20 hours under conditions of 10 nM protease, 50 μg / mL antibody, PBS, and 37°C, and then subjected to reduced SDS-PAGE. The results are shown in Figures 19A and 19B. Both the MabCXCL10 variant and the MabCXCL10_G7 variant shown in Table 4 showed the generation of a new band around 37 kDa after hMT-SP1 treatment. That is, it was confirmed that the protease cleavage sequence shown in Table 2 is cleaved by hMT-SP1. Furthermore, it was confirmed that the protease cleavage sequence shown in Table 2 is also cleaved by human uPA and mouse uPA using a similar method.

[0242] Example 11: Preparation and evaluation of polypeptides into which diverse protease cleavage sequences have been introduced. 11-1 Creation of polypeptides into which recognition sequences of various proteases have been introduced. Expression vectors for MRA (heavy chain: MRAH-G1T4 (SEQ ID NO: 147), light chain: MRAL-k0 (SEQ ID NO: 148)), a neutralizing antibody against human IL-6R, were prepared by methods known to the art. The CDR sequences of MRA are as follows: H-CDR1 (SDHAWS, SEQ ID NO: 398), H-CDR2 (YISYSGITTYNPSLKS, SEQ ID NO: 399), H-CDR3 (SLARTTAMDY, SEQ ID NO: 400), L-CDR1 (RASQDISSYLN, SEQ ID NO: 401), L-CDR2 (YTSRLHS, SEQ ID NO: 402), L-CDR3 (QQGNTLPYT, SEQ ID NO: 403). Table 5 shows peptide sequences known to be cleaved at MMP-2, MMP-7, and MMP-9, as well as peptide sequences containing a movable linker made of a glycine-serine polymer near these sequences.

[0243] [Table 5]

[0244] These insertion sequences were inserted near the boundary between the variable and constant regions of the heavy chain of the MRA antibody, resulting in the modified heavy chains MEIVHG4SMP2MP9G4S-MEIVHG4SMP2MP9G4SG1T4 (SEQ ID NO: 153), MEIVHG4SMP2.2G4S-MEIVHG4SMP2.2G4SG1T4 (SEQ ID NO: 154), MEIVHG4SMP2.4G4S-MEIVHG4SMP2.4G4SG1T4 (SEQ ID NO: 155), MEIVHG4SMP9G4S-MEIVHG4SMP9G4SG1T4 (SEQ ID NO: 156), MEIVHMP2.1-MEIVHMP2.1G1T4 (SEQ ID NO: 157), MEIVHMP2.3-MEIVHMP2.3G1T4 (SEQ ID NO: 158), and MEIVHMP7.2-MEIVHMP7.2G1T4. We designed (heavy chain sequence number: 159) and prepared expression vectors encoding these modified heavy chains using methods known to the art. These modified heavy chains and MRA light chains were combined to express the MRA variants shown in Table 6 by transient expression using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) using methods known to those skilled in the art, and then purified using methods known to those skilled in the art with Protein A.

[0245] [Table 6]

[0246] 11-2. Evaluation of protease cleavage of polypeptides into which diverse protease recognition sequences have been introduced. We investigated whether the MRA variant prepared in 11-1 could be cleaved by proteases. Recombinant human MMP-2 (R&D Systems, 902-MP-010), recombinant human MMP-7 (R&D Systems, 907-MP-010), and recombinant human MMP-9 (R&D Systems, 911-MP-010) were used as proteases. The proteases were mixed with 1 mM p-aminophenylmercuric acetate (APMA; abcam, ab112146) and activated at 37°C for 1 and 24 hours, respectively, before use. Figures 20A, 20B, and 21 show the results of evaluating protease cleavage by reducing SDS-PAGE after reacting with 50 nM, 100 nM, or 500 nM protease, 50 μg / mL antibody, and either an assay buffer (MMP Activity Assay Kit (Fluorometric - Green) (ab112146), Component C: Assay Buffer) or 20 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, pH 7.2 (hereinafter referred to as Tris) at 37°C for 20 hours. The MRA modified antibodies were reacted with the proteases shown in Table 6. In MMP-2, the cuttings are MEIVHG4SMP2MP9G4S-MEIVHG4SMP2MP9G4SG1T4 / MRAL-k0, MEIVHG4SMP2.2G4S-MEIVHG4SMP2.2G4SG1T4 / MRAL-k0, MEIVHG4SMP2.4G4S-MEIVHG4SMP2.4G4SG1T4 / MRAL-k0, MEIVHMP2.1-MEIVHMP2.1G1T4 / MRAL-k0, MEIVHMP2.3-MEIVHMP2.3G1T4 / MRAL-k0; in MMP-7, the cutting is MEIVHMP7.2-MEIVHMP7.2G1T4 / MRAL-k0; in MMP-9, MEIVHG4SMP2MP9G4S-MEIVHG4SMP2MP9G4SG1T4 / MRAL-k0, Cutting of MEIVHG4SMP9G4S-MEIVHG4SMP9G4SG1T4 / MRAL-k0 was observed.

[0247] Example 12 Evaluation of antibodies in which protease cleavage sequences have been introduced at various positions in the heavy chain. 12-1 Production of antibodies in which protease cleavage sequences are introduced at various positions in the heavy chain. Peptide sequence B (SEQ ID NO: 160), which has been reported to be cleaved by urokinase (uPA) and matryptase (MT-SP1), was inserted at different positions within the MRA heavy chain variable region (MRAH, SEQ ID NO: 161) to create modified MRA heavy chain regions as shown in Table 7. These modified MRA heavy chain variable region sequences were ligated to the MRA heavy chain constant region (G1T4, SEQ ID NO: 162) to create modified MRA heavy chains, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art. Furthermore, peptide sequence B (SEQ ID NO: 160) was inserted at different positions within the MRA heavy chain constant region (G1T4, SEQ ID NO: 162) to create modified MRA heavy chain regions as shown in Table 8. These modified MRA heavy chain constant region sequences were ligated to the MRA heavy chain variable region (MRAH, SEQ ID NO: 161) to create modified MRA heavy chains, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art. Tables 7 and 8 show the insertion sites of protease cleavage sequences in the modified MRA heavy chain variable region and the modified MRA heavy chain constant region. In Table 7, the insertion site refers to the position adjacent to the constant region side of the description position (Kabat numbering) in the antibody heavy chain variable region, and in Table 8, the insertion site refers to the position adjacent to the variable region side of the description position (EU numbering) in the antibody heavy chain constant region.

[0248] [Table 7] TIFF2026108884000019.tif135147

[0249] [Table 8]

[0250] The MRA heavy chain variants and MRA light chains prepared as described above were combined to produce the MRA variants shown in Table 9. These variants were expressed by transient expression using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) using methods known to those skilled in the art, and then purified using methods known to those skilled in the art with Protein A.

[0251] [Table 9] TIFF2026108884000022.tif229139

[0252] 12-2. Evaluation of protease cleavage of anti-human IL-6R neutralizing antibodies in which protease cleavage sequences have been introduced into the antibody heavy chain. We investigated whether the MRA variant prepared in 12-1 could be cleaved by a protease. Recombinant human matotryptase / ST14 catalytic domain (human MT-SP1, hMT-SP1) (R&D Systems, 3946-SE-010) was used as the protease. The mixture was reacted with 10 nM protease, 50 μg / mL antibody, PBS, and 37°C for 20 hours, and then subjected to reduced SDS-PAGE. The results are shown in Figures 22A, 22B, 22C, 22D, 22E, 22F, 22G, 22H, 22I, and Figures 23A, 23B, and 23C. In the MRA variant after protease treatment, the heavy chain is cleaved, and the heavy chain band appears at a lower molecular weight position compared to the MRA variant that has not undergone protease treatment (the band appearing around 50 kDa in the MT-SP1(-) lane in the figure). From this result, it was confirmed that the MRA variant prepared in 12-1 is cleaved by hMT-SP1.

[0253] Example 13 Evaluation of antibodies into which protease cleavage sequences have been introduced at various positions on the light chain. 13-1 Production of antibodies in which protease cleavage sequences are introduced at various positions on the light chain. Peptide sequence B (SEQ ID NO: 160), which has been reported to be cleaved by urokinase (uPA) and matryptase (MT-SP1), was inserted at different positions within the MRA light chain variable region (MRAL, SEQ ID NO: 230) to create modified MRA light chain regions as shown in Table 10. These modified MRA light chain variable regions were ligated to the MRA light chain constant region (k0, SEQ ID NO: 231) to create MRA light chain modifiers, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art. Furthermore, peptide sequence B (SEQ ID NO: 160) was inserted at different positions...

Claims

[Claim 1] The invention described herein.

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