Ligand-binding molecule capable of adjusting ligand binding activity

Abstract

To provide a ligand-binding molecule whose binding activity to a ligand is attenuated by cutting off of a cleavage site, and a method for producing the same, a composite, a fusion protein and a pharmaceutical composition.SOLUTION: A ligand-binding molecule includes antibody VH and antibody VL, where the ligand-binding molecule is bindable to ligand, and includes at least one protease cleavage sequence, the antibody VL is associated with the antibody VH in the ligand-binding molecule, the association is dissolved when the protease cleavage sequence is cut off by protease, binding of the ligand-binding molecule to the ligand in a state where the protease cleavage sequence is cut off is weaker than binding of the ligand-binding molecule to the ligand in a state where the protease cleavage sequence is not cut off.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention provides ligand-binding molecules that have at least one cleavage site and whose binding to a ligand is weakened when the cleavage site is cleaved, methods for producing the ligand-binding molecules, and pharmaceutical compositions containing the ligand-binding molecules. [Background technology]

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

[0003] Approved antibody drugs for cancer treatment include Rituxan, which targets the CD20 antigen, cetuximab, which targets the EGFR antigen, and Herceptin, which targets the HER2 antigen (Non-Patent Document 3). These antibody molecules bind to antigens expressed on cancer cells and exert cytotoxic activity against cancer cells through ADCC, signal inhibition, etc.

[0004] Another known method involves delivering a ligand to solid tumors using immunocytokines, which are antibodies that bind to cancer antigens highly expressed in cancer cells and are fused with physiologically active ligands such as cytokines. The cytokines delivered to solid tumors by immunocytokines activate the immune system, thereby exerting antitumor effects. 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 and enhance efficacy (Non-Patent Documents 4, 5, 6). However, these cytokines have issues such as insufficient clinical efficacy when administered systemically, a narrow therapeutic window, and high toxicity that precludes systemic administration, and therefore have not yet been approved as pharmaceuticals.

[0005] The main reason for this is that even immunocytokines, when administered systemically, are exposed to the entire body and may act systemically and exert toxicity, or they can only be administered in extremely low doses to avoid toxicity. There is also a report that there was no difference in the antitumor effect between an immunocytokine in which IL-2 was fused to an antibody that binds to a cancer antigen and an immunocytokine in which IL-2 was fused to an antibody that does not bind to a cancer antigen (Non-Patent Document 7).

[0006] As a way to avoid the above problems, molecules have been reported in which cytokines and cytokine receptors are linked via linkers that are cleaved by proteases highly expressed in cancer. Cytokines are inhibited by the cytokine receptors linked via linkers, but when the linker is cleaved by a protease, the cytokines are released from the cytokine receptors and become active. For example, a molecule has been reported in which TNF-alpha and TNF-R are linked via a linker that is cleaved by uPA (Non-Patent Document 8), and a molecule has been reported in which IL-2 and IL-2R are linked via a linker that is cleaved by MMP-2 (Non-Patent Document 9). However, in these molecules, the cytokines retain activity even before the linker is cleaved, and cleavage of the linker only increases activity by approximately 10-fold. Another molecule has been reported in which an anti-IL-2 scFv is linked to IL-2 instead of IL-2R via a linker that is cleaved by MMP-2 (Non-Patent Document 9). [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 light of the above circumstances, and one of its objects is to provide a ligand-binding molecule that selectively activates a ligand such as a cytokine or a chemokine in a target tissue, a pharmaceutical composition containing the ligand-binding molecule, and methods for producing the pharmaceutical composition and the active ingredient. [Means for solving the problem]

[0009] The present inventors have conducted extensive research to achieve the above-mentioned object and have created a ligand-binding molecule whose binding activity to a ligand is attenuated by cleavage of the cleavage site. Furthermore, the present inventors have found that the ligand-binding molecule or a pharmaceutical composition containing the ligand-binding molecule is useful for treating diseases using the ligand, and that the ligand-binding molecule is useful for treating diseases that involve administering the ligand-binding molecule, and that the ligand-binding molecule is useful in the manufacture of pharmaceuticals for treating diseases. Furthermore, the present inventors have created a method for producing the ligand-binding molecule, thereby completing the present invention.

[0010] The present invention is based on these findings and specifically includes the following exemplary embodiments. (1) A ligand-binding molecule, which is a molecule capable of binding to a ligand, the molecule being a polypeptide having at least one cleavage site, and in which the binding to the ligand is weakened when the molecule is cleaved at the 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 comprises a protease cleavage sequence. (4) The ligand-binding molecule according to (3), wherein the protease is a target tissue-specific protease. (5) The ligand-binding molecule according to (4), wherein the target tissue is a cancer tissue and the target tissue-specific protease is a cancer tissue-specific protease. (6) The ligand-binding molecule according to (4), wherein the target tissue is an inflamed tissue and the target tissue-specific protease is an inflamed 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 matriptase, urokinase (uPA), and metalloproteases. (8) The ligand-binding molecule according to (3), wherein the protease cleavage sequence is a sequence selected from the sequences shown in SEQ ID NOs: 3, 34, 66, 70, 71, 72, 73, 35, 75, 76, 335 to 345, 1161 to 1180, and 1392 to 1411, and the sequences listed in Table 1. (9) The ligand-binding molecule according to any one of (3) to (8), further comprising a first flexible linker attached to one end of the protease cleavage sequence. (10) The ligand-binding molecule according to (9), further comprising a second flexible linker attached to the other end of the protease cleavage sequence. (11) The ligand-binding molecule according to (9), wherein the first flexible linker is a flexible linker consisting of a glycine-serine polymer. (12) The ligand-binding molecule according to (10), wherein the second flexible linker is a flexible linker consisting of a glycine-serine polymer. (13) The ligand-binding molecule according to any one of (1) to (12), which comprises an antibody VH, an antibody VL, and an antibody constant region. (14) The ligand-binding molecule according to (13), wherein 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. (15) The ligand-binding molecule according to (14), wherein 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 118 (EU numbering) to amino acid 140 (EU numbering) of the antibody heavy chain constant region. (16) The ligand-binding molecule according to (14), wherein 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 108 (EU numbering) (Kabat numbering: 108) to amino acid 131 (EU numbering) (Kabat numbering: 131) of the antibody light chain constant region. (17) The ligand-binding molecule according to (13), wherein 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. (18) 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, may be selected from the group consisting of amino acids 7 (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), and amino acids 74 (Kabat numbering). The ligand-binding molecule according to (17), wherein the amino acid sequence is inserted at any position in a sequence selected from the group consisting of amino acids 101 (Kabat numbering) to 79 (Kabat numbering), 83 (Kabat numbering) to 89 (Kabat numbering), 95 (Kabat numbering) to 99 (Kabat numbering), and 101 (Kabat numbering) 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 the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker, are inserted at any position in a sequence selected from the group consisting of amino acids 7 (Kabat numbering) to 19 (Kabat numbering), 39 (Kabat numbering) to 46 (Kabat numbering), 49 (Kabat numbering) to 62 (Kabat numbering), and 96 (Kabat numbering) to 107 (Kabat numbering) of the antibody VL. (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 flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible 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 ligand-binding molecule according to (20), wherein 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 (Kabat numbering) of the antibody VH to amino acid 122 (EU numbering) of the antibody heavy chain constant region. (22) The ligand-binding molecule according to (20), wherein 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 (Kabat numbering) of the antibody VL to amino acid 113 (EU numbering) of the antibody light chain constant region (position 113 according to Kabat numbering). (23) The ligand-binding molecule according to any one of (13) to (22), wherein the antibody VL and the antibody VH in the ligand-binding molecule are associated with each other, and the association is dissolved by cleavage of the cleavage site or by cleavage of the protease cleavage sequence by a protease. (24) The ligand-binding molecule according to any one of (1) to (23), wherein 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. (25) The ligand-binding molecule according to any one of (1) to (24), wherein the ligand is a cytokine or a chemokine. (26) The ligand-binding molecule according to any one of (1) to (24), wherein the ligand is selected from interleukins, interferons, hematopoietic factors, the TNF superfamily, chemokines, cell growth factors, and the TGF-β family. (27) The ligand-binding molecule according to any one of (1) to (24), wherein the ligand is CXCL10, IL-12, PD-1, or IL-6R. (28) The ligand is CXCL10, and the ligand-binding molecule comprises an antibody VH and an antibody VL, and the ligand-binding molecule comprises: (a) an antibody VH comprising an H-CDR1 of SEQ ID NO: 374, an H-CDR2 of SEQ ID NO: 375, and an H-CDR3 of SEQ ID NO: 376, and an antibody VL comprising an L-CDR1 of SEQ ID NO: 377, an L-CDR2 of SEQ ID NO: 378, and an L-CDR3 of SEQ ID NO: 379; or (b) an antibody VH comprising an H-CDR1 of SEQ ID NO: 380, an H-CDR2 of SEQ ID NO: 381, and an H-CDR3 of SEQ ID NO: 382, and an antibody VL comprising an L-CDR1 of SEQ ID NO: 383, an L-CDR2 of SEQ ID NO: 384, and an L-CDR3 of SEQ ID NO: 385; or (c) having an antibody VH and an antibody VL that compete with (a) or (b); or (d) A ligand-binding molecule according to (27), which has an antibody VH and an 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 the sequences shown in SEQ ID NOs: 4 to 14, 23 to 27, 33, 59, 60, and 346 to 367, or an antibody light chain selected from the sequences shown in SEQ ID NOs: 15 to 22, 1146 to 1160, 1282 to 1380, and 1386 to 1389. (30) The ligand is IL-12, and the ligand-binding molecule comprises an antibody VH and an antibody VL, and the ligand-binding molecule comprises: (a) an antibody VH comprising an H-CDR1 of SEQ ID NO: 386, an H-CDR2 of SEQ ID NO: 387, and an H-CDR3 of SEQ ID NO: 388, and an antibody VL comprising an L-CDR1 of SEQ ID NO: 389, an L-CDR2 of SEQ ID NO: 390, and an L-CDR3 of SEQ ID NO: 391; or (b) having an antibody VH and an antibody VL that compete with (a); or (c) A ligand-binding molecule according to (27), which has an antibody VH and an antibody VL that bind to the same epitope as (a). (31) The ligand-binding molecule according to (30), wherein the ligand-binding molecule is an antibody comprising the antibody heavy chain shown in SEQ ID NO: 146. (32) The ligand is PD-1, and the ligand-binding molecule comprises an antibody VH and an antibody VL, and the ligand-binding molecule comprises: (a) an antibody VH comprising an H-CDR1 of SEQ ID NO: 392, an H-CDR2 of SEQ ID NO: 393, and an H-CDR3 of SEQ ID NO: 394, and an antibody VL comprising an L-CDR1 of SEQ ID NO: 395, an L-CDR2 of SEQ ID NO: 396, and an L-CDR3 of SEQ ID NO: 397; or (b) having an antibody VH and an antibody VL that compete with (a); or (c) A ligand-binding molecule according to (27), which has an antibody VH and an antibody VL that bind to the same epitope as (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), and the ligand-binding molecule comprises an antibody VH and an antibody VL, and the ligand-binding molecule comprises: (a) an antibody VH comprising an H-CDR1 of SEQ ID NO: 398, an H-CDR2 of SEQ ID NO: 399, and an H-CDR3 of SEQ ID NO: 400, and an antibody VL comprising an L-CDR1 of SEQ ID NO: 401, an L-CDR2 of SEQ ID NO: 402, and an L-CDR3 of SEQ ID NO: 403; or (b) having an antibody VH and an antibody VL that compete with (a); or (c) A ligand-binding molecule according to (27), which has an antibody VH and an antibody VL that bind to the same epitope as (a). (35) The ligand-binding molecule according to (34), wherein the ligand-binding molecule is an antibody comprising an antibody heavy chain selected from the sequences shown in SEQ ID NOs: 153 to 156, 157 to 159, and 404 to 470, or an antibody light chain selected from the sequences shown in SEQ ID NOs: 471 to 535. (36) The ligand-binding molecule according to any one of (1) to (35), wherein the ligand-binding molecule is an IgG antibody. (37) The ligand-binding molecule according to any one of (1) to (36), which is bound to the ligand. (38) The ligand-binding molecule according to any one of (1) to (36), which is fused to the ligand. (39) The ligand-binding molecule according to (38), wherein the ligand-binding molecule does not bind to another ligand when fused to the ligand. (40) The ligand-binding molecule according to (38) or (39), wherein the ligand-binding molecule is fused to 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 to the ligand. (43) The ligand-binding molecule according to (42), wherein the cleavage site is contained 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 to the ligand, and the ligand-binding molecule comprises: (a) an antibody heavy chain comprising an H-CDR1 having SEQ ID NO: 374, an H-CDR2 having SEQ ID NO: 375, and an H-CDR3 having SEQ ID NO: 376, and an antibody light chain comprising an L-CDR1 having SEQ ID NO: 377, an L-CDR2 having SEQ ID NO: 378, and an L-CDR3 having SEQ ID NO: 379; or (b) an antibody heavy chain comprising an H-CDR1 of SEQ ID NO: 380, an H-CDR2 of SEQ ID NO: 381, and an H-CDR3 of SEQ ID NO: 382, and an antibody light chain comprising an L-CDR1 of SEQ ID NO: 383, an L-CDR2 of SEQ ID NO: 384, and an L-CDR3 of SEQ ID NO: 385; The ligand-binding molecule according to (42) or (43). (45) The ligand-binding molecule according to any one of (42) to (44), wherein the ligand is a modified CXCL10 shown in SEQ 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 to the ligand, and the series of polypeptides in which CXCL10 and the antibody light chain are fused include 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 to the ligand. (48) The ligand-binding molecule according to (47), wherein the cleavage site is contained in the antibody light chain or the antibody heavy chain. (49) The ligand-binding molecule of (47) or (48), wherein the ligand is PD-1, the antibody light chain has an L-CDR1 of SEQ ID NO: 395, an L-CDR2 of SEQ ID NO: 396, and an L-CDR3 of SEQ ID NO: 397, and the antibody heavy chain has an H-CDR1 of SEQ ID NO: 392, an H-CDR2 of SEQ ID NO: 393, and an H-CDR3 of 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 SEQ ID NO: 320. (51) The ligand-binding molecule according to any one of (47) to (50), wherein the ligand is PD-1, an antibody heavy chain contained in the ligand-binding molecule is fused to 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, an antibody light chain contained in the ligand-binding molecule is fused to 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 to the ligand. (54) The ligand-binding molecule according to (53), wherein the cleavage site is contained 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 has an L-CDR1 of SEQ ID NO: 389, an L-CDR2 of SEQ ID NO: 390, and an L-CDR3 of SEQ ID NO: 391, and the antibody heavy chain has an H-CDR1 of SEQ ID NO: 386, an H-CDR2 of SEQ ID NO: 387, and an 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 to the ligand. (57) The ligand-binding molecule according to (56), wherein the cleavage site is contained 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 has an L-CDR1 of SEQ ID NO: 401, an L-CDR2 of SEQ ID NO: 402, and an L-CDR3 of SEQ ID NO: 403, and the antibody heavy chain has an H-CDR1 of SEQ ID NO: 398, an H-CDR2 of SEQ ID NO: 399, and an H-CDR3 of SEQ ID NO: 400. (59) A complex formed of the ligand and the 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 is fused to the ligand-binding molecule according to any one of (1) to (36). (61) The fusion protein according to (60), wherein the ligand-binding molecule does not bind to another ligand when fused to the ligand. (62) The fusion protein according to (60) or (61), wherein the ligand-binding molecule is fused to 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 consisting 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 to the ligand. (66) The fusion protein according to (65), wherein the cleavage site is contained in the antibody light chain or the 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 to the ligand, and the ligand-binding molecule comprises: (a) an antibody heavy chain comprising an H-CDR1 having SEQ ID NO: 374, an H-CDR2 having SEQ ID NO: 375, and an H-CDR3 having SEQ ID NO: 376, and an antibody light chain comprising an L-CDR1 having SEQ ID NO: 377, an L-CDR2 having SEQ ID NO: 378, and an L-CDR3 having SEQ ID NO: 379; or (b) an antibody heavy chain comprising an H-CDR1 of SEQ ID NO: 380, an H-CDR2 of SEQ ID NO: 381, and an H-CDR3 of SEQ ID NO: 382, and an antibody light chain comprising an L-CDR1 of SEQ ID NO: 383, an L-CDR2 of SEQ ID NO: 384, and an L-CDR3 of SEQ ID NO: 385; The fusion protein according to (65) or (66). (68) The fusion protein according to any one of (65) to (67), wherein the ligand is a variant of CXCL10 shown in SEQ 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 to the ligand, and the series of polypeptides in which CXCL10 and the antibody light chain are fused comprises 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 to the ligand. (71) The ligand-binding molecule according to (70), wherein the cleavage site is 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 has an L-CDR1 of SEQ ID NO: 395, an L-CDR2 of SEQ ID NO: 396, and an L-CDR3 of SEQ ID NO: 397, and the antibody heavy chain has an H-CDR1 of SEQ ID NO: 392, an H-CDR2 of SEQ ID NO: 393, and an 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 SEQ ID NO: 320. (74) The fusion protein according to any one of (70) to (73), wherein the ligand is PD-1, the antibody heavy chain contained in the ligand-binding molecule is fused to 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. (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 to 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. (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 to the ligand. (77) The fusion protein according to (76), wherein the cleavage site is contained in the antibody light chain or the antibody heavy chain. (78) The fusion protein according to (76) or (77), wherein the ligand is IL-12, the antibody light chain has an L-CDR1 of SEQ ID NO: 389, an L-CDR2 of SEQ ID NO: 390, and an L-CDR3 of SEQ ID NO: 391, and the antibody heavy chain has an H-CDR1 of SEQ ID NO: 386, an H-CDR2 of SEQ ID NO: 387, and an 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 to the ligand. (80) The fusion protein according to (79), wherein the cleavage site is 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 has an L-CDR1 of SEQ ID NO: 401, an L-CDR2 of SEQ ID NO: 402, and an L-CDR3 of SEQ ID NO: 403, and the antibody heavy chain has an H-CDR1 of SEQ ID NO: 398, an H-CDR2 of SEQ ID NO: 399, and an H-CDR3 of SEQ ID NO: 400. (82) A pharmaceutical composition comprising the ligand-binding molecule according to any one of (1) to (58). (83) A pharmaceutical composition comprising the ligand-binding molecule according to any one of (1) to (37) and a ligand. (84) A pharmaceutical composition comprising the complex according to (59). (85) A pharmaceutical composition comprising the fusion protein according to any one of (60) to (81). (86) A method for producing a ligand-binding molecule according to any one of (1) to (58). (87) The method for production according to (86), which comprises introducing a protease cleavage sequence into a molecule capable of binding to a ligand. (88) A method for producing the fusion protein according to any one of (60) to (81), comprising fusing a ligand-binding molecule having a protease cleavage sequence with the ligand. (89) A polynucleotide encoding the ligand-binding molecule according to any one of (1) to (58). (90) A vector comprising the polynucleotide according to (89). (91) A host cell comprising the polynucleotide according to (89) or the vector according to (90). (92) A method for producing the ligand-binding molecule according to any one of (1) to (58), comprising the step of culturing the host cell according to (91). (93) A polynucleotide encoding the fusion protein according to any one of (60) to (81). (94) A vector comprising the polynucleotide according to (93). (95) A host cell comprising the polynucleotide according to (93) or the vector according to (94). (96) A method for producing the fusion protein according to any one of (60) to (81), comprising a step of culturing the host cell according to (95). (97) A protease substrate comprising a sequence selected from the sequences shown in SEQ ID NOs: 1161 to 1180 and 1392 to 1411 and the sequences listed in Table 1. (98) The protease substrate according to (97), wherein the protease is matriptase 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 shown in SEQ ID NOs: 1161 to 1180, 1392 to 1411, and the sequences listed in Table 1.

[0011] The present invention can also specifically include the embodiments exemplified below. (B1) A ligand-binding molecule, the ligand-binding molecule being capable of binding to a ligand, the molecule being a polypeptide comprising at least one protease cleavage sequence comprising one or more sequences selected from the sequences shown in SEQ ID NOs: 1161 to 1180, 1392 to 1411, and the sequences listed in Table 1, wherein the binding of the ligand-binding molecule to a ligand when the protease cleavage sequence is cleaved is weaker than the binding of the ligand-binding molecule to a ligand when the protease cleavage sequence is uncleaved. (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 a 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 an inflamed tissue and the target tissue-specific protease is an inflamed 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 matriptase, urokinase (uPA), and metalloproteases. (B7) The ligand-binding molecule according to any one of (B1) to (B6), further comprising a first flexible linker attached to one end of the protease cleavage sequence. (B8) The ligand-binding molecule according to (B7), wherein the first flexible linker is a flexible linker consisting of a glycine-serine polymer. (B9) The ligand-binding molecule according to (B7) or (B8), further comprising a second flexible linker attached to the other end of the protease cleavage sequence. (B10) The ligand-binding molecule according to (B9), wherein the second flexible linker is a flexible linker consisting of a glycine-serine polymer. (B11) The ligand-binding molecule according to any one of (B1) to (B10), wherein the ligand-binding molecule comprises an antibody VH, an antibody VL, and an antibody constant region. (B12) The ligand-binding molecule according to (B11), wherein 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. (B13) The ligand-binding molecule according to (B12), wherein 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 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 according to (B12), wherein 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 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 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. (B16) The ligand-binding molecule according to (B15), wherein 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 introduced at any position in the antibody VH sequence selected from the group consisting of amino acids 7 (Kabat numbering) to 16 (Kabat numbering), 40 (Kabat numbering) to 47 (Kabat numbering), 55 (Kabat numbering) to 69 (Kabat numbering), 73 (Kabat numbering) to 79 (Kabat numbering), 83 (Kabat numbering) to 89 (Kabat numbering), 95 (Kabat numbering) to 99 (Kabat numbering), and 101 (Kabat numbering) to 113 (Kabat numbering). (B17) The ligand-binding molecule according to (B15), wherein 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 introduced at any position in a sequence selected from the group consisting of amino acids 7 (Kabat numbering) to 19 (Kabat numbering), 39 (Kabat numbering) to 46 (Kabat numbering), 49 (Kabat numbering) to 62 (Kabat numbering), and 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 the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible 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 according to (B18), wherein 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 introduced at any position in the sequence from amino acid 109 (Kabat numbering) of the antibody VH to amino acid 122 (EU numbering) of the antibody heavy chain constant region. (B20) The ligand-binding molecule according to (B18), wherein 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 introduced at any position in the sequence from amino acid 104 (Kabat numbering) of the antibody VL to amino acid 113 (EU numbering) of the antibody light chain constant region (position 113 according to Kabat numbering). (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 with each other, and the association is dissolved by cleavage of the protease cleavage sequence with a protease. (B22) The ligand-binding molecule according to any one of (B1) to (B21), wherein 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. (B23) The ligand-binding molecule according to any one of (B1) to (B22), wherein the ligand is a cytokine or a 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, and the ligand-binding molecule comprises an antibody VH and an antibody VL, and the ligand-binding molecule is: (a) an antibody VH comprising an H-CDR1 of SEQ ID NO: 374, an H-CDR2 of SEQ ID NO: 375, and an H-CDR3 of SEQ ID NO: 376, and an antibody VL comprising an L-CDR1 of SEQ ID NO: 377, an L-CDR2 of SEQ ID NO: 378, and an L-CDR3 of SEQ ID NO: 379; or (b) an antibody VH comprising an H-CDR1 of SEQ ID NO: 380, an H-CDR2 of SEQ ID NO: 381, and an H-CDR3 of SEQ ID NO: 382, and an antibody VL comprising an L-CDR1 of SEQ ID NO: 383, an L-CDR2 of SEQ ID NO: 384, and an L-CDR3 of SEQ ID NO: 385; or (c) having an antibody VH and an antibody VL that compete with (a) or (b); or (d) A ligand-binding molecule according to (B25), which has an antibody VH and an 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 to 14, 23 to 27, 33, 59, 60, and 346 to 367, or an antibody light chain selected from the sequences shown in SEQ ID NOs: 15 to 22, 1146 to 1160, 1282 to 1380, and 1386 to 1389. (B28) The ligand is IL-12, and the ligand-binding molecule comprises an antibody VH and an antibody VL, and the ligand-binding molecule comprises: (a) an antibody VH comprising an H-CDR1 of SEQ ID NO: 386, an H-CDR2 of SEQ ID NO: 387, and an H-CDR3 of SEQ ID NO: 388, and an antibody VL comprising an L-CDR1 of SEQ ID NO: 389, an L-CDR2 of SEQ ID NO: 390, and an L-CDR3 of SEQ ID NO: 391; or (b) having an antibody VH and an antibody VL that compete with (a); or (c) A ligand-binding molecule according to (B25), which has an antibody VH and an antibody VL that bind to the same epitope as (a). (B29) The ligand-binding molecule according to (B28), which is an antibody comprising the antibody heavy chain shown in SEQ ID NO: 146. (B30) The ligand is PD-1, and the ligand-binding molecule comprises an antibody VH and an antibody VL, and the ligand-binding molecule comprises: (a) an antibody VH comprising an H-CDR1 of SEQ ID NO: 392, an H-CDR2 of SEQ ID NO: 393, and an H-CDR3 of SEQ ID NO: 394, and an antibody VL comprising an L-CDR1 of SEQ ID NO: 395, an L-CDR2 of SEQ ID NO: 396, and an L-CDR3 of SEQ ID NO: 397; or (b) having an antibody VH and an antibody VL that compete with (a); or (c) A ligand-binding molecule according to (B25), which has an antibody VH and an antibody VL that bind to the same epitope as (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 to 315, and 322. (B32) The ligand is IL-6R (IL-6 receptor), and the ligand-binding molecule comprises an antibody VH and an antibody VL, and the ligand-binding molecule is: (a) an antibody VH comprising an H-CDR1 of SEQ ID NO: 398, an H-CDR2 of SEQ ID NO: 399, and an H-CDR3 of SEQ ID NO: 400, and an antibody VL comprising an L-CDR1 of SEQ ID NO: 401, an L-CDR2 of SEQ ID NO: 402, and an L-CDR3 of SEQ ID NO: 403; or (b) having an antibody VH and an antibody VL that compete with (a); or (c) A ligand-binding molecule according to (B25), which has an antibody VH and an antibody VL that bind to the same epitope as (a). (B33) The ligand-binding molecule according to (B32), wherein the ligand-binding molecule is an antibody comprising an antibody heavy chain selected from the sequences shown in SEQ ID NOs: 153 to 156, 157 to 159, and 404 to 470, or an antibody light chain selected from the sequences shown in SEQ ID NOs: 471 to 535. (B34) The ligand-binding molecule according to any one of (B1) to (B33), wherein the ligand-binding molecule is an IgG antibody. (B35) The ligand-binding molecule according to any one of (B1) to (B34), which is bound to the ligand. (B36) The ligand-binding molecule according to any one of (B1) to (B34), which is fused to the ligand. (B37) The ligand-binding molecule according to (B36), which does not bind to another ligand when fused to the ligand. (B38) The ligand-binding molecule according to (B36) or (B37), wherein the ligand-binding molecule is fused to 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 comprises an antibody light chain and an antibody heavy chain, and the antibody light chain or the antibody heavy chain is fused to the ligand. (B41) The ligand-binding molecule according to (B40), wherein the protease cleavage sequence is contained 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 to the ligand, and the ligand-binding molecule comprises: (a) an antibody heavy chain comprising an H-CDR1 having SEQ ID NO: 374, an H-CDR2 having SEQ ID NO: 375, and an H-CDR3 having SEQ ID NO: 376, and an antibody light chain comprising an L-CDR1 having SEQ ID NO: 377, an L-CDR2 having SEQ ID NO: 378, and an L-CDR3 having SEQ ID NO: 379; or (b) an antibody heavy chain comprising an H-CDR1 of SEQ ID NO: 380, an H-CDR2 of SEQ ID NO: 381, and an H-CDR3 of SEQ ID NO: 382, and an antibody light chain comprising an L-CDR1 of SEQ ID NO: 383, an L-CDR2 of SEQ ID NO: 384, and an L-CDR3 of SEQ ID NO: 385; A ligand-binding molecule according to (B40) or (B41). (B43) A ligand-binding molecule according to any one of (B40) to (B42), wherein the ligand is a modified CXCL10 shown in SEQ ID NO: 370. (B44) A ligand-binding molecule described in any one of (B40) to (B43), wherein the antibody light chain contained in the ligand-binding molecule is fused to the ligand, and the series of polypeptides in which CXCL10 and the antibody light chain are fused include the sequence shown in SEQ ID NO: 372. (B45) The ligand-binding molecule of any one of (B36) to (B39), 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 to the ligand. (B46) The ligand-binding molecule according to (B45), wherein the protease cleavage sequence is contained in the antibody light chain or the antibody heavy chain. (B47) The ligand-binding molecule of (B45) or (B46), wherein the ligand is PD-1, the antibody light chain has an L-CDR1 of SEQ ID NO: 395, an L-CDR2 of SEQ ID NO: 396, and an L-CDR3 of SEQ ID NO: 397, and the antibody heavy chain has an H-CDR1 of SEQ ID NO: 392, an H-CDR2 of SEQ ID NO: 393, and an H-CDR3 of SEQ ID NO: 394. (B48) A ligand-binding molecule according to any one of (B45) to (B47), wherein the ligand is PD-1 represented by SEQ ID NO: 320. (B49) The ligand-binding molecule of any one of (B45) to (B48), wherein the ligand is PD-1, the antibody heavy chain contained in the ligand-binding molecule is fused to 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 of any one of (B45) to (B48), wherein the ligand is PD-1, an antibody light chain contained in the ligand-binding molecule is fused to 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 comprises an antibody light chain and an antibody heavy chain, and the antibody light chain or the antibody heavy chain is fused to the ligand. (B52) The ligand-binding molecule according to (B51), wherein the protease cleavage sequence is contained 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 has an L-CDR1 of SEQ ID NO: 389, an L-CDR2 of SEQ ID NO: 390, and an L-CDR3 of SEQ ID NO: 391, and the antibody heavy chain has an H-CDR1 of SEQ ID NO: 386, an H-CDR2 of SEQ ID NO: 387, and an H-CDR3 of SEQ ID NO: 388. (B54) The ligand-binding molecule according to any one of (B36) to (B39), 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 to the ligand. (B55) The ligand-binding molecule according to (B54), wherein the protease cleavage sequence is contained 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 has an L-CDR1 of SEQ ID NO: 401, an L-CDR2 of SEQ ID NO: 402, and an L-CDR3 of SEQ ID NO: 403, and the antibody heavy chain has an H-CDR1 of SEQ ID NO: 398, an H-CDR2 of SEQ ID NO: 399, and an H-CDR3 of SEQ ID NO: 400. (B57) A complex formed of the ligand and the ligand-binding molecule according to any one of (B1) to (B34) that is bound to the ligand. (B58) A fusion protein in which the ligand is fused to the ligand-binding molecule according to any one of (B1) to (B34). (B59) The fusion protein according to (B58), wherein the ligand-binding molecule does not bind to another ligand when fused to the ligand. (B60) The fusion protein according to (B58) or (B59), wherein the ligand-binding molecule is fused to 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 consisting of a glycine-serine polymer. (B63) A fusion protein according to any one of (B58) to (B62), 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 to the ligand. (B64) The fusion protein according to (B63), wherein the protease cleavage sequence is contained in the antibody light chain or the 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 to the ligand, and the ligand-binding molecule comprises: (a) an antibody heavy chain comprising an H-CDR1 having SEQ ID NO: 374, an H-CDR2 having SEQ ID NO: 375, and an H-CDR3 having SEQ ID NO: 376, and an antibody light chain comprising an L-CDR1 having SEQ ID NO: 377, an L-CDR2 having SEQ ID NO: 378, and an L-CDR3 having SEQ ID NO: 379; or (b) an antibody heavy chain comprising an H-CDR1 of SEQ ID NO: 380, an H-CDR2 of SEQ ID NO: 381, and an H-CDR3 of SEQ ID NO: 382, and an antibody light chain comprising an L-CDR1 of SEQ ID NO: 383, an L-CDR2 of SEQ ID NO: 384, and an L-CDR3 of SEQ ID NO: 385; A fusion protein according to (B63) or (B64). (B66) A fusion protein according to any one of (B63) to (B65), wherein the ligand is a modified CXCL10 shown in SEQ ID NO: 370. (B67) A fusion protein described in any one of (B63) to (B66), in which the antibody light chain contained in the ligand-binding molecule is fused to the ligand, and the series of polypeptides in which CXCL10 and the antibody light chain are fused include the sequence shown in SEQ ID NO: 372. (B68) A fusion protein according to any one of (B58) to (B62), 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 to the ligand. (B69) The ligand-binding molecule according to (B68), wherein the protease cleavage sequence is contained in the antibody light chain or the antibody heavy chain. (B70) The fusion protein according to (B68) or (B69), wherein the ligand is PD-1, the antibody light chain has an L-CDR1 of SEQ ID NO: 395, an L-CDR2 of SEQ ID NO: 396, and an L-CDR3 of SEQ ID NO: 397, and the antibody heavy chain has an H-CDR1 of SEQ ID NO: 392, an H-CDR2 of SEQ ID NO: 393, and an H-CDR3 of SEQ ID NO: 394. (B71) The fusion protein according to any one of (B68) to (B70), wherein the ligand is PD-1 represented by SEQ ID NO: 320. (B72) A fusion protein according to any one of (B68) to (B71), wherein the ligand is PD-1, the antibody heavy chain contained in the ligand-binding molecule is fused to 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. (B73) The fusion protein according to any one of (B68) to (B71), wherein the ligand is PD-1, the antibody light chain contained in the ligand-binding molecule is fused to 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. (B74) The fusion protein according to any one of (B58) to (B62), 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 to the ligand. (B75) The fusion protein according to (B74), wherein the protease cleavage sequence is contained in the antibody light chain or the antibody heavy chain. (B76) The fusion protein according to (B74) or (B75), wherein the ligand is IL-12, the antibody light chain has an L-CDR1 of SEQ ID NO: 389, an L-CDR2 of SEQ ID NO: 390, and an L-CDR3 of SEQ ID NO: 391, and the antibody heavy chain has an H-CDR1 of SEQ ID NO: 386, an H-CDR2 of SEQ ID NO: 387, and an H-CDR3 of SEQ ID NO: 388. (B78) The fusion protein according to any one of (B58) to (B62), 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 to the ligand. (B78) The fusion protein according to (B77), wherein the protease cleavage sequence is contained in the antibody light chain or the antibody heavy chain. (B79) The fusion protein according to (B77) or (B78), wherein the ligand is IL-6R, the antibody light chain has 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 has H-CDR1 of SEQ ID NO: 398, H-CDR2 of SEQ ID NO: 399, and H-CDR3 of SEQ ID NO: 400. (B80) A pharmaceutical composition comprising the ligand-binding molecule according to any one of (B1) to (B56). (B81) A pharmaceutical composition comprising a ligand-binding molecule according to any one of (B1) to (B35) and a ligand. (B82) A pharmaceutical composition comprising the complex according to (B57). (B83) A pharmaceutical composition comprising the fusion protein according to any one of (B58) to (B79). (B84) A method for producing a ligand-binding molecule according to any one of (B1) to (B56). (B85) A method for producing according to (B84), which comprises introducing a protease cleavage sequence into a molecule capable of binding to a ligand. (B86) A method for producing the fusion protein according to any one of (B58) to (B79), which comprises fusing a ligand-binding molecule having a protease cleavage sequence with the ligand. (B87) A polynucleotide encoding the ligand-binding molecule according to any one of (B1) to (B56). (B88) A vector comprising the polynucleotide according to (B87). (B89) A host cell comprising the polynucleotide according to (B87) or the vector according to (B88). (B90) A method for producing the ligand-binding molecule according to any one of (B1) to (B56), comprising the step of culturing the host cell according to (B89). (B92) A polynucleotide encoding the fusion protein according to any one of (B58) to (B79). (B92) A vector comprising the polynucleotide according to (B91). (B93) A host cell comprising the polynucleotide according to (B91) or the vector according to (B92). (B94) A method for producing the fusion protein according to any one of (B58) to (B79), comprising the step of culturing the host cell according to (B93). [Brief explanation of the drawings]

[0012] [Figure 1]

[0033] Figure 1 shows a fusion protein of an IgG antibody and a ligand, which contains a ligand-linker-anti-ligand antibody VH molecule that is specifically released in target tissues, 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 tissues and one mode of its activation. An anti-ligand antibody with a protease cleavage sequence inserted near the boundary between VH and CH1 is mixed with the ligand and administered to an individual. [Figure 3] This figure shows an IgG antibody that specifically releases a ligand in target tissues, and one mode of its activation. An anti-ligand antibody with a protease cleavage sequence inserted near the boundary between VH and CH1 is administered to an individual. The administered antibody binds to a ligand that is originally present in the body, and the subsequent activation mode is the same as that shown in Figure 2. [Figure 4] FIG. 1 shows the results of evaluating the interaction between MabCXCL10 and human CXCL10 using Biacore. [Figure 5A] FIG. 1 shows a model of an antibody molecule created by inserting a protease cleavage sequence near the boundary between the antibody variable and constant regions of MabCXCL10. [Figure 5B]1 shows the name of each heavy chain variant prepared, the position where the protease cleavage sequence was inserted, and the inserted amino acid sequence, with the insertion site indicated by [insert]. [Figure 5C]

[0033] Figure 1 shows the name of each light chain variant prepared, the position where the protease cleavage sequence was inserted, and the inserted amino acid sequence, with the insertion site indicated by [insert]. [Figure 6A] FIG. 1 shows the results of an evaluation using Biacore of the interaction between human CXCL10 and 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. [Figure 6B] FIG. 1 shows the results of an evaluation using Biacore of the interaction between human CXCL10 and an antibody molecule created by inserting a protease cleavage sequence near the boundary between the variable and constant regions of the light chain of MabCXCL10. [Figure 7-1] (A) An antibody molecule constructed by inserting a protease cleavage sequence near the boundary between the variable and constant regions of the heavy chain of MabCXCL10 was treated with protease (MT-SP1), then subjected to reduced SDS-PAGE and the extent of cleavage was assessed by Coomassie Brilliant Blue (CBB) detection. Two new bands were generated by protease treatment; the band appearing at approximately 15 kDa is derived from the VH, and the band appearing at 25-50 kDa is derived from the constant region. [Figure 7-2] (A) Continued, and (B) shows the results of reducing SDS-PAGE analysis of the extent of cleavage of an antibody molecule constructed by inserting protease cleavage sequences into the variable and constant regions of the light chain of MabCXCL10 after protease (MT-SP1) treatment. Two new bands originating from the light chain cleaved by protease treatment are observed. [Figure 7-3] This is a continuation of (B). [Figure 8]

[0033] Figure 1 shows the names of heavy chain variants created by inserting a protease cleavage sequence and a flexible linker sequence near the boundary between the variable and constant regions of MabCXCL10, the positions where the protease cleavage sequence and the flexible linker sequence were inserted, and the inserted amino acid sequences. The insertion sites are indicated by [insert]. [Figure 9] This figure shows the results of an evaluation using Biacore of the interaction between human CXCL10 and an antibody molecule created by inserting a protease cleavage sequence and a flexible linker sequence near the boundary between the variable and constant regions of the heavy chain of MabCXCL10. [Figure 10A] This figure shows the results of evaluating the degree of cleavage of antibody molecules constructed 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 after protease (uPA, MT-SP1) treatment and subsequent electrophoresis on reducing SDS-PAGE followed by detection with CBB. Of the two new bands that emerged upon protease treatment, the band appearing at approximately 15 kDa is derived from the VH, and the band appearing at 25-50 kDa is derived from the constant region. [Figure 10B] FIG. 10B is a diagram showing a continuation of FIG. 10A. [Figure 11A] FIG. 1 shows the results of evaluating whether CXCL10 is released by treating a complex of MabCXCL10a and CXCL10 with a protease (MT-SP1). [Figure 11B] FIG. 1 shows the results of evaluating whether CXCL10 is released by treating the complex of EEIVHC006a / EEIVL and CXCL10 with protease (MT-SP1). [Figure 12]This figure shows the name of each heavy chain created by replacing part of the amino acid sequence near the boundary between the variable and constant regions of MabCXCL10 with a protease cleavage sequence and a flexible linker sequence, the site of amino acid insertion and modification, the inserted sequence, and the amino acid sequence after insertion and modification. The insertion site is indicated by [insert]. The amino acid residues indicated by strikethrough in the "Insertion and modification positions" column were deleted during insertion of the inserted sequence, i.e., were replaced with the amino acid at the most C-terminus of the inserted sequence. [Figure 13] This figure shows the results of evaluating the degree of cleavage of antibody molecules constructed by substituting a protease cleavage sequence and a flexible linker for a portion of the amino acid sequence near the boundary between the variable and constant regions of MabCXCL10. The antibody molecules were treated with proteases (uPA, MT-SP1), then electrophoresed on reducing SDS-PAGE and detected with CBB. Of the two new bands that emerged upon protease treatment, the band appearing at approximately 15 kDa is derived from the VH, and the band appearing at 25-50 kDa is derived from the constant region. [Figure 14] FIG. 1 shows luciferase activity (luminescence value). [Figure 15] This shows the results of SDS-PAGE before and after protease cleavage of the CXCL10-anti-CXCL10 antibody fusion protein. [Figure 16] FIG. 1 shows luciferase activity (luminescence value). [Figure 17] FIG. 10 shows a reducing SDS-PAGE image in which protease cleavage of an anti-IL-12 neutralizing antibody into which a protease cleavage sequence and a flexible linker sequence have been introduced was evaluated. [Figure 18] This graph shows the production of interferon gamma when IL-12 and an antibody were added. NoAb is a sample to which only IL-12 was added without any antibody, and NoIL-12 is a sample to which neither IL-12 nor an antibody was added. [Figure 19A] FIG. 1 shows protease cleavage of an antibody. [Figure 19B] FIG. 1 shows protease cleavage of an antibody. [Figure 20A]FIG. 1 shows the results of cleavage with various proteases. [Figure 20B] FIG. 1 shows the results of cleavage with various proteases. [Figure 21] FIG. 1 shows the results of cleavage with various proteases. [Figure 22A] FIG. 1 shows the results of protease cleavage of modified MRA. [Figure 22B] FIG. 1 shows the results of protease cleavage of modified MRA. [Figure 22C] FIG. 1 shows the results of protease cleavage of modified MRA. [Figure 22D] FIG. 1 shows the results of protease cleavage of modified MRA. [Figure 22E] FIG. 1 shows the results of protease cleavage of modified MRA. [Figure 22F] FIG. 1 shows the results of protease cleavage of modified MRA. [Figure 22G] FIG. 1 shows the results of protease cleavage of modified MRA. [Figure 22H] FIG. 1 shows the results of protease cleavage of modified MRA. [Figure 22I] FIG. 1 shows the results of protease cleavage of modified MRA. [Figure 23A] FIG. 1 shows the results of protease cleavage of modified MRA. [Figure 23B] FIG. 1 shows the results of protease cleavage of modified MRA. [Figure 23C] FIG. 1 shows the results of protease cleavage of modified MRA. [Figure 24A] FIG. 1 shows the results of protease cleavage of modified MRA. [Figure 24B] FIG. 1 shows the results of protease cleavage of modified MRA. [Figure 24C] FIG. 1 shows the results of protease cleavage of modified MRA. [Figure 24D] FIG. 1 shows the results of protease cleavage of modified MRA. [Figure 24E] FIG. 1 shows the results of protease cleavage of modified MRA. [Figure 25A] FIG. 1 shows the results of protease cleavage of modified MRA. [Figure 25B] FIG. 1 shows the results of protease cleavage of modified MRA. [Figure 26] This figure compares real-time graphs showing the binding of PD-1 to 5C4-bio in binding assessment samples containing protease-treated / protease-untreated antibodies and PD-1. The thick black line represents the binding assessment sample containing protease-treated antibody, and the thin gray line represents the binding assessment sample containing protease-untreated antibody. The X axis represents measurement time (seconds), with the start of measurement set to 0 seconds. The Y axis represents binding. The name of each graph indicates the antibody contained in the assessment sample; the None (antigen only) graph shows that only antigen was used as the assessment sample, with no antibody mixed in. [Figure 27] Electrophoresis results of protease-treated and non-protease-treated antibodies. The protease (+) lane is the protease-treated antibody, and the protease (-) lane is the protease-untreated antibody. [Figure 28] This figure compares real-time graphs showing the binding of protease-treated and protease-untreated antibodies to PD-1. The thick black line represents the protease-treated antibody, and the thin gray line represents the protease-untreated antibody. The X axis represents the measurement time (seconds), with the start of measurement set to 0 seconds. The Y axis represents binding. The name of each graph indicates the antibody used; in the graph marked "None," only PBS buffer was used, with no antibody used. [Figure 29]A comparison of 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 measurement time (seconds), with the start of measurement set to 0 seconds. The Y-axis represents binding. The name of each graph indicates the antibody contained in the sample; the "Antigen and Protease" graph shows samples containing only PD-1 and no antibody. [Figure 30] Real-time graphs comparing the binding of free PD-1 to 5C4-bio in protease-treated fusion protein and protease-untreated protein solutions. The thick black line represents the protease-treated sample, while the thin gray line represents the untreated sample. The X-axis represents measurement time (seconds), with the start of measurement set at 0 seconds. The Y-axis represents binding. The name of each graph indicates the antibody in the fusion protein. For the graph labeled "None (antigen only)," no fusion protein was used as the evaluation sample; only the antigen PD-1 was used. For the graph labeled "5C4H-G1T4 / 5C4L-KT0," no fusion protein was used; only the 5C4H-G1T4 / 5C4L-KT0 antibody was used. [Figure 31] Electrophoresis results of protease-treated antibody-PD-1 fusion proteins. The protease (+) lane shows the protease-treated fusion protein, and the protease (-) lane shows the protease-untreated fusion protein. [Figure 32] FIG. 1 shows the results of evaluating the in vivo cleavage efficiency of an antibody molecule into which a protease cleavage sequence has been inserted, when the antibody molecule was administered to mice. DETAILED DESCRIPTION OF THE INVENTION

[0013] The term "polypeptide" as used herein generally refers to peptides and proteins having a length of about four amino acids or more. The polypeptide of the present invention is generally a polypeptide consisting of an artificially designed sequence, but is not particularly limited thereto and may be, for example, a polypeptide derived from a living organism. It may also be a natural polypeptide, a synthetic polypeptide, a recombinant polypeptide, or the like. Furthermore, fragments of the above polypeptides are also included in the polypeptide of the present invention.

[0014] As used herein, amino acids are represented by one-letter or three-letter codes, or both, such as 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, and Val / V.

[0015] To modify an amino acid in the amino acid sequence of a polypeptide, 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 used. Furthermore, several known methods can also be used to modify amino acids by substituting amino acids other than natural amino acids (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 a tRNA in which a non-natural amino acid is bound to an amber suppressor tRNA complementary to the UAG codon (amber codon), which is a type of stop codon, can be suitably used.

[0016] As used herein, the meaning of the term "and / or" used to describe amino acid modification sites includes any combination of "and" and "or." Specifically, for example, "amino acids 37, 45, and / or 47 are substituted" includes the following amino acid modification variations: (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] Herein, expressions for amino acid modifications may be appropriately expressed by listing the one-letter or three-letter codes for the amino acid before and after the modification before and after a number representing a specific position. For example, the modification F37V or Phe37Val used when substituting an amino acid in an antibody variable region represents a substitution of Phe at position 37 according to the Kabat numbering system with Val. That is, the number represents the amino acid position according to the Kabat numbering system, the one-letter or three-letter code preceding the number represents the amino acid before substitution, and the one-letter or three-letter code following the number represents the amino acid after substitution. Similarly, the modification P238A or Pro238Ala used when substituting an amino acid in the Fc region of an antibody constant region represents a substitution of Pro at position 238 according to the EU numbering system with Ala. That is, the number represents the amino acid position according to the EU numbering system, the one-letter or three-letter code preceding the number represents the amino acid before substitution, and the one-letter or three-letter code following the number represents the amino acid after substitution.

[0018] The present invention relates to a ligand-binding molecule that has a cleavage site and whose binding to a ligand is weakened when the cleavage site is cleaved. The ligand-binding molecule of the present invention is a polypeptide that can bind to a ligand.

[0019] The ligand-binding molecules of the present invention are molecules capable of binding to ligands, particularly molecules capable of binding to ligands in an uncleaved state. Here, "binding" typically refers to binding through interactions primarily based on non-covalent bonds such as electrostatic forces, van der Waals forces, and hydrogen bonds. Suitable examples of ligand-binding modes of the ligand-binding molecules of the present invention include, but are not limited to, antigen-antibody reactions in which antigen-binding regions, antigen-binding molecules, antibodies, and antibody fragments bind to antigens.

[0020] Note that "capable of binding to a ligand" means that the ligand-binding molecule is capable of binding 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 connected by a covalent bond. For example, the fact that a ligand and a ligand-binding molecule are covalently linked via a linker does not mean that the molecule is capable of binding to the ligand. Furthermore, "attenuated binding to a ligand" means that the binding ability is attenuated. For example, when a ligand and a ligand-binding molecule are covalently linked via a linker, cleavage of the linker is not considered to be attenuated binding to the ligand. Note that in the present invention, the ligand-binding molecule may be connected to the ligand via a linker or the like, as long as the ligand-binding molecule is capable of binding to the ligand.

[0021] The ligand-binding molecule of the present invention is limited only by its ability to bind to a ligand in an uncleaved state, and any molecule of any structure can be used as long as it can bind to the target ligand in an uncleaved state. Examples of ligand-binding molecules include, but are not limited to, antibody heavy chain variable regions (VH) and antibody light chain variable regions (VL), single domain antibodies (sdAb), modules called A domains of about 35 amino acids contained in Avimer, a cell membrane protein present in living organisms (International Publication Nos. WO2004 / 044011 and WO2005 / 040229), Adnectin (International Publication No. WO2002 / 032925) containing the Fn3 domain, which is a domain that binds to proteins in fibronectin, a glycoprotein expressed on cell membranes, Affibody (International Publication No. WO1995 / 001937) using an IgG-binding domain consisting of a 58-amino acid three-helix bundle of Protein A as a scaffold, and ankyrin repeats (ankyrin repeats) having a structure in which subunits of a 33-amino acid turn, two antiparallel helices, and a loop are repeatedly stacked. Examples of such proteins include DARPins (Designed Ankyrin Repeat proteins), which are regions exposed on the molecular surface of ankyrin repeat (AR) molecules (International Publication WO 2002 / 020565); Anticalin, which is a four-loop region supporting one side of a barrel structure in which eight highly conserved antiparallel strands twist toward the center in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) (International Publication WO 2003 / 029462); and a concave region of a parallel sheet structure within a horseshoe-shaped structure formed by repeated stacking of leucine-rich-repeat (LRR) modules in the variable lymphocyte receptor (VLR), which does not have an immunoglobulin structure and is part of the adaptive immune system of jawless fish such as lampreys and hagfish (International Publication WO 2008 / 016854).

[0022] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so 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. Methods for producing antibodies that bind to IL-6R (anti-IL-6R antibodies) are exemplified below. Antibodies that bind to antigens other than IL-6R can also be produced appropriately according to the following examples.

[0024] Anti-IL-6R antibodies can be obtained as polyclonal or monoclonal antibodies using known methods. Monoclonal antibodies derived from mammals 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 by genetic engineering techniques. Antibodies referred to in this application include "humanized antibodies" and "chimeric antibodies."

[0025] Monoclonal antibody-producing hybridomas can be prepared using known techniques, for example, as follows: A mammal is immunized using an IL-6R protein as a sensitizing antigen according to a conventional immunization method. The resulting immune cells are fused with known parent cells by a conventional cell fusion method. Next, monoclonal antibody-producing cells can be screened using conventional screening methods to select hybridomas that produce anti-IL-6R antibodies.

[0026] Specifically, monoclonal antibodies can be produced, for example, as follows. First, the IL-6R gene can be expressed to obtain the IL-6R protein used as a sensitizing antigen for antibody production. Specifically, a suitable host cell is transformed by inserting a gene sequence encoding IL-6R into a known expression vector. The desired human IL-6R protein is purified from the host cell or culture supernatant by a known method. To obtain soluble IL-6R from the culture supernatant, for example, soluble IL-6R is expressed as described by Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968). 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 of 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. Alternatively, it can be obtained by incorporating a portion of the IL-6R gene into an expression vector and expressing it. It can also 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. The number of amino acids constituting the peptide used as a sensitizing antigen is preferably at least 5 or more, 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] Alternatively, a fusion protein obtained by fusing a desired partial polypeptide or peptide of the IL-6R protein with a different polypeptide can be used as a sensitizing antigen. For example, an antibody Fc fragment or a peptide tag can be suitably used to produce a fusion protein used as a sensitizing antigen. A vector expressing a fusion protein can be prepared by fusing genes encoding two or more desired polypeptide fragments in frame and inserting the fusion gene into an expression vector as described above. Methods for producing fusion proteins are described in Molecular Cloning, 2nd ed. (Sambrook, J et al., Molecular Cloning, 2nd ed., pp. 9:47-9:58 (1989) Cold Spring Harbor Lab. Press). Methods for obtaining IL-6R to be used as a sensitizing antigen and immunization methods using the same are also specifically described in WO2003 / 000883, WO2004 / 022754, WO2006 / 006693, etc.

[0029] The mammal to be immunized with the sensitizing antigen is not limited to a specific animal, but is preferably selected in consideration of compatibility with the parent cells used in cell fusion. Generally, rodents such as mice, rats, hamsters, rabbits, and monkeys are preferably used.

[0030] The above-mentioned animals are immunized with the sensitizing antigen according to known methods. For example, a common method for immunization is to administer the sensitizing antigen intraperitoneally or subcutaneously to a mammal. Specifically, the sensitizing antigen is diluted at an appropriate dilution ratio with PBS (Phosphate-Buffered Saline) or physiological saline, and optionally mixed with a conventional adjuvant, such as Freund's complete adjuvant, and emulsified. The sensitizing antigen is then administered to the mammal several times every 4 to 21 days. A suitable carrier can 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] Hybridomas producing the desired antibodies can also be prepared 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 to the immunized animal, and a sensitizing antigen is expressed in the immunized animal's body, thereby conferring immune stimulation. Compared to general immunization methods in which a protein antigen is administered to the immunized animal, DNA immunization is expected to have the following advantages: -Maintaining the structure of membrane proteins such as IL-6R can provide immune stimulation -No need to purify the immunogen

[0032] To obtain the monoclonal antibody of the present invention by DNA immunization, first, DNA expressing the IL-6R protein is administered to an animal to be immunized. DNA encoding IL-6R can be synthesized by known methods such as PCR. The obtained DNA is inserted into an appropriate expression vector and administered to the animal to be immunized. Commercially available expression vectors, such as pcDNA3.1, can be suitably used as the expression vector. Commonly used methods can be used to administer the vector to a living body. For example, DNA immunization can be performed by introducing gold particles adsorbed with the expression vector into the cells of an animal to be immunized 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 a mammal is immunized in this manner and an increase in the titer of an antibody that binds to IL-6R is confirmed in the serum, immune cells are collected from the mammal and subjected to cell fusion. Splenocytes are particularly preferred as immune cells.

[0034] Mammalian myeloma cells are used as the cells to be fused with the immune cells. The myeloma cells preferably contain an appropriate selection marker 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) and thymidine kinase deficiency (hereinafter abbreviated as TK deficiency). Cells deficient in 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 by utilizing the salvage pathway of normal cells, allowing them to grow even in HAT selective medium.

[0035] HGPRT-deficient or TK-deficient cells can be selected on media containing 6-thioguanine, 8-azaguanine (hereafter abbreviated as 8AG), or 5'-bromodeoxyuridine, respectively. Normal cells that incorporate these pyrimidine analogs into their DNA die. On the other hand, cells lacking these enzymes and unable to incorporate these pyrimidine analogs can survive in selective media. Another selectable marker, called G418 resistance, confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) via the neomycin resistance gene. Various myeloma cell lines 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), and 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] Basically, cell fusion between the immune cells and myeloma cells is carried out according to known methods, such as the method of Kohler and Milstein et al. (Methods Enzymol. (1981) 73, 3-46). More specifically, the cell fusion can be carried out in a conventional nutrient medium in the presence of a cell fusion promoter, such as polyethylene glycol (PEG) or Sendai virus (HVJ), with the addition of an adjuvant such as dimethyl sulfoxide, if desired, to further enhance the fusion efficiency.

[0038] The ratio of immune cells to myeloma cells can be set arbitrarily. For example, the ratio of immune cells to myeloma cells is preferably 1 to 10. The culture medium used for the cell fusion may be, for example, RPMI1640 culture medium, MEM culture medium, or other conventional culture medium suitable for growing the myeloma cell line, and may further be suitably supplemented with serum supplements such as fetal calf serum (FCS).

[0039] For cell fusion, predetermined amounts of the immune cells and myeloma cells are thoroughly mixed in the culture medium, and a PEG solution (e.g., an average molecular weight of approximately 1000 to 6000) preheated to approximately 37°C is added, usually at a concentration of 30 to 60% (w / v). The mixture is gently mixed to form the desired fused cells (hybridomas). Next, an appropriate culture medium such as those listed above is successively added, and the mixture is centrifuged and the supernatant is removed. This procedure is repeated to remove cell fusion agents and other substances that are undesirable for hybridoma growth.

[0040] The hybridomas thus obtained can be selected by culturing them in a conventional selective culture medium, such as HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Culture can be continued using the HAT culture medium for a sufficient period of time (usually several days to several weeks) for cells other than the desired hybridoma (unfused cells) to die. Hybridomas producing the desired antibody are then screened and single-cloned by the conventional limiting dilution method.

[0041] The hybridomas thus obtained can be selected using a selective medium corresponding to the selection marker possessed by the myeloma used in cell fusion. For example, cells lacking HGPRT or TK can be selected by culturing them in HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells that have successfully fused with normal cells can selectively grow in HAT medium. Culture in the above HAT medium is continued for a period of time sufficient for cells other than the desired hybridoma (non-fused cells) to die. Specifically, the desired hybridoma can generally be selected by culturing for several days to several weeks. Hybridomas producing the desired antibody can then be screened and single-cell cloned by the conventional limiting dilution method.

[0042] Screening and monocloning of the desired antibody can be preferably carried out by known screening methods based on antigen-antibody reactions. 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 analyzes cells contacted with a fluorescent antibody using laser light and measures the fluorescence emitted by individual cells, thereby enabling measurement of antibody binding to the cell surface.

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

[0044] Alternatively, the binding activity of an antibody to 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 hybridoma culture supernatant is contacted 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 that binds to the cells can be detected with an anti-mouse immunoglobulin antibody. Hybridomas that produce the desired antibody capable of binding to the antigen and are selected by these screening methods can be cloned by limiting dilution or other methods.

[0045] The hybridomas producing the monoclonal antibodies thus prepared can be subcultured in a conventional culture medium and can be stored for a long period of time in liquid nitrogen.

[0046] The hybridomas are cultured according to conventional methods, and the desired monoclonal antibodies can be isolated from the culture supernatant. Alternatively, the hybridomas can be administered to a compatible mammal to grow, and the monoclonal antibodies can be isolated from the ascites. The former method is suitable for obtaining highly purified antibodies.

[0047] Antibodies encoded by antibody genes cloned from antibody-producing cells such as hybridomas can also be suitably used. The cloned antibody genes are incorporated into an appropriate vector and introduced into a host, whereby the antibodies encoded by the genes are 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 an anti-IL-6R antibody is obtained from hybridoma cells that produce the anti-IL-6R antibody. To do this, total RNA is usually first extracted from the hybridoma. The following methods 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) or similar. Alternatively, kits for directly extracting total mRNA from cells, such as the QuickPrep mRNA Purification Kit (GE Healthcare Biosciences), are commercially available. Using such kits, mRNA can be isolated from hybridomas. cDNA encoding antibody V regions can be synthesized from the resulting mRNA using reverse transcriptase. cDNA can be synthesized using an AMV Reverse Transcriptase First-Strand cDNA Synthesis Kit (Seikagaku Corporation) or similar. Alternatively, 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) can be used appropriately for cDNA synthesis and amplification. Furthermore, during the process of synthesizing such cDNA, appropriate restriction enzyme sites, as described below, can be introduced at both ends of the cDNA.

[0050] The desired cDNA fragment is purified from the resulting PCR product and then ligated to vector DNA. The recombinant vector thus constructed is introduced into E. coli or other bacteria, and colonies are selected. The desired recombinant vector can then be prepared from the E. coli that formed the colonies. Whether the recombinant vector contains the nucleotide sequence of the desired cDNA is then confirmed by known methods, such as the dideoxynucleotide chain termination method.

[0051] A convenient way to obtain genes encoding variable regions is to use the 5'-RACE method, which uses primers specifically designed for amplifying variable region genes. First, cDNA is synthesized using RNA extracted from hybridoma cells as a template, and a 5'-RACE cDNA library is obtained. A commercially available kit, such as the SMART RACE cDNA Amplification Kit, can be used to synthesize the 5'-RACE cDNA library.

[0052] The resulting 5'-RACE cDNA library is used as a template for PCR amplification of antibody genes. Primers for amplifying mouse antibody genes can be designed based on known antibody gene sequences. These primers have different base sequences for each immunoglobulin subclass. Therefore, it is recommended that the subclass be determined in advance using a commercially available kit such as the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche Diagnostics).

[0053] Specifically, for example, when the goal is to obtain a gene encoding mouse IgG, primers capable of amplifying genes encoding γ1, γ2a, γ2b, and γ3 heavy chains and κ and λ light chains can be used. To amplify IgG variable region genes, the 3' primer generally anneals to a region corresponding to the constant region close to the variable region. Meanwhile, the 5' primer used is a primer included in the 5' RACE cDNA library construction kit.

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

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

[0056] Panning methods using phage vectors are also suitable for screening antibodies using binding activity as an index. When antibody genes are obtained as a library of heavy and light chain subclasses from a polyclonal antibody-expressing cell population, screening methods using phage vectors are advantageous. Genes encoding the heavy and light chain variable regions can be linked with an appropriate linker sequence to form single-chain Fvs (scFvs). Phages expressing scFvs on their surface can be obtained by inserting a gene encoding an scFv into a phage vector. After contacting this phage with a desired antigen, DNA encoding an scFv with the desired binding activity can be recovered by recovering the phage bound to the antigen. By repeating this procedure as necessary, scFvs with the desired binding activity can be enriched.

[0057] After obtaining cDNA encoding the V region of the desired anti-IL-6R antibody, the cDNA is digested with restriction enzymes that recognize restriction enzyme sites inserted at both ends of the cDNA. Preferred restriction enzymes recognize and digest nucleotide sequences that appear infrequently in the nucleotide sequence constituting the antibody gene. Furthermore, to insert one copy of the digested fragment into a vector in the correct orientation, it is preferable to insert a restriction enzyme that generates cohesive ends. An antibody expression vector can be obtained by inserting the cDNA encoding the V region of the anti-IL-6R antibody digested as described above into an appropriate expression vector. In this case, a chimeric antibody can be obtained by fusing a gene encoding the antibody constant region (C region) with a gene encoding the V region in frame. Here, a chimeric antibody refers to an antibody in which the constant region and variable region are derived from different sources. Therefore, in addition to heterogeneous chimeric antibodies such as mouse-human, human-human allogeneic chimeric antibodies are also included in the chimeric antibodies of the present invention. A chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector that already contains a constant region. Specifically, for example, a restriction enzyme recognition sequence for a restriction enzyme that digests the V region gene can be appropriately positioned at the 5' end of an expression vector carrying DNA encoding the desired antibody constant region (C region). A chimeric antibody expression vector is constructed by fusion in-frame of both DNAs 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 its expression is controlled by an expression control region. Expression control regions for antibody expression include, for example, enhancers and promoters. Furthermore, an appropriate signal sequence can 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) can be used as a signal sequence, although other suitable signal sequences can also be added. The expressed polypeptide is cleaved at the carboxyl terminal of the sequence, and the cleaved polypeptide can be secreted extracellularly as a mature polypeptide. Next, appropriate host cells can be transformed with this expression vector to obtain recombinant cells expressing DNA encoding the anti-IL-6R antibody.

[0059] An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, 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 an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0061] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain 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 (FR) and three complementarity-determining regions (CDR). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). One VH or VL domain may be sufficient to confer antigen-binding specificity.

[0062] As used herein, the term "complementarity determining region" or "CDR" refers to each region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops ("hypervariable loops") and / or antigen contact residues ("antigen contacts"). Typically, antibodies contain six CDRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary CDRs herein include the following: (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) antigenic contacts 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), comprising 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 (eg, FR residues) are numbered herein according to Kabat et al., supra.

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

[0064] As used herein, 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-bonded light chains and two identical heavy chains. From the N-terminus to the C-terminus, each heavy chain contains 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 a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain. Similarly, from the N-terminus to the C-terminus, each light chain contains 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 can 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 major 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 the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0066] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, for human IgG1, the heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain, except that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) residues of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also referred to as the EU index) as 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 photolyzed. The cleavage site can be located anywhere in the polypeptide, as long as cleavage attenuates 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 molecules of the present invention have weaker (i.e., attenuated) ligand binding in the cleaved state compared to the uncleaved state. In embodiments in which the binding between the ligand-binding molecule and the ligand is an antigen-antibody reaction, attenuation of ligand binding can be evaluated by the ligand-binding activity of the ligand-binding molecule.

[0069] The binding activity of a ligand-binding molecule and a ligand can be evaluated by well-known methods such as FACS, ELISA format, ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), BIACORE method using 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 uses two beads, donor and acceptor, based on the following principle: A luminescent signal is detected only when a molecule bound to a donor bead interacts with a molecule bound to an acceptor bead and the two beads are in close proximity. A photosensitizer inside the donor bead, excited by a laser, converts surrounding oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and, when it reaches a nearby acceptor bead, triggers a chemiluminescent reaction within the bead, ultimately emitting light. If 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 no chemiluminescent reaction occurs.

[0070] For example, biotin-labeled ligand-binding molecules are bound to donor beads, and glutathione S-transferase (GST)-tagged ligands are bound to acceptor beads. In the absence of competing untagged ligand-binding molecules, the ligand-binding molecules interact with the ligand, generating a signal at 520-620 nm. The untagged ligand-binding molecules compete with the interaction between the tagged ligand-binding molecules and the ligand. Relative binding affinity can be determined by quantifying the decrease in fluorescence that occurs as a result of competition. Biotinylation of ligand-binding molecules such as antibodies using sulfo-NHS-biotin or similar is well known. GST-tagging of ligands can be achieved by expressing the GST-fused ligand in cells harboring a vector capable of expressing a fusion gene in which a polynucleotide encoding the ligand and a polynucleotide encoding GST are fused in frame, followed by purification using a glutathione column. The resulting signals are suitably analyzed by fitting to a one-site competition model using non-linear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

[0071] One of the substances (ligand) whose interaction is to be observed is immobilized on a thin gold film on a sensor chip. When light is shone from the back of the sensor chip so that it is totally reflected at the interface between the gold film and the glass, a portion of the reflected light exhibits a reduced reflection intensity (SPR signal). When the other substance (analyte) whose interaction is to be observed is passed over the surface of the sensor chip, binding occurs between the ligand and the analyte, increasing the mass of the immobilized ligand molecule and changing the refractive index of the solvent on the sensor chip surface. This change in refractive index shifts the position of the SPR signal (conversely, dissociation returns the signal position). The Biacore system plots the amount of 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). The kinetics (association rate constant (ka) and dissociation rate constant (kd)) can be determined from the sensorgram curve, and the dissociation constant (KD) can be calculated from the ratio of these constants. Inhibition assays and equilibrium analysis are also suitable for use with the BIACORE method. An example of an inhibition assay is described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010, and an example of an equilibrium value analysis is described in Methods Enzymol. 2000;323:325-40.

[0072] A ligand-binding molecule's ability to bind to a ligand is attenuated when, for example, the amount of ligand binding 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, or 15% or less, and 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 a control ligand-binding molecule, as measured by the above-described measurement method. Any suitable indicator of binding activity may be used, such as the dissociation constant (KD). When the dissociation constant (KD) is used as an indicator of binding activity, a larger KD for the test ligand-binding molecule relative to the ligand indicates that the binding activity of the test ligand-binding molecule relative to the ligand is weaker than that of the control ligand-binding molecule. The ligand-binding function is attenuated when, for example, the dissociation constant (KD) of the test ligand-binding molecule for the ligand is at least 2-fold, preferably at least 5-fold, at least 10-fold, and particularly preferably at least 100-fold, compared to the dissociation constant (KD) of the control ligand-binding molecule for the ligand. The control ligand-binding molecule may be, for example, an uncleaved form of the ligand-binding molecule.

[0073] In one embodiment of the present invention, the ligand-binding molecule of the present invention is released from the ligand-binding molecule by cleavage at the cleavage site. Here, if the ligand is bound to a portion of the ligand-binding molecule via a linker and the linker does not have a cleavage site, the ligand will be released while still connected to that portion of the ligand-binding molecule via the linker (see, for example, Figure 1). Thus, even if the ligand is released together with a portion of the ligand-binding molecule, it can be said that the ligand has been released from the ligand-binding molecule as long as it is released from the majority of the ligand-binding molecules.

[0074] One method for detecting the release of a ligand from a ligand-binding molecule upon cleavage at the cleavage site is to detect the ligand using a ligand-detecting antibody that recognizes the ligand. When the ligand-binding molecule is an antibody fragment, the ligand-detecting antibody preferably binds to the same epitope as the ligand-binding molecule. Ligand detection using a ligand-detecting antibody can be confirmed by well-known methods such as FACS, ELISA format, ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), BIACORE method using 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, a ligand-detecting antibody that recognizes the ligand can be biotinylated and contacted with a biosensor, followed by measuring binding to the ligand in the sample, thereby detecting ligand release. Specifically, the amount of ligand can be measured using a ligand-detecting antibody in a sample containing a ligand-binding molecule and a ligand before or after protease treatment, and the amount of ligand detected in the sample before and after protease treatment can be compared to detect ligand release. Alternatively, the amount of ligand can be measured using a ligand-detecting antibody 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 protease, and the amount of ligand detected in the sample with and without protease can be compared to detect ligand release. More specifically, ligand release can be detected using the methods described in the Examples of the present application. When a ligand-binding molecule is fused with a ligand to form a fusion protein, the amount of ligand can be measured using a ligand-detecting antibody in a sample containing the fusion protein before or after protease treatment, and the amount of ligand detected in the sample before and after protease treatment can be compared to detect ligand release. Furthermore, the amount of ligand can be measured using a ligand-detecting antibody for a sample containing a protease and a fusion protein and a sample containing the fusion protein but not the protease, and the amount of ligand detected in the samples with and without the protease can be compared to detect the release of the ligand. More specifically, the release of the ligand can be detected by the method described in the Examples of the present application.

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

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

[0077] As used herein, the term "protease" refers to an enzyme, such as an endopeptidase or exopeptidase, that hydrolyzes peptide bonds, typically an endopeptidase. The protease used in the present invention is limited only by its ability to cleave the protease cleavage sequence, and its type is not particularly limited. In some embodiments, a target tissue-specific protease is used. The target tissue-specific protease may be, for example, (1) a protease that is expressed at a higher level in target tissue than in normal tissue; (2) a protease that has higher activity in target tissues than in normal tissues; (3) a protease that is expressed at a higher level in target cells than in normal cells; (4) a protease that has higher activity in target cells than in normal cells; In a more specific embodiment, a cancer tissue-specific protease or an inflamed tissue-specific protease is used.

[0078] As used herein, the term "target tissue" refers to a tissue that contains 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 inflamed tissue.

[0079] The term "cancer tissue" refers to tissue containing at least one cancer cell. Thus, 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. As used herein, a tumor mass refers to a foci of tumor tissue. The term "tumor" is generally used to refer to benign or malignant neoplasms.

[0080] As used herein, "inflamed tissue" includes, for example, the following: Joints in rheumatoid arthritis and osteoarthritis Lungs (alveoli) in bronchial asthma and COPD Digestive tract in inflammatory bowel disease, Crohn's disease, and ulcerative colitis Fibrotic tissue in liver, kidney, and lung fibrosis -Tissues undergoing rejection 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 dermatitis Spinal nerve damage in herniated discs and chronic lower back pain

[0081] Proteases that are specifically expressed or specifically activated in several types of target tissues or that are thought to be associated with the disease state of the target tissues (target tissue-specific proteases) are known. For example, International Publication Nos. WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846 disclose proteases that are specifically expressed in cancer tissues. 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 can be expressed in an inactive form and then become active. In many tissues, substances that inhibit active proteases exist, and their activity is controlled by the activation process and the presence of inhibitors (Nat Rev Cancer. 2003 Jul;3(7):489-501.). In target tissues, active proteases can escape inhibition and become specifically activated. Active proteases can be measured using an antibody that recognizes the active form of the protease (PNAS 2013 Jan 2; 110(1): 93-98.) or a method in which a peptide recognized by the protease is fluorescently labeled, and the fluorescent light is quenched before cleavage but emits 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" means: (i) a protease that is expressed at a higher level in target tissue than in normal tissue; (ii) a protease that has higher activity in target tissue than in normal tissue; (iii) a protease that is expressed at a higher level in target cells than in normal cells; (iv) a protease that has higher activity in target cells than in normal cells; It can refer to either of the following.

[0084] Specific proteases include, but are not limited to, cysteine proteases (including cathepsin family B, L, S, etc.), aspartyl proteases (cathepsin D, E, K, O, etc.), serine proteases (including matriptase (MT-SP1), cathepsin A and G, thrombin, plasmin, urokinase (uPA), tissue plasminogen activator (tPA), elastase, proteinase 3, thrombin, kallikrein, tryptophan, etc.), and the like. metalloproteases (including membrane-bound (MMP14-17 and MMP24-25) and secreted (MMP1-13, MMP18-23, and MMP26-28) metalloproteases (MMP1-28); proteases A disintegrin and metalloprotease (ADAM), metalloproteases with A disintegrin or thrombospondin motifs (ADAMTS), meprin (meprin α alpha), meprin beta), CD10 (CALLA), as well as prostate-specific antigen (PSA), legumain, TMPRSS3, TMPRSS4, neutrophil elastase (HNE), beta-secretase (BACE), fibroblast activation protein alpha (FAP), granzyme B, guanidinobenzoatase (GB), hepsin, neprilysin, NS3 / 4A, HCV-NS3 / 4, calpain, ADAMDEC1, renin, cathepsin C, cathepsin V / L2, cathepsin X / Z / P, cruzipain, otubain 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, marapsin, 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-LikeThese include proteases, AMSH, gamma secretase, antiplasmin cleaving enzyme (APCE), decysin 1, N-Acetylated Alpha-Linked Acidic Dipeptidase-Like 1 (NAALADL1), and furin.

[0085] From another perspective, the target tissue-specific protease can refer to a cancer tissue-specific protease or an inflamed tissue-specific protease.

[0086] Examples of cancer tissue-specific proteases include proteases that are specifically expressed in cancer tissues, such as those disclosed in International Publication Nos. WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846.

[0087] The more specific the type of cancer tissue-specific protease is expressed in the cancer tissue to be treated, the more effective it is in reducing side effects. The concentration of the cancer tissue-specific protease in cancer tissue is preferably at least 5 times higher than the concentration in normal tissue, more preferably at least 10 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, the activity of the cancer tissue-specific protease in cancer tissue is preferably at least 2 times higher than the activity in normal tissue, more preferably at least 3 times higher, more preferably at least 4 times higher, more preferably at least 5 times higher, even more preferably at least 10 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, the cancer tissue-specific protease may be one that is bound to the cell membrane of the cancer cell, or one that is not bound to the cell membrane and is secreted extracellularly. When the cancer tissue-specific protease is not bound to the cell membrane of the cancer cell, it is preferable that the cancer tissue-specific protease is present inside or near the cancer tissue so that cytotoxicity by immune cells is specific to the cancer cell. As used herein, "near the cancer tissue" means within a range in which the cancer tissue-specific protease cleavage sequence is cleaved and the effect of reducing ligand binding activity is exerted. However, it is preferable that the range be one in which normal cells are not damaged as much as possible. From another perspective, cancer tissue-specific proteases are (i) proteases that are expressed at higher levels in cancer tissues than in normal tissues; (ii) a protease that has higher activity in cancer tissue than in normal tissue; (iii) proteases that are expressed at higher levels in cancer cells than in normal cells; (iv) proteases that have higher activity in cancer cells than in normal cells; Either: The cancer tissue-specific protease may be used alone or in combination of two or more types. The number of types of cancer tissue-specific proteases can be appropriately determined by those skilled in the art, taking into consideration the type of cancer to be treated.

[0088] From the above viewpoints, among the proteases exemplified above, serine proteases and metalloproteases are preferred as cancer tissue-specific proteases, matriptase (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 more specific the type of inflammatory tissue-specific protease is expressed in the inflamed tissue of the treatment target, the more effective it is in reducing side effects. The concentration of the inflammatory tissue-specific protease in the inflamed tissue is preferably at least 5 times higher than the concentration in normal tissue, more preferably at least 10 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, the activity of the inflammatory tissue-specific protease in the inflamed tissue is preferably at least 2 times higher than the activity in normal tissue, more preferably at least 3 times higher, at least 4 times higher, at least 5 times higher, or at least 10 times higher, more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. Furthermore, the inflammatory tissue-specific protease may be bound to the cell membrane of inflammatory cells, or may be secreted extracellularly without being bound to the cell membrane. When the inflammatory tissue-specific protease is not bound to the cell membrane of inflammatory cells, it is preferable that the inflammatory tissue-specific protease is present inside or near the inflammatory tissue, so that cytotoxicity by immune cells is specific to inflammatory cells. As used herein, "near the inflammatory tissue" means within a range in which the inflammatory tissue-specific protease cleavage sequence is cleaved to exert the effect of reducing ligand binding activity. However, it is preferable that the range be within which normal cells are not damaged as much as possible. From another perspective, inflammatory tissue-specific proteases are (i) proteases that are expressed at higher levels in inflamed tissues than in normal tissues; (ii) proteases that have higher activity in inflamed tissue than in normal tissue; (iii) proteases that are expressed at higher levels in inflammatory cells than in normal cells; (iv) proteases that have higher activity in inflammatory cells than in normal cells; Either: The inflammatory tissue-specific protease may be used alone 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 condition of the target to be treated.

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

[0091] A protease cleavage sequence is a particular amino acid sequence that is specifically recognized by a target tissue-specific protease when a polypeptide is hydrolyzed by the target tissue-specific protease in an aqueous solution. From the viewpoint 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 or more specifically activated in the target tissue / cells to be treated. Specific examples of protease cleavage sequences include target sequences that are specifically hydrolyzed by the proteases specifically expressed in cancer tissues and proteases specific to inflammatory tissues, as disclosed in International Publication Nos. WO 2013 / 128194, WO 2010 / 081173, WO 2009 / 025846, etc. Artificially modified sequences, such as those obtained by introducing appropriate amino acid mutations into target sequences that are specifically hydrolyzed by known proteases, can also be used. Furthermore, protease cleavage sequences identified by methods known to those skilled in the art, such as those described in Nature Biotechnology 19, 661-667 (2001), may also be used. Furthermore, a naturally occurring protease cleavage sequence may be used, for example, a sequence in a protein that changes its molecular shape upon protease cleavage, such as the protease cleavage of TGF-β that converts it to its latent form.

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

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

[0094] [Table 1] TIFF2025114603000003.tif245150TIFF2025114603000004.tif245150TIFF2025114603 000005.tif245150TIFF2025114603000006.tif245150TIFF2025114603000007.tif24515 0TIFF2025114603000008.tif245150TIFF2025114603000009.tif245150TIFF2025114603 000010.tif231150TIFF2025114603000011.tif229153TIFF2025114603000012.tif55153

[0095] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1161) X1 to X8 each represent an amino acid, and 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; and X6 is 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.

[0096] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1162) X1 to X8 each represent an amino acid, and 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; and X7 is A, 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.

[0097] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1163) X1 to X8 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, 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; and X7 is an amino acid selected from A, D, E, 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.

[0098] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1164) X1 to X8 each represent an amino acid, and 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; and X7 is A, 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.

[0099] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1165) X1 to X8 each represent an amino acid, and 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; and X7 is A, 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.

[0100] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1166) X1 to X8 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, 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; and 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 protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1167) X1 to X8 each represent an amino acid, and 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; and X6 is 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.

[0102] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1168) X1 to X8 each represent an amino acid, and 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; and X6 is 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.

[0103] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1169) 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 protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1170) 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 protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1171) X1 to X9 each represent an amino acid, and 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; and X6 is 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; and X9 is an amino acid selected from A, G, H, I, L and R.

[0106] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1172) X1 to X9 each represent an 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; and X7 is A, 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.

[0107] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1173) 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, 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; and X7 is an amino acid selected from A, D, E, 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.

[0108] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1174) X1 to X9 each represent an amino acid, and 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; and X7 is A, 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 protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1175) X1 to X9 each represent an amino acid, and 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; and X7 is A, 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 protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1176) 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, 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; and X7 is A, D, E, F, G, 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.

[0111] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1177) X1 to X9 each represent an amino acid, and 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; and X6 is 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, 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; and X9 is an amino acid selected from A, G, H, I, L and R.

[0112] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1178) X1 to X9 each represent an amino acid, and 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; and X6 is 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; and X9 is an amino acid selected from A, G, H, I, L and R.

[0113] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1179) 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 protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1180) 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 protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1392) X1 to X11 each represent an 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, 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.

[0116] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1393) X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; 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 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; and 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 protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1394) X1 to X11 each represent an 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, 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 A, D, E, F, H, I, K, L, M, N, P, Q, R, 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.

[0118] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1395) X1 to X11 each represent an 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, 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.

[0119] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1396) X1 to X11 each represent an 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, 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, 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.

[0120] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1397) X1 to X11 each represent an 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 E, F, K, M, N, P, Q, R, 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.

[0121] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1398) X1 to X11 each represent an 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, X7 is an amino acid selected from A, D, F, G, L, M, P, Q, V, and W; and 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 protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1399) X1 to X11 each represent an 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, 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 protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1400) X1 to X11 each represent an 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; and X8 is an amino acid selected from H, V, and Y.

[0124] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1401) X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; 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.

[0125] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 1402) X1 to X11 each represent an 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, 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; and X9 is an amino acid selected from A, G, H, I, L and R.

[0126] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1403) X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; 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 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; and X9 is an amino acid selected from A, G, H, I, L and R.

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

[0128] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 1405) X1 to X11 each represent an 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, 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; and X9 is an amino acid selected from A, G, H, I, L and R.

[0129] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 1406) X1 to X11 each represent an 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, 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, 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; and X9 is an amino acid selected from A, G, H, I, L and R.

[0130] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1407) X1 to X11 each represent an 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 E, F, K, M, N, P, Q, R, 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; and X9 is an amino acid selected from A, G, H, I, L and R.

[0131] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1408) X1 to X11 each represent an 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, X7 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; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0132] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 1409) X1 to X11 each represent an 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, 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; and X9 is an amino acid selected from A, G, H, I, L and R.

[0133] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1410) X1 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.

[0134] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 1411) 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.

[0135] In addition to using the protease cleavage sequences described above, new protease cleavage sequences can be obtained through screening. For example, new protease cleavage sequences can be identified by altering the interactions between the cleavage sequence and the enzyme's active and recognition residues based on the results of crystal structure analysis of known protease cleavage sequences. New protease cleavage sequences can also be identified by modifying amino acids in known protease cleavage sequences and confirming their interaction with the protease. Another example is the discovery of sequences cleaved by proteases by displaying peptide libraries using in vitro display methods such as phage display and ribosome display, or by using peptide arrays immobilized on chips or beads to confirm their interaction with the protease. The interaction between a protease cleavage sequence and a protease can be confirmed by confirming protease cleavage in vitro or in vivo.

[0136] By quantifying the amount of cleaved fragments separated by electrophoresis such as SDS-PAGE after protease treatment, it is possible to evaluate the protease cleavage sequence, protease activity, and cleavage rate of a molecule into which a protease cleavage sequence has been introduced. A non-limiting example of a method for evaluating the cleavage rate of a molecule into which a protease cleavage sequence has been introduced is as follows. For example, when evaluating the cleavage rate of an antibody variant incorporating a protease cleavage sequence 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 antibody variant is incubated with 40 nM huPA or 3 nM hMT-SP1, 100 μg / mL PBS, and 37°C for 1 hour before being subjected to capillary electrophoresis immunoassay. Capillary electrophoresis immunoassays can be performed using Protein Simple (Wes), but are not limited to this method. Alternatively, detection can be performed by Western blotting after separation by SDS-PAGE or other methods. An anti-human lambda chain HRP-conjugated antibody (Abcam; ab9007) can be used to detect the light chain before and after cleavage, but any antibody capable of detecting cleaved fragments can be used. The area of each peak obtained after protease treatment can be output using Wes-specific software (Compass for SW; Protein Simple) to calculate the cleavage rate (%) of the modified antibody using the formula: (cleaved light chain peak area) * 100 / (cleaved light chain peak area + uncleaved light chain peak area). The cleavage rate can be calculated as long as protein fragments can be detected before and after protease treatment. This calculation is possible for various proteins, not just modified antibodies, that incorporate a protease cleavage sequence.

[0137] After administering a molecule incorporating 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 a modified antibody incorporating 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 modified antibody can be evaluated by subjecting it to capillary electrophoresis immunoassay. Capillary electrophoresis immunoassays can be performed using Protein Simple (Wes), but are not limited to this. Alternatively, Western blotting can be used after separation by SDS-PAGE or other methods. Light chains of modified antibodies recovered from mice can be detected using an anti-human lambda chain HRP-labeled antibody (abcam; ab9007), but any antibody capable of detecting cleavage fragments can be used. The area of each peak obtained by capillary electrophoresis immunoassay was output using Wes-specific software (Compass for SW; Protein Simple), and the remaining light chain ratio (light chain peak area) / (heavy chain peak area) was calculated, allowing the proportion of full-length light chain remaining uncleaved in the mouse body to be calculated. Calculation of in vivo cleavage efficiency is possible as long as protein fragments recovered from the body can be detected. This method allows for the calculation of cleavage rates for various proteins, including those containing protease cleavage sequences, in addition to modified antibodies. Calculating the cleavage rate using the above-described method makes it possible to compare the in vivo cleavage rates of modified antibodies containing different cleavage sequences, for example, and also to compare the cleavage rates of the same modified antibody between different animal models, such as normal mouse models and tumor-bearing 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 useful as protease substrates that are hydrolyzed by the action of proteases. Specifically, the present invention provides protease substrates comprising sequences selected from the sequences set forth in SEQ ID NOS: 1161-1180, 1392-1411, and the sequences set forth in Table 1. The protease substrates of the present invention can be used, for example, as a library for selecting substrates with desired properties for incorporation into ligand-binding molecules. Specifically, the protease sensitivity of ligand-binding molecules can be evaluated in order to selectively cleave them with proteases localized in lesions. After administration to a living body, ligand-binding molecules may contact various proteases and reach lesions. Therefore, it is desirable for the substrate to be sensitive to proteases localized in lesions while exhibiting as high resistance as possible to other proteases. To select a desired protease cleavage sequence depending on the purpose, the protease resistance of each protease substrate can be determined by comprehensively analyzing the sensitivity of each substrate to various proteases in advance. Based on the obtained protease resistance spectrum, a protease cleavage sequence having the required sensitivity and resistance can be found. Alternatively, ligand-binding molecules incorporating a protease cleavage sequence may reach the lesion site not only through the enzymatic action of a protease but also through various environmental stresses such as changes in pH, temperature, redox stress, etc. Based on information comparing the resistance of each protease substrate to such external factors, a protease cleavage sequence with desired properties can be selected according to the purpose.

[0139] In one embodiment of the invention, the protease cleavage sequence further comprises a flexible linker attached to either or both ends. The flexible linker at one end of the protease cleavage sequence can be referred to as a first flexible linker, and the flexible linker at the other end can be referred to as a second flexible linker. In certain embodiments, the protease cleavage sequence and flexible linker comprise one of the following formulas: (protease cleavage sequence) (first flexible linker)-(protease cleavage sequence) (protease cleavage sequence)-(second flexible linker) (first flexible linker)-(protease cleavage sequence)-(second flexible linker) In this embodiment, the flexible linker is preferably a peptide linker. The first and second flexible linkers are independently and optionally present and may be the same or different flexible linkers containing at least one flexible amino acid (e.g., Gly). For example, the linker may contain a sufficient number of residues to provide the desired protease accessibility to the protease cleavage sequence (amino acids 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, particularly Gly, etc.).

[0140] Flexible linkers suitable for use on either end of a protease cleavage sequence typically improve protease access to the protease cleavage sequence and increase the cleavage efficiency of the protease. Suitable flexible linkers can be easily selected and can range in length from 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, or 3 to 12 amino acids, including 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 flexible linker is a peptide linker of 1 to 7 amino acids.

[0141] Examples of flexible 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 an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Of these, glycine and glycine-serine polymers have attracted attention because these amino acids are relatively unstructured and tend to function as neutral tethers between components. Examples of flexible linkers made of glycine-serine polymers include, but are not limited to, 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, SEQ ID NO: 36) Gly·Gly·Ser·Gly (GGSG, SEQ ID NO: 37) Gly·Ser·Gly·Gly (GSGG, SEQ ID NO: 38) Ser·Gly·Gly·Gly (SGGG, SEQ ID NO: 39) Gly·Ser·Ser·Gly (GSSG, SEQ ID NO: 40) Gly·Gly·Gly·Gly·Ser (GGGGS, SEQ ID NO: 41) Gly·Gly·Gly·Ser·Gly (GGGSG, SEQ ID NO: 42) Gly·Gly·Ser·Gly·Gly (GGSGG, SEQ ID NO: 43) Gly·Ser·Gly·Gly·Gly (GSGGG, SEQ ID NO: 44) Gly·Ser·Gly·Gly·Ser (GSGGS, SEQ ID NO: 45) Ser·Gly·Gly·Gly·Gly (SGGGG, SEQ ID NO: 46) Gly·Ser·Ser·Gly·Gly (GSSGG, SEQ ID NO: 47) Gly·Ser·Gly·Ser·Gly (GSGSG, SEQ ID NO: 48) Ser·Gly·Gly·Ser·Gly (SGGSG, SEQ ID NO: 49) Gly·Ser·Ser·Ser·Gly (GSSSG, SEQ ID NO: 50) Gly·Gly·Gly·Gly·Gly·Ser (GGGGGS, SEQ ID NO: 51) Ser·Gly·Gly·Gly·Gly·Gly (SGGGGG, SEQ ID NO: 52) Gly·Gly·Gly·Gly·Gly·Gly·Ser (GGGGGGS, SEQ ID NO: 53) Ser·Gly·Gly·Gly·Gly·Gly·Gly·Gly (SGGGGGG, SEQ ID NO: 54) (Gly·Gly·Gly·Gly·Ser (GGGGS, SEQ ID NO: 41))n (Ser·Gly·Gly·Gly·Gly (SGGGG, SEQ ID NO: 46)) [n is an integer of 1 or more], etc. 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 an antibody VH and an antibody VL. Examples of ligand-binding molecules comprising a VH and a VL include, but are not limited to, Fv, scFv, Fab, Fab', Fab'-SH, F(ab'), and complete antibodies.

[0143] In some embodiments of the present invention, the ligand-binding molecule comprises an Fc region. When an Fc region of an IgG antibody is used, the type is not limited, and Fc regions such as IgG1, IgG2, IgG3, and IgG4 can be used. For example, an Fc region comprising a sequence selected from the amino acid sequences set forth in SEQ ID NOs: 55, 56, 57, and 58, or an Fc region mutant obtained by modifying these Fc regions, can be used. Furthermore, in some embodiments of the present invention, the ligand-binding molecule comprises an 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 via the 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 via the variable region, and ligand binding can be achieved via either or both of the two variable regions of the IgG antibody.

[0145] In some embodiments of the present invention, cleavage of a cleavage site / protease cleavage sequence in a ligand-binding molecule disrupts the domain in the ligand-binding molecule that has ligand-binding activity, thereby attenuating ligand binding. For example, when an IgG antibody is used as the ligand-binding molecule, an embodiment includes providing a cleavage site / protease cleavage sequence in the antibody variable region, such that in the cleaved state, the antibody variable region is no longer able to form a complete antibody variable region, thereby attenuating ligand binding.

[0146] As used herein, "association" can be rephrased as, for example, a state in which two or more polypeptide regions interact with each other. Generally, hydrophobic bonds, hydrogen bonds, ionic bonds, etc. are formed between the target polypeptide regions to form an association. As a commonly observed example of an association, it is known that in antibodies, such as natural antibodies, the heavy chain variable region (VH) and the light chain variable region (VL) maintain a paired structure through non-covalent bonds between them.

[0147] In some embodiments of the present invention, the VH and VL contained in the ligand-binding molecule are associated. Furthermore, the association between the antibody VH and VL can be dissolved, for example, by cleavage of the cleavage site / protease cleavage sequence. Dissolution of the association can be expressed, for example, as dissolving all or part of the interaction between two or more polypeptide domains. Dissolution of the association between VH and VL may mean dissolving all or part of the interaction between VH and VL. Ligand-binding molecules of the present invention include those in which the association between an antibody VL or a portion thereof and an antibody VH or a portion thereof in the ligand-binding molecule is abolished by cleavage of the cleavage site or by cleavage of the protease cleavage sequence with a protease.

[0148] In some embodiments of the present invention, a ligand-binding molecule comprises an antibody VH and an antibody VL, and when the cleavage site / protease cleavage sequence of the ligand-binding molecule is not cleaved, the antibody VH and antibody VL in the ligand-binding molecule are associated, and cleavage of the cleavage site / protease cleavage sequence eliminates the association of the 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 of the cleavage site / protease cleavage sequence can attenuate the binding of the ligand-binding molecule to a ligand.

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

[0150] In some embodiments of the present invention, a cleavage site / protease cleavage sequence is provided in a ligand-binding molecule comprising an antibody VH and an antibody VL, and a ligand-binding molecule is designed in which, before cleavage, all heavy-chain-light-chain interactions occur between the two peptides in the Fab structure, whereas upon cleavage of the cleavage site / protease cleavage sequence, the interaction between the peptide comprising VH (or a portion of VH) and the peptide comprising VL (or a portion of VL) is weakened and the association between VH and VL is dissolved.

[0151] In one embodiment of the present invention, the cleavage site / protease cleavage sequence is located in the antibody constant region. In a more specific embodiment, the cleavage site / protease cleavage sequence is located closer to the variable region than amino acid 140 (EU numbering) in the antibody heavy chain constant region, preferably closer to the variable region than 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 closer to the variable region than amino acid 130 (EU numbering) (Kabat numbering number 130) in the antibody light chain constant region, preferably closer to the variable region than amino acid 113 (EU numbering) (Kabat numbering number 113) in the antibody light chain constant region, or closer to the variable region than amino acid 112 (EU numbering) (Kabat numbering number 112) in the antibody light chain constant region. 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 number 108) to amino acid 131 (EU numbering) (Kabat numbering number 131) in the antibody light chain constant region.

[0152] In one embodiment of the present invention, the cleavage site / protease cleavage sequence is located in the antibody VH or VL. In a more specific embodiment, the cleavage site / protease cleavage sequence is located closer to the antibody constant region than amino acid 7 (Kabat numbering) of the antibody VH, preferably closer to the antibody constant region than amino acid 40 (Kabat numbering) of the antibody VH, more preferably closer to the antibody constant region than amino acid 101 (Kabat numbering) of the antibody VH, even more preferably closer to the antibody constant region than amino acid 109 (Kabat numbering) of the antibody VH, or closer to the antibody constant region than amino acid 111 (Kabat numbering) of the antibody VH. In a more specific embodiment, the cleavage site / protease cleavage sequence is located closer to the antibody constant region than amino acid 7 (Kabat numbering) of the antibody VL, preferably closer to the antibody constant region than amino acid 39 (Kabat numbering) of the antibody VL, more preferably closer to the antibody constant region than amino acid 96 (Kabat numbering) of the antibody VL, even more preferably closer to the antibody constant region than amino acid 104 (Kabat numbering) of the antibody VL, or closer to the antibody constant region than amino acid 105 (Kabat numbering) of the antibody VL. In some more specific embodiments, the cleavage site / protease cleavage sequence is introduced into antibody VH or VL at residues forming a loop structure or residues close to the loop structure. The loop structure in antibody VH or VL refers to a portion of antibody VH or VL that does not form a secondary structure such as an α-helix or β-sheet. The positions of residues forming a loop structure or residues close to the loop structure are specifically: from amino acid 7 (Kabat numbering) to amino acid 16 (Kabat numbering), 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 amino acid 95 (Kabat numbering). It can refer to the ranges from amino acid 7 (Kabat numbering) to amino acid 99 (Kabat numbering), amino acid 101 (Kabat numbering) to amino acid 113 (Kabat numbering), antibody VL amino acid 7 (Kabat numbering) to amino acid 19 (Kabat numbering), amino acid 39 (Kabat numbering) to amino acid 46 (Kabat numbering), amino acid 49 (Kabat numbering) to amino acid 62 (Kabat numbering), and 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 antibody VH sequence from amino acid 7 (Kabat numbering) to amino acid 16 (Kabat numbering), 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), or 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 antibody VL sequence from amino acid 7 (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), or 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 the antibody VH and the antibody constant region. Near the boundary between the antibody VH and the antibody heavy chain constant region refers to the region between amino acid 101 (Kabat numbering) of the antibody VH and amino acid 140 (EU numbering) of the antibody heavy chain constant region, preferably the region between amino acid 109 (Kabat numbering) of the antibody VH and amino acid 122 (EU numbering) of the antibody heavy chain constant region, or the region between amino acid 111 (Kabat numbering) of the antibody VH and amino acid 122 (EU numbering) of the antibody heavy chain constant region. Furthermore, when an antibody VH and an antibody light chain constant region are linked, the vicinity of the boundary between the antibody VH and the antibody light chain constant region can refer to the region between amino acid 101 (Kabat numbering) of the antibody VH and amino acid 130 (EU numbering (Kabat numbering 130)) of the antibody light chain constant region, and preferably refers to the region between amino acid 109 (Kabat numbering) of the antibody VH and amino acid 113 (EU numbering) (Kabat numbering 113) of the antibody light chain constant region, or can refer to the region between amino acid 111 (Kabat numbering) of the 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. Near the boundary between the antibody VL and the antibody light chain constant region can refer to the region between amino acid 96 (Kabat numbering) of the antibody VL and amino acid 130 (EU numbering) of the antibody light chain constant region (Kabat numbering number 130), preferably the region between amino acid 104 (Kabat numbering) of the antibody VL and amino acid 113 (EU numbering) of the antibody light chain constant region (Kabat numbering number 113), or the region between amino acid 105 (Kabat numbering) of the antibody VL and amino acid 112 (EU numbering) of the antibody light chain constant region (Kabat numbering number 112). When an antibody VL and an antibody heavy chain constant region are linked, the vicinity of the boundary between the antibody VL and the antibody heavy chain constant region can refer to the region between amino acid 96 (Kabat numbering) of the antibody VL and amino acid 140 (EU numbering) of the antibody heavy chain constant region, preferably the region between amino acid 104 (Kabat numbering) of the antibody VL and amino acid 122 (EU numbering) of the antibody heavy chain constant region, or the region 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 in a ligand-binding molecule, for example, at multiple locations selected from within the antibody constant region, within the antibody VH, within the antibody VL, near the boundary between the antibody VH and the antibody constant region, and near the boundary between the antibody VL and the antibody constant region. Furthermore, those skilled in the art who have read this invention can change the shape of a molecule comprising the antibody VH, antibody VL, and antibody constant region, for example by swapping the antibody VH and antibody VL, and such molecular shapes do not depart from the scope of the present invention.

[0156] As used herein, the term "ligand" refers to a biologically active molecule that typically functions by interacting with a cell surface receptor and thereby stimulating, inhibiting, or otherwise modulating biological activity, and typically appears to be involved in a signal transduction pathway within the cell that possesses the receptor.

[0157] As used herein, the term "ligand" encompasses a desired molecule that exhibits biological activity by interacting with a biomolecule. For example, the term "ligand" does not only refer to a molecule that interacts with a receptor, but also includes a molecule that exhibits biological activity by interacting with the molecule; for example, a receptor that interacts with the molecule and a binding fragment thereof are also included as a ligand. For example, a protein containing the ligand-binding site of a protein known as a receptor or the site where the receptor interacts with another molecule are included as ligands in the present invention. Specifically, soluble receptors, soluble fragments of receptors, the extracellular domain of a transmembrane receptor, and polypeptides containing them are included as ligands in the present invention.

[0158] The ligands of the present invention typically exert their desired biological activity by binding to one or more binding partners. The binding partner of a ligand can be an extracellular, intracellular, or transmembrane protein. In one embodiment, the binding partner of a ligand is an extracellular protein, e.g., a soluble receptor. In another embodiment, the binding partner of a ligand is a membrane-bound receptor. A ligand of the invention can specifically bind to its binding partner with a dissociation constant (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 inducers, PAMPs, DAMPs, nucleic acids, or fragments thereof. In particular embodiments, the ligand may be an interleukin, an interferon, a hematopoietic factor, a TNF superfamily member, a chemokine, a cell growth factor, a TGF-β family member, a myokine, an adipokines, or a neurotrophic factor. In more specific embodiments, the ligand may be 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 contain four characteristic cysteines (Cys) and are classified into CXC (alpha), CC (beta), C (gamma), and CX3C (delta) chemokine classes, depending on the motif represented by the first two cysteines. Two disulfide bonds are formed between the first and third cysteines and between the second and fourth cysteines. Disulfide bridges are generally considered necessary, and Clark-Lewis and coworkers reported that, at least for CXCL10, disulfide bonds are crucial for chemokine activity (Clark-Lewis et al., J. Biol. Chem. 269:16075-16081, 1994). The only exception to having four cysteines is lymphotactin, which has only two cysteine residues and thus manages to maintain a functional structure with only one disulfide bond. The CXC or alpha subfamily has been further divided into two groups: ELR-CXC chemokines and non-ELR-CXC chemokines, depending on the presence of an ELR motif (Glu-Leu-Arg) preceding the first cysteine (see, e.g., Clark-Lewis, supra, 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 tumor necrosis factor-α and is 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-γ-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. 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 in stem cell engraftment following transplantation (Nagasawa, T., Int. J. Hematol. 72:408-11, 2000), stem cell mobilization (Gazitt, Y., J. Hematother Stem Cell Res 10:229-36, 2001; Hattori et al., Blood 97:3354-59, 2001), and enhancing 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 activities of chemokines have 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, pp. 129-134; Raphaele, B. et al., "Calcium Mobilization," Methods in Molecular Biology (2000) vol. 138, pp. 143-148; Paul D. Ponath et al., "Transwell Chemotaxis," Methods in Molecular Biology (2000) vol. 138, pp. 113-120, Humana Press, Totowa, New Jersey).

[0163] The biological activities of CXCL10 include, for example, binding to the CXCL10 receptor (CXCR3), CXCL10-induced calcium flux, CXCL10-induced cell chemotaxis, CXCL10 binding to glycosaminoglycans, and CXCL10 oligomerization. The physiological activity of CXCL10 can be measured using a method that measures the cell migration activity of CXCL10, a Reporter assay using a CXCR3 stable expressing cell line (see PLoS One. 2010 Sep 13;5(9):e12700), and a PathHunter assay that utilizes B-Arrestin recruitment induced in the early stage of GPCR signaling. TM Examples include β-Arrestin recruitment assay.

[0164] Interleukin-12 (IL-12) is a heterodimeric cytokine consisting of disulfide-linked glycosylated polypeptide chains of 30 and 40 kD. The cytokine is 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 binds to IL-12 receptors expressed on the plasma membrane of cells (e.g., T cells, NK cells), thereby altering (e.g., initiating or blocking) biological processes. For example, IL-12 binding to the receptor stimulates the proliferation of preactivated T cells and NK cells, enhances the cytolytic activity of cytotoxic T cells (CTLs), NK cells, and lymphokine-activated killer (LAK) cells, induces the production of gamma interferon (IFNγ) by T cells 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 necessary for the generation of cytolytic cells (e.g., NK cells, CTLs) and for initiating cellular immune responses (e.g., Th1 cell-mediated immune responses). Thus, IL-12 is absolutely important in the generation and regulation of both preventive 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 assay, cell activation by IL-12 (cell surface marker expression, cytokine production, etc.), and promotion of cell differentiation by IL-12.

[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. 14:391-779-82; Bennett et al. (2003) J Immunol 170:711-8). The first members of this family, CD28 and ICOS, were discovered by 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 differential expression 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 differential expression in cytotoxic T lymphocytes and TH1 cells, respectively. CD28, ICOS, and CTLA-4 all contain unpaired cysteine residues that allow for 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 encodes a 55-kDa type I transmembrane protein that is part of the Ig gene superfamily. PD-1 contains a membrane-proximal immunoreceptor tyrosine-based inhibitory 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 important for B7-1 and B7-2 binding. Two ligands for PD-1, PD-L1 and PD-L2, have been identified, and binding to PD-1 has been shown to negatively regulate T cell activation (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 other CD28 family members. PD-L1, a ligand for PD-1, is abundant in various human cancers (Dong et al. (2002) Nat. Med. 8:787-9). PD-1 / PD-L1 interaction results in a reduction of tumor-infiltrating lymphocytes, reduced T cell receptor-mediated proliferation, and immune evasion by cancerous 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). Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1, and the effect is additive when the interaction of PD-2 with PD-L2 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 various autoimmune phenotypes, including autoimmune cardiomyopathy and lupus-like syndromes accompanied by 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, 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, ITSM of PD-1 inhibits BCR-mediated Ca 2+ It has been shown that ATP is essential for inhibiting the flow of ATP and the tyrosine phosphorylation of downstream effector molecules (Okazaki et al. (2001) PNAS 98:13866-71).

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

[0171] In some embodiments of the present invention, the ligand is a chemokine. Chemokines generally act as chemoattractants, recruiting immune effector cells to the site of chemokine expression. This may be beneficial for expressing specific chemokine genes, for example, along with cytokine genes, in order to recruit 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 are also known to have chemoattractant properties and may be classified under the term chemokine.

[0172] In some embodiments of the present invention, modified forms of 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. CXCL10, PD-1, IL-12, IL-6R, IL-1R1, IL-1R2, IL-1RAcP, and IL-1Ra may have the same sequence as naturally occurring CXCL10, PD-1, IL-12, IL-6R, IL-1R1, IL-1R2, IL-1RAcP, or IL-1Ra, or may be a variant that has a different sequence from naturally occurring CXCL10, PD-1, IL-12, IL-6R, IL-1R1, IL-1R2, IL-1RAcP, or IL-1Ra but retains the physiological activity of the corresponding naturally occurring ligand. To obtain a modified ligand, the ligand sequence may be artificially modified for various purposes, and preferably, a modified ligand is obtained by adding a modification that is not susceptible to 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. Non-limiting examples of embodiments in which the biological activity of a ligand is inhibited include, for example, embodiments in which the binding of the uncleaved ligand-binding molecule to the ligand substantially or significantly interferes with or competes with the binding of the ligand to its binding partner. When an antibody or a 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 preferred that the uncleaved ligand-binding molecule can sufficiently neutralize the biological activity of the ligand upon binding to the ligand. That is, the biological activity of the ligand bound to the uncleaved ligand-binding molecule is preferably lower than the biological activity of the ligand not bound to the uncleaved ligand-binding molecule. For example, but not limited to, 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, and 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 of 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 the ligand will not exert its biological activity before reaching the target tissue when the ligand-binding molecule is administered. Alternatively, the present invention provides a method for neutralizing the biological activity of a ligand. The method of the present invention comprises the steps of contacting a ligand whose biological activity is to be neutralized with a ligand-binding molecule of the present invention and recovering the binding product between the two. By cleaving the ligand-binding molecule of the recovered binding product, the biological activity of the ligand that has been neutralized thereby can be restored. In other words, the method of neutralizing the biological activity of a ligand of the present invention can further additionally comprise 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., to cancel 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 natural binding partner in vivo (e.g., a natural receptor for the ligand). For example, but not limited to, the binding activity of the cleaved ligand-binding molecule to the ligand is 90% or less, preferably 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, and 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 of the binding amount of the natural binding partner to the ligand in vivo (per unit binding partner). Any desired indicator of binding activity may be used, such as the dissociation constant (KD). When the dissociation constant (KD) is used as an index for evaluating binding activity, a larger KD of the cleaved ligand-binding molecule for the ligand compared to the KD of its natural binding partner in vivo indicates that the binding activity of the cleaved ligand-binding molecule for the ligand is weaker than that of the natural binding partner in vivo. The KD of the cleaved ligand-binding molecule for the ligand is, for example, 1.1-fold or more, preferably 1.5-fold or more, 2-fold or more, 5-fold or more, 10-fold or more, and particularly preferably 100-fold or more, compared to the KD of the natural binding partner in vivo. Since the cleaved ligand-binding molecule has only low or almost no binding activity for the ligand, it is expected that the ligand-binding molecule will be able to release the ligand after cleavage and will be prevented from rebinding to another ligand molecule.

[0177] After the ligand-binding molecule is cleaved, it is desirable that the inhibited biological activity of the ligand is restored. It is desirable that the binding of the cleaved ligand-binding molecule to the ligand is attenuated, thereby also attenuating the function of the ligand-binding molecule in inhibiting the biological activity of the ligand. Those skilled in the art can confirm the biological activity of a ligand using known methods, such as methods for detecting the binding of a ligand to its binding partner.

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

[0179] In some embodiments of the present invention, an uncleaved ligand-binding molecule is fused to a ligand to form a fusion protein, and the ligand-binding molecule portion and the ligand portion in the fusion protein further interact with each other through antigen-antibody binding. The ligand-binding molecule and the ligand can be fused via a linker or without a linker. Whether the ligand-binding molecule and the ligand in the fusion protein are fused via a linker or without a linker, the non-covalent bond between the ligand-binding molecule portion and the ligand portion still exists. In other words, even in embodiments in which the ligand-binding molecule is fused to the ligand, the non-covalent bond between the ligand-binding molecule portion and the ligand portion is similar to that in embodiments in which the ligand-binding molecule and the ligand are not fused. Cleavage of the ligand-binding molecule weakens the non-covalent bond, i.e., the bond between the ligand-binding molecule and the ligand is weakened. In a preferred embodiment of the present invention, the ligand-binding molecule and the ligand are fused via a linker. The linker used to fuse the ligand-binding molecule and the ligand may be 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), but in this embodiment, a peptide linker is preferred. 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. Examples of peptide linkers include, but are not limited to: 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, SEQ ID NO: 36) Gly·Gly·Ser·Gly (GGSG, SEQ ID NO: 37) Gly·Ser·Gly·Gly (GSGG, SEQ ID NO: 38) Ser·Gly·Gly·Gly (SGGG, SEQ ID NO: 39) Gly·Ser·Ser·Gly (GSSG, SEQ ID NO: 40) Gly·Gly·Gly·Gly·Ser (GGGGS, SEQ ID NO: 41) Gly·Gly·Gly·Ser·Gly (GGGSG, SEQ ID NO: 42) Gly·Gly·Ser·Gly·Gly (GGSGG, SEQ ID NO: 43) Gly·Ser·Gly·Gly·Gly (GSGGG, SEQ ID NO: 44) Gly·Ser·Gly·Gly·Ser (GSGGS, SEQ ID NO: 45) Ser·Gly·Gly·Gly·Gly (SGGGG, SEQ ID NO: 46) Gly·Ser·Ser·Gly·Gly (GSSGG, SEQ ID NO: 47) Gly·Ser·Gly·Ser·Gly (GSGSG, SEQ ID NO: 48) Ser·Gly·Gly·Ser·Gly (SGGSG, SEQ ID NO: 49) Gly·Ser·Ser·Ser·Gly (GSSSG, SEQ ID NO: 50) Gly·Gly·Gly·Gly·Gly·Ser (GGGGGS, SEQ ID NO: 51) Ser·Gly·Gly·Gly·Gly·Gly (SGGGGG, SEQ ID NO: 52) Gly·Gly·Gly·Gly·Gly·Gly·Ser (GGGGGGS, SEQ ID NO: 53) Ser·Gly·Gly·Gly·Gly·Gly·Gly·Gly (SGGGGGG, SEQ ID NO: 54) (Gly·Gly·Gly·Gly·Ser (GGGGS, SEQ ID NO: 41))n (Ser·Gly·Gly·Gly·Gly (SGGGG, SEQ ID NO: 46)) [n is an integer of 1 or more], etc. However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0180] The synthetic compound linker (chemical crosslinker) is a crosslinker commonly used for crosslinking peptides, such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES), and the like. These crosslinkers are commercially available.

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

[0182] As used herein, "treatment" (and its grammatical derivatives, such as "treat," "treating," etc.) refers to a clinical intervention intended to alter the natural course of the individual being treated and can be performed prophylactically or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the ligand-binding molecules of the present invention can regulate the biological activity of the ligand and are used to delay the onset of disease or slow the progression of disease.

[0183] In the present invention, the pharmaceutical composition generally refers to an agent for treating or preventing a disease, or for testing or diagnosing a disease. Furthermore, in the present invention, the term "pharmaceutical composition comprising a ligand-binding molecule" can be rephrased as "a method for treating a disease, comprising administering a ligand-binding molecule to a subject," or as "use of a ligand-binding molecule in the manufacture of a medicament for treating a disease." The term "pharmaceutical composition comprising a ligand-binding molecule" can also be rephrased as "use of a ligand-binding molecule for treating a disease." The term "pharmaceutical composition comprising a ligand-binding molecule and a ligand" can be rephrased as "a method for treating a disease, comprising administering the ligand-binding molecule and the ligand to a subject," or as "use of the ligand-binding molecule and the ligand in the manufacture of a medicament for treating a disease." The term "pharmaceutical composition comprising a ligand-binding molecule and a ligand" can be rephrased as "use of the ligand-binding molecule and the ligand for treating a disease." The term "pharmaceutical composition comprising a fusion protein" can be rephrased as "a method for treating a disease, comprising administering the fusion protein to a subject," or as "use of the fusion protein in the manufacture of a medicament for treating a disease." The term "pharmaceutical composition comprising a fusion protein" can be rephrased as "use of the 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 an individual binds to a ligand originally present in the individual in, for example, blood, tissue, etc., and is transported further in the body while bound to the ligand. The ligand-binding molecule delivered 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 exerts biological activity in the target tissue and can treat diseases caused by the target tissue. In embodiments in which the ligand-binding molecule suppresses the biological activity of the ligand when bound to the ligand and is cleaved specifically in the target tissue, the biological activity of the ligand is not exerted during delivery, but is only exerted after cleavage in the target tissue, allowing the disease to be treated and systemic side effects to be suppressed.

[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 in the body while the ligand remains bound. Once delivered to a target tissue, the ligand-binding molecule is cleaved in the target tissue, weakening its binding to the ligand and releasing the bound ligand in the target tissue. The released ligand exerts biological activity in the target tissue, allowing the treatment of diseases caused by the target tissue. In embodiments in which the ligand-binding molecule suppresses the biological activity of the ligand when bound to the ligand and is cleaved specifically in the target tissue, the biological activity of the ligand is not exerted during delivery, but is only exerted upon cleavage in the target tissue, thereby treating diseases and reducing systemic side effects. A ligand-binding molecule administered to an individual can bind not only to the ligand administered to the individual but also to a ligand originally present in the individual, and can transport the ligand originally present in the individual or the ligand administered to the individual in the body while still bound. Specifically, the present invention provides a method for producing a ligand conjugate, which comprises the steps of contacting a ligand-binding molecule with a ligand and recovering the complex consisting of the ligand-binding molecule and the ligand. The conjugate of the present invention can be prepared into a pharmaceutical composition by, for example, combining it with a pharmaceutically acceptable carrier.

[0186] In some embodiments of the present invention, a fusion protein comprising a ligand-binding molecule fused with a ligand can be administered to an individual. In some of these embodiments, the ligand-binding molecule and the ligand in the fusion protein form a fusion protein with or without a linker, but a non-covalent bond between the ligand-binding molecule portion and the ligand portion still exists. When a fusion protein comprising a ligand-binding molecule and a ligand fused with a ligand is administered to an individual, the fusion protein is delivered in vivo, 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 a portion of the ligand and the ligand-binding molecule are released 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, thereby treating a disease caused by the target tissue. In embodiments in which the ligand-binding molecule suppresses the biological activity of the ligand when bound to the ligand and the ligand-binding molecule is cleaved specifically in the target tissue, the biological activity of the ligand in the fusion protein is not exerted during delivery, but is exerted only after cleavage in the target tissue, thereby treating a disease and minimizing systemic side effects. Thus, in accordance with the present invention, there is provided a method of administering a ligand to a subject in need thereof, comprising the steps of: [1] contacting a ligand with the ligand conjugate of the present invention to obtain a conjugated product comprising the two; and [2] A step of administering the binding product of [1] to a subject in need of administration of the ligand.

[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 used parenterally in the form of injections of sterile solutions or suspensions in water or other pharmaceutically acceptable liquids. For example, they can be formulated by appropriately combining them with pharmacologically acceptable carriers or vehicles, specifically, sterile water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, etc., and blending them into unit dosage forms required for generally accepted pharmaceutical practice. The amount of active ingredient in these preparations is set so that an appropriate volume within the specified range is obtained.

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

[0189] Oily liquids include sesame oil and soybean oil, and may also contain benzyl benzoate and / or benzyl alcohol as a solubilizing agent. They may also contain buffers (e.g., phosphate buffer and sodium acetate buffer), soothing agents (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants. The prepared injection solution is usually filled into an appropriate ampule.

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

[0191] The administration method can be selected appropriately depending on the patient's age and symptoms. The dosage of a pharmaceutical composition containing a ligand-binding molecule can be set, for example, in the range of 0.0001 mg to 1,000 mg per kg of body weight per administration. Alternatively, the dosage can be set, for example, in the range of 0.001 to 100,000 mg per patient, although the present invention is not necessarily limited to these values. The dosage and administration method vary depending on the patient's body weight, age, symptoms, etc., but those skilled in the art can determine an appropriate dosage and administration method taking these conditions into consideration.

[0192] The present invention also relates to a method for producing a ligand-binding molecule whose binding to a ligand is attenuated in a cleaved state, or a fusion protein in which the ligand-binding molecule is fused with a ligand. In one embodiment, the present invention provides a method for producing a ligand-binding molecule or a fusion protein, which comprises introducing a protease cleavage sequence into a molecule capable of binding to a ligand.

[0193] Examples of methods for introducing a protease cleavage sequence into a molecule capable of binding to a ligand include a method in which a protease cleavage sequence is inserted into the amino acid sequence of a polypeptide capable of binding to a ligand, or a method in which part of the amino acid sequence of a polypeptide capable of binding to a ligand is replaced 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 it 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 part of amino acid sequence B with amino acid sequence A).

[0195] An example of a method for obtaining a molecule capable of binding to a ligand is to obtain a ligand-binding domain that has the ability to bind to the ligand. The ligand-binding domain can be obtained, for example, by a method using a known antibody production method. The antibody obtained by this production method may be used as is as the ligand-binding domain, or only the Fv domain of the obtained antibody may be used.If the Fv domain is a single chain (also referred to as "sc") that can recognize an antigen, only the single chain may be used.Alternatively, a Fab domain containing the Fv domain may also be used.

[0196] Specific methods for producing antibodies are well known to those skilled in the art. For example, monoclonal antibodies may be produced by hybridoma methods (Kohler and Milstein, Nature 256:495 (1975)), recombinant methods (U.S. Patent No. 4,816,567), or isolated from phage antibody libraries (Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1991)). Alternatively, antibodies may 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, humanized antibodies in which the CDRs of non-human animals, such as mouse antibodies, are grafted onto human antibodies are well known. Common genetic recombination techniques for obtaining humanized antibodies are also known. Specifically, overlap extension PCR is a well-known method for grafting the CDRs of a mouse antibody onto human FRs.

[0198] A vector for expressing a humanized antibody can be prepared by inserting DNA encoding an antibody variable region in which three CDRs and four FRs are linked together with DNA encoding a human antibody constant region into an expression vector so that they are fused in frame. After introducing the integration vector into a host to establish recombinant cells, the recombinant cells are cultured to express the DNA encoding the humanized antibody, and the humanized antibody is produced in the cultured cells (see European Patent Application Publication No. 239400 and International Publication No. WO 1996 / 002576).

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

[0200] Transgenic animals carrying the entire repertoire of human antibody genes (see WO 1993 / 012227, WO 1992 / 003918, WO 1994 / 002602, WO 1994 / 025585, WO 1996 / 034096, and WO 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 referred to as "scFv") by phage display. Phages expressing scFvs that bind to an antigen can be selected. The DNA sequence encoding the Fv region of a human antibody that binds to an antigen can be determined by analyzing the gene of the selected phage. After determining the DNA sequence of the scFv that binds to the antigen, the Fv region sequence can be fused in frame with the sequence of the C region of a desired human antibody and then inserted into an appropriate expression vector to prepare an expression vector. The expression vector is introduced into a suitable expression cell such as those listed above, and the gene encoding the human antibody is expressed to obtain the human antibody. These methods are already known (see WO 1992 / 001047, WO 1992 / 020791, WO 1993 / 006213, WO 1993 / 011236, WO 1993 / 019172, WO 1995 / 001438, WO 1995 / 015388).

[0202] A molecule capable of binding to a ligand, into which a protease cleavage sequence has been introduced, becomes a ligand-binding molecule of the present invention. Optionally, it is possible to confirm whether the ligand-binding molecule is cleaved by treatment with a protease corresponding to the protease cleavage sequence. For example, whether the protease cleavage sequence has been cleaved can be confirmed by contacting a molecule capable of binding to a ligand, into which a protease cleavage sequence has been introduced, with a protease and confirming the molecular weight of the product after protease treatment by electrophoresis such as SDS-PAGE.

[0203] Furthermore, by quantifying the amount of cleaved fragments after protease treatment separated by electrophoresis such as SDS-PAGE, it is possible to evaluate the protease activity and the cleavage rate of a molecule into which a protease cleavage sequence has been introduced. Non-limiting examples of methods for evaluating the cleavage rate of a molecule into which a protease cleavage sequence has been introduced include the following method. For example, when evaluating the cleavage rate of an antibody variant incorporating a protease cleavage sequence 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 antibody variant is incubated with 40 nM huPA or 3 nM hMT-SP1, 100 μg / mL PBS, and 37°C for 1 hour before being subjected to capillary electrophoresis immunoassay. Capillary electrophoresis immunoassays can be performed using Protein Simple (Wes), but are not limited to this method. Alternatively, detection can be performed by Western blotting after separation by SDS-PAGE or other methods. An anti-human lambda chain HRP-conjugated antibody (Abcam; ab9007) can be used to detect the light chain before and after cleavage, but any antibody capable of detecting cleaved fragments can be used. The area of each peak obtained after protease treatment can be output using Wes-specific software (Compass for SW; Protein Simple) to calculate the cleavage rate (%) of the modified antibody using the formula: (cleaved light chain peak area) * 100 / (cleaved light chain peak area + uncleaved light chain peak area). The cleavage rate can be calculated as long as protein fragments can be detected before and after protease treatment. This calculation is possible for various proteins, not just modified antibodies, that incorporate a protease cleavage sequence.

[0204] After administering a molecule incorporating 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 a modified antibody incorporating 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 modified antibody can be evaluated by subjecting it to capillary electrophoresis immunoassay. Capillary electrophoresis immunoassays can be performed using Protein Simple (Wes), but are not limited to this. Alternatively, Western blotting can be used after separation by SDS-PAGE or other methods. Light chains of modified antibodies recovered from mice can be detected using an anti-human lambda chain HRP-labeled antibody (abcam; ab9007), but any antibody capable of detecting cleavage fragments can be used. The area of each peak obtained by capillary electrophoresis immunoassay was output using Wes-specific software (Compass for SW; Protein Simple), and the remaining light chain ratio (light chain peak area) / (heavy chain peak area) was calculated, allowing the proportion of full-length light chain remaining uncleaved in the mouse body to be calculated. Calculation of in vivo cleavage efficiency is possible as long as protein fragments recovered from the body can be detected. This method allows for the calculation of cleavage rates for various proteins, including those containing protease cleavage sequences, in addition to modified antibodies. Calculating the cleavage rate using the above-described method makes it possible to compare the in vivo cleavage rates of modified antibodies containing different cleavage sequences, for example, and also to compare the cleavage rates of the same modified antibody between different animal models, such as normal mouse models and tumor-bearing mouse models.

[0205] The present invention also relates to polynucleotides encoding ligand-binding molecules whose binding to a ligand is weakened in a cleaved state, or polynucleotides encoding fusion proteins in which the ligand-binding molecule is fused with a ligand.

[0206] The polynucleotides of the present invention are typically carried (inserted) into an appropriate vector and introduced into host cells. There is no particular limitation on the vector, so long as it stably retains the inserted nucleic acid. For example, when Escherichia coli is used as the host, a preferred cloning vector is the pBluescript vector (Stratagene), although various commercially available vectors can also be used. When using a vector for the purpose of producing the ligand-binding molecule or fusion protein of the present invention, an expression vector is particularly useful. There is no particular limitation on the expression vector, so long as it expresses the ligand-binding molecule in a test tube, in Escherichia coli, in cultured cells, or in an individual organism. Preferred examples of the expression vector include the pBEST vector (Promega) for in vitro expression, the pET vector (Invitrogen) for Escherichia coli, the pME18S-FL3 vector (GenBank Accession No. AB009864) for cultured cells, and the pME18S vector (Mol Cell Biol. 8:466-472 (1988)) for individual organisms. The DNA of the present invention can be inserted into a vector by conventional methods, for example, by ligase reaction using a restriction enzyme site (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Sections 11.4-11.11).

[0207] The host cell is not particularly limited, and various host cells can be used depending on the purpose. Examples of cells for expressing a ligand-binding molecule or a fusion protein 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. Vectors can be introduced into host cells by known methods, such as calcium phosphate precipitation, electroporation (Current protocols in Molecular Biology, edited by Ausubel et al. (1987) Published by John Wiley & Sons, Sections 9.1-9.9), lipofectamine (GIBCO-BRL), and microinjection.

[0208] Appropriate secretion signals can be incorporated into the ligand-binding molecule or fusion protein of interest to secrete the ligand-binding molecule or fusion protein expressed in host cells into the lumen of the endoplasmic reticulum, the periplasmic space, or the extracellular environment. These signals can be endogenous or heterologous to the ligand-binding molecule or fusion protein of interest.

[0209] In the above production methods, the ligand-binding molecule or fusion protein of the present invention can be recovered by collecting the medium if the ligand-binding molecule or fusion protein of the present invention is secreted into the medium, or by lysing the cells if the ligand-binding molecule or fusion protein of the present invention is produced intracellularly, followed by recovering the ligand-binding molecule or fusion protein.

[0210] Ligand binding molecules or fusion proteins of the invention can be recovered and purified from recombinant cell culture using known methods, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite 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 there is no technical inconsistency based on the common general knowledge of those skilled in the art. Furthermore, an invention that excludes any combination of one or more embodiments described herein from the present invention should also be considered as the invention contemplated and described in this specification, provided that there is no technical inconsistency based on the common general knowledge of those skilled in the art. [Example]

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

[0213] Example 1: Issues with previously reported immunocytokines and protease-activated cytokines Immunocytokines targeting antigens expressed in cancer tissues have generally been produced by fusing the target cytokine to the terminus of a 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, local delivery of these cytokines to the tumor site using antibodies is expected to enhance efficacy while reducing side effects (Non-Patent Documents 4, 5, 6). However, these cytokines have limitations, such as insufficient clinical efficacy when administered systemically, a narrow therapeutic window, and high toxicity that precludes systemic administration. The primary reasons for this include the fact that even immunocytokines, when administered systemically, are exposed to the entire body and may exert toxicity through systemic action, or that only extremely low doses can be administered to avoid toxicity. Furthermore, immunocytokines that bind to cancer antigens are internalized by cancer cells and disappear within the tumor, making it difficult to expose the cytokines to the tumor site in some cases. There is also a report that the antitumor effect of an immunocytokine in which IL-2 is fused to an antibody that binds to a cancer antigen is the same as that of an immunocytokine in which IL-2 is fused to an antibody that does not bind to a cancer antigen (Non-Patent Document 7).

[0214] As a method to reduce the systemic effects of immunocytokines, a major issue, molecules have been reported in which the cytokine and cytokine receptor are linked by a linker that is cleaved by a protease highly expressed in cancer. Cytokines are inhibited by the cytokine receptor linked by the linker, but when the linker is cleaved by a protease, the cytokine receptor is released and the cytokine becomes active. Examples include a molecule in which TNFalpha and TNFR are linked by a linker that is cleaved by uPA (Non-Patent Document 8) and a molecule in which IL-2 and IL-2R are linked by a linker that is cleaved by MMP-2 (Non-Patent Document 9). However, in these molecules, the cytokines retain biological activity even before the linker is cleaved, and linker cleavage only increases activity by approximately 10-fold. This is due to two reasons: first, the affinity between cytokines and cytokine receptors is not strong, so that cytokines retain some activity even before protease cleavage; second, cytokine receptors can still bind to cytokines even after the linker is cleaved by proteases, thereby inhibiting the biological activity of the cytokines.

[0215] A molecule has been reported in which an anti-IL-2 scFv, instead of IL-2R, is linked to IL-2 via a linker that is cleaved by MMP-2 (Non-Patent Document 9). Considering that this molecule in which IL-2 and anti-IL-2 scFv are linked via a protease-cleavable linker also releases IL-2 upon linker cleavage, just like molecules in which a cytokine is linked to a cytokine receptor, it is natural to use an anti-IL-2 scFv with a weak affinity for IL-2. Furthermore, unlike the IgG-IL-2 fusion described above, these reported protease-activated cytokines lack an Fc region, which is expected to result in a short half-life, making it difficult to maintain high exposure. 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 extend the therapeutic window.

[0216] Example 2: Issues in applying chemokines to cancer immunotherapy Chemokines (Nature Immunology 9, 949-952 (2008)) are basic proteins that exert their effects via G protein-coupled receptors and are a group of cytokines. They act on specific leukocytes that express the receptor, causing the leukocytes to migrate in the direction of the substance's concentration gradient (chemotaxis) (Nat Cell Biol. 2016 Jan;18(1):43-53.). Chemokines are produced in large quantities at sites of inflammation and are known to induce leukocyte migration from within blood vessels into inflamed tissues. Since chemokine regulation can control leukocyte migration, it is thought that it could be used in cancer immunotherapy. If T cells, antigen-presenting cells, M1 macrophages, etc. can be migrated to the local area of solid tumors, it is thought that an anti-tumor effect can be induced. Cytokines can also exert their effects through systemic administration, but chemokines induce cell migration to tissues with high concentrations due to a concentration gradient, so the expected effect cannot be obtained by systemic administration of chemokines. Therefore, cancer immunotherapy (chemokine therapy) using systemic administration of chemokines is thought to be unrealistic.

[0217] Example 3: Concept of a ligand-binding molecule capable of releasing target tissue-specific ligands by introducing a protease cleavage sequence As shown in Examples 1 and 2, previously reported cytokine and chemokine therapies have the following problems. 1. In the case of immunocytokines, even if cytokines are targeted to solid tumors using antibodies, cytokines act throughout the body, causing side effects, or they can only be administered in low doses to avoid side effects, preventing high exposure within the tumor. 2. In the case of protease-activated cytokines, where a linker that can be cleaved by a protease connects the cytokine receptor (or antibody) and the cytokine, neutralization of cytokine activity is insufficient, and the cytokine retains some activity even before protease cleavage. 3. In the case of cytokines that are activated by proteases, the cytokine receptor (or antibody) can still bind to the cytokine even after the linker is cleaved by the protease, thereby inhibiting the biological activity of the cytokine. 4. In the case of cytokines activated by proteases, the half-life of the inactive cytokines is short and their retention time in the blood is short, so the required dosage is large.

[0218] To solve this problem, it was considered important to meet the following conditions: 1. In the whole body, ligands such as cytokines or chemokines are sufficiently inhibited (biological activity is minimized) by ligand-binding molecules. 2. The biological activity of the ligand is restored by protease cleavage (becoming an active ligand). 3. Ligand-binding molecules lose their ligand-binding activity due to cleavage by proteases. 4. The ligand that has been cleaved by a protease and has become active has a shorter half-life than the ligand bound to the ligand-binding molecule before being cleaved by a protease.

[0219] We have devised a pharmaceutical composition that satisfies the above conditions: a molecule whose binding to a ligand is weakened by cleavage of the cleavage site. First, a binding molecule for the ligand is obtained, and then the cleavage site is inserted into the binding molecule to create a ligand-binding molecule.

[0220] Example 4: Examples of anti-ligand antibodies incorporating protease cleavage sequences Figures 1, 2, and 3 show examples of molecules that use antibodies as molecules that bind to ligands. 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 anti-ligand antibody retains its ligand-binding activity even after the protease cleavage sequence is introduced. It is confirmed that the ligand dissociates by cleaving it with a protease while it is 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 of the anti-ligand antibody are linked via a linker, and a protease cleavage sequence is introduced near the interface 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. Systemic administration of this ligand-anti-ligand antibody fusion neutralizes the ligand, preventing its biological activity. Furthermore, the fusion possesses an Fc region, resulting in a long half-life. When the systemically administered ligand-anti-ligand antibody fusion is cleaved at the protease cleavage sequence near the interface between VH and CH1 by a protease highly expressed in tumor tissue, the VH molecule of the ligand-linker-anti-ligand antibody is released. Because VH or VL alone cannot bind to the ligand (both VH and VL are required for ligand binding), neutralization of the ligand is reversed, allowing it to exert its biological effect in tumor tissue. Furthermore, since the VH molecule of this released ligand-linker-antiligand antibody does not have an Fc region and has a small molecular weight, it has a very short half-life and is rapidly eliminated from the body, thereby 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 ligand is administered as a mixture with an anti-ligand antibody that has a protease cleavage sequence introduced near the boundary between VH and CH1. If the affinity of the anti-ligand antibody for the ligand is sufficiently strong and there is a sufficient amount of anti-ligand antibody 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 does not exert its biological activity. Furthermore, the ligand-anti-ligand antibody complex has a long half-life due to its Fc region. When the systemically administered ligand-anti-ligand antibody complex is cleaved at the protease cleavage sequence near the boundary between VH and CH1 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 for ligand binding), the neutralization of the ligand is reversed, allowing it to exert its biological effect in tumor tissue. Furthermore, since the released ligand molecule does not have an Fc region and has a small molecular weight, it has a very short half-life and is rapidly eliminated from the body, minimizing systemic side effects caused by the ligand. In Figure 3, an anti-ligand antibody with a protease cleavage sequence introduced near the boundary between VH and CH1 is systemically administered. The administered antibody binds to a ligand originally present in the body, and the subsequent process is the same as described above for Figure 2. In this way, by using an anti-ligand antibody with a protease cleavage sequence introduced near the boundary between VH and CH1, it is possible to selectively release the ligand in tissues where the protease is expressed, allowing it to exert its biological effect. If the ligand is a cytokine, the cytokine can be selectively activated in tissues where the protease is expressed. If the ligand is a chemokine, the chemokine is present at high concentrations in tissues where the protease is expressed, resulting in a low chemokine concentration in peripheral blood, allowing cells expressing the chemokine receptor to migrate to tissues where the protease is expressed.

[0221] Example 5: Production and evaluation of CXCL10-releasing antibodies 5-1. Introduction of a protease cleavage sequence into anti-CXCL10 neutralizing antibodies CXCL10 is a chemokine that induces migration of 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 those skilled in the art. MabCXCL10 was expressed and purified using a FreeStyle 293 (Life Technologies) by methods known to those skilled in 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), and 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) by amine coupling using NHS·EDC. The running buffer was 20 mM ACES, 0.05% Tween 20, 200 mM NaCl, pH 7.4. 1.563 nM human CXCL10 was applied as the analyte, and antibody binding was assessed at 37°C. Figure 4 shows the sensorgram representing the amount of binding over time, calculated as the difference from the blank value using running buffer alone. The starting point of the horizontal axis is the time at which the analyte flow began. The vertical axis represents the response (amount of binding) at each time point, with the response at the start of analyte flow set to 0. As shown in the sensorgram in Figure 4, binding of MabCXCL10 to human CXCL10 was confirmed. We investigated the insertion of a protease cleavage sequence near the boundary between the variable and constant regions of the heavy or light chain of MabCXCL10. Peptide sequence A (SEQ ID NO: 3), which has been reported to be cleaved by urokinase (uPA) and matriptase (MT-SP1), which are expressed specifically in cancer cells, was inserted into seven sites near the boundary between the variable and constant regions of the heavy or light chain. We also designed a variant that would not undergo glycosylation due to the insertion of the cleavage sequence. Expression vectors encoding 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), and EEIVHEG (SEQ ID NO: 14), and 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. These heavy chain variants were combined with native light chains, or native heavy chains and light chain variants, and protease cleavage sequences were inserted near the boundary between the heavy chain variable and constant regions to produce the following IgG1 antibodies: 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 SEQ ID NO: 7, light chain SEQ ID NO: 2), EEIVHE / EEIVL (heavy chain SEQ ID NO: 8, light chain SEQ ID NO: 2), EEIVHF / EEIVL (heavy chain SEQ ID NO: 9, light chain SEQ ID NO: 2), EEIVHG / EEIVL (heavy chain SEQ ID NO: 10, light chain SEQ ID NO: 2), EEIVHBG / EEIVL (heavy chain SEQ ID NO: 11, light chain SEQ ID NO: 2), EEIVHCG / EEIVL (heavy chain SEQ ID NO: 12, light chain SEQ ID NO: 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 SEQ ID NO: 17), EEIVH / EEIVLD (heavy chain SEQ ID NO: 1, light chain SEQ ID NO: 18), EEIVH / EEIVLE (heavy chain SEQ ID NO: 1, light chain SEQ ID NO: 19), EEIVH / EEIVLF (heavy chain SEQ ID NO: 1, light chain SEQ ID NO: 20), EEIVH / EEIVLG (heavy chain SEQ ID NO: 1, light chain SEQ ID NO: 21), and EEIVH / EEIVLEG (heavy chain SEQ ID NO: 1, light chain SEQ ID NO: 22) were transiently expressed 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.

[0222] 5-2. Evaluation of the binding activity of anti-CXCL10 neutralizing antibodies incorporating protease cleavage sequences The interaction between the antibody prepared in 5-1 and human CXCL10 (266-IP-010 / CF, R&D Systems) was evaluated using Biacore. The results are shown in Figure 6. 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. The running buffer was 20 mM ACES, 0.05% Tween 20, 150 mM NaCl, pH 7.4. Human CXCL10 (3.125, 1.563, and 0.781 nM) was run as the analyte, and antibody binding to the antigen was assessed at 25°C. The sensorgrams showing the amount of binding over time, calculated as the difference from a blank using running buffer alone, are shown in Figure 6. The horizontal axis represents the starting point of the analyte flow. The vertical axis represents the response (amount of binding) at each time point, with the response at the start of analyte flow set to 0. As shown in the sensorgrams in Figure 6, all antibodies bound to human CXCL10. In other words, we were able to insert a protease cleavage sequence near the boundary between the antibody variable and constant regions without losing antigen-binding activity.

[0223] 5-3. Evaluation of protease cleavage of anti-CXCL10 neutralizing antibodies incorporating protease cleavage sequences We tested whether the antibodies prepared in 5-1 could be cleaved by proteases. Using recombinant human matriptase / ST14 catalytic domain (MT-SP1) (R&D Systems, 3946-SE-010) as the protease, the antibodies were incubated for 20 hours at 37°C using 20 nM protease and 60 or 100 μg / mL antibody in PBS. After incubation, the protease cleavage was assessed by reducing SDS-PAGE. The results are shown in Figure 7. Following protease treatment, new bands between 25 kDa and 50 kDa were observed for EEIVHA / EEIVL, EEIVHE / EEIVL, EEIVHF / EEIVL, EEIVHG / EEIVL, EEIVHEG / EEIVL, and EEIVHBG / EEIVL. Furthermore, protease treatment of EEIVH / EEIVLEG, EEIVH / EEIVLF, and EEIVH / EEIVLG produced bands of 25 kDa or less, confirming 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 flexible linker sequence near the protease cleavage sequence of an anti-CXCL10 neutralizing antibody In 5-3, we investigated inserting a sequence containing a linker consisting of a glycine-serine polymer near the protease cleavage sequence of EEIVHC / EEIVL that was not cleaved by the recombinant human matriptase / 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 constructed using methods known to those skilled in the art. These heavy chain variants were combined with native light chains to insert a protease cleavage sequence near the boundary between the heavy chain variable and constant regions to produce the following IgG1 antibodies: 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 transiently expressed 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.

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

[0226] 5-6. Evaluation of protease cleavage of anti-CXCL10 neutralizing antibodies incorporating protease cleavage sequences and flexible linker sequences We examined whether the antibody prepared in 5-5 could be cleaved by proteases. Human urokinase (uPA) (R&D Systems, 1310-SE-010) and recombinant human matriptase / ST14 catalytic domain (MT-SP1) (R&D Systems, 3946-SE-010) were used as proteases. After incubation for 2 and 20 hours at 37°C with 12.5 nM protease and 133 μg / mL antibody in PBS, the antibody was cleaved by proteases using reducing SDS-PAGE. The results are shown in Figure 10. As a result, new bands between 25 kDa and 50 kDa were generated by protease treatment in EEIVHC002 / EEIVL, EEIVHC003 / EEIVL, EEIVHC004 / EEIVL, EEIVHC005 / EEIVL, and EEIVHEC006 / EEIVL, confirming that the antibodies in EEIVHC002 / EEIVL, EEIVHC003 / EEIVL, EEIVHC004 / EEIVL, EEIVHC005 / EEIVL, and EEIVHEC006 / EEIVL were cleaved by proteases. These results demonstrate that even antibodies that are not susceptible to protease cleavage when only a protease cleavage site is introduced near the boundary between the variable and constant regions, such as EEIVHC / EEIVL, can be made susceptible to protease cleavage by introducing a flexible linker sequence near the cleavage site. Therefore, it was demonstrated that antibodies that are susceptible to protease cleavage can be made by arbitrarily combining protease cleavage sites and flexible linkers.

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

[0228] 5-8. Generation of anti-CXCL10 neutralizing antibodies in which a portion of the amino acid sequence near the boundary between the antibody variable and constant regions is replaced with a protease cleavage sequence and a portion of the flexible linker sequence, and evaluation of protease cleavage We investigated replacing a portion of the amino acid sequence near the boundary between the variable and constant regions of the heavy chain of MabCXCL10 with a protease cleavage sequence and a portion of the flexible linker sequence. The heavy chain shown in Figure 12 was designed by substituting a portion of the amino acids in the heavy chain with peptide sequence A (SEQ ID NO: 3), a sequence reported to be cleaved by urokinase (uPA) and matriptase (MT-SP1), which are expressed in a cancer-specific manner. Expression vectors encoding the heavy chain variants EESVHA009 (SEQ ID NO: 59) and EESVHA012 (SEQ ID NO: 60) were constructed using methods known to those skilled in the art. These heavy chain variants were combined with native light chains 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 antibodies were transiently expressed 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. 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 matriptase / ST14 catalytic domain (MT-SP1) (R&D Systems, 3946-SE-010) were used as proteases. After incubation for 20 hours at 37°C with 12.5 nM protease and 100 μg / mL antibody in PBS, the protease cleavage was evaluated by reducing SDS-PAGE. The results are shown in Figure 13. After protease treatment, EESVHA009 / EEIVL and EESVHA012 / EEIVL showed new bands between 25 and 50 kDa. Therefore, it was confirmed that the antibodies in EESVHA009 / EEIVL and EESVHA012 / EEIVL were cleaved by proteases.

[0229] Example 6 Consideration of permissible sites for inserting cleavage sequences to eliminate antigen-binding ability by protease cleavage There has been a report on the creation of an antibody in which a protease cleavage sequence has been inserted just before aspartic acid 216 of the heavy chain of a human IgG1 antibody, and on its in vitro functional evaluation (International Publication No. WO2004 / 021861A2). Although no experimental data is provided, it is claimed that mixing this antibody with an antigen and then treating it with a medium containing the corresponding protease releases the antigen from the antigen-antibody complex. The amino acid at position 216 in the heavy chain of the human IgG1 antibody, in which the report claimed a protease cleavage sequence had been inserted, is not an aspartic acid according to any of the numbering systems (Kabat, E. et al., Sequences of Proteins of Immunological Interest, 5th edition)—Kabat, E. et al., Sequences of Proteins of Immunological Interest, 5th edition—which are the Kabat, EU, or OU numbering systems. However, according to a different reference, the amino acid at position 216 in the heavy chain of a human IgG1 antibody is thought to be the aspartic acid immediately following the cysteine that forms the disulfide bond 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 aspartic acid at position 216, the antibody cleaved by protease is thought to form a Fab region similar to that formed when the hinge region of an antibody is cleaved by papain. It is generally recognized that cleavage of the hinge region of an antibody by papain is unlikely to result in a loss of antigen-binding ability. Therefore, even if an antibody in which a protease cleavage sequence has been inserted immediately before aspartic acid 216 is cleaved with the corresponding protease, it is unlikely to lose its antigen-binding ability. Let us consider the case where a protease cleavage sequence is inserted immediately before amino acid 216 (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-terminal to cysteine 220 (Kabat numbering), where the disulfide bond between the heavy and light chains is formed. Therefore, the effects of protease cleavage of the heavy chain at this site are assumed to be similar to those of loss of the disulfide bond between the heavy and light chains. Previous literature suggests that antigen binding is unlikely to be lost even in Fab regions that are unable to form disulfide bonds 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) in the heavy chain of a human IgG1 antibody according to the Kabat numbering 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 by protease cleavage.

[0230] Example 7 Evaluation of migration activity following protease cleavage of anti-CXCL10 neutralizing antibody / CXCL10 complexes incorporating a protease cleavage sequence It was evaluated whether a complex formed between CXCL10 and the CXCL10 neutralizing antibody into which a protease cleavage sequence was introduced, prepared in Example 5, would release CXCL10 upon protease cleavage, and whether CXCL10 would exert cell migration activity. The cell migration activity of CXCL10 was investigated by preparing Ba / F3 transfectant cells expressing mouse CXCR3 (mCXCR3) (hereafter referred to as BaF3 / mCXCR3) and then measuring the activity of these cells with HTS Transwell. TMThe assay was performed using a 96-well permeable support with 5.0 μm pore polycarbonate membrane (Cat. 3387, Corning). Five analytes were prepared: CXCL10 + protease, EEIVHC006a / EEIVL + CXCL10, EEIVHC006a / EEIVL + CXCL10 + protease, EEIVHC006a / EEIVL + protease, and MabCXCL10 + CXCL10 + protease. In a ProteoSave SS 1.5 mL microtube (Cat. MS-4265M, Sumitomo Bakelite), antibody (MabCXCL10 or EEIVHC006a / EEIVL) at a final concentration of 10 μg / mL, hCXCL10 (Cat. 300-12, Peprotech) at a final concentration of 100 ng / mL, or both 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 added to a final concentration of 12.5 nM after the reaction. 235 μL of each analyte was transferred to the lower chamber, and 2.0 × 10 BaF3 / mCXCR3 cells were transferred to the upper chamber. 5 The cells were seeded at 75 μL / well to achieve a total of 100 cells / well, and the reaction was carried out for 6 hours. The reaction was carried out under 5% carbon dioxide gas at 37°C. After 6 hours of reaction, 100 μL of the solution in the lower chamber was transferred to a 96-well fluorescent luminescence plate (Cat. 3912, Corning) and analyzed by CellTiter-Glo. TM 100 μL of Luminescent Cell Viability Assay solution (Cat. G7571, Promega) was added. After incubation at room temperature for 10 minutes, luminescence was measured using a SpectraMax M3 multimode microplate reader (Molecular Devices) to assess cell migration into the lower chamber. The results are shown in Figure 14. Compared with the CXCL10 + protease analyte, the EEIVHC006a / EEIVL + CXCL10 analyte reduced luminescence intensity. The luminescence intensity reflected the amount of migrated cells, indicating that EEIVHC006a / EEIVL formed a complex with CXCL10 and neutralized the effect of CXCL10. On the other hand, the EEIVHC006a / EEIVL + CXCL10 + protease analyte restored luminescence intensity compared with the EEIVHC006a / EEIVL + CXCL10 analyte, demonstrating that it induces cell migration similarly to the CXCL10 + protease analyte. The luminescence intensity did not recover when the MabCXCL10 antibody lacking the cleavage sequence was added to the MabCXCL10 + CXCL10 + protease analyte. These results indicated that the ability of EEIVHC006a / EEIVL to neutralize CXCL10 was reduced following antibody cleavage by proteases.

[0231] Example 8 Evaluation of the migration activity following protease cleavage of an anti-CXCL10 neutralizing antibody-CXCL10 fusion protein incorporating a protease cleavage sequence 8-1 Preparation of anti-CXCL10 neutralizing antibody-CXCL10 fusion protein incorporating a protease cleavage sequence and evaluation of protease cleavage The light chain of MabCXCL10_G7 (heavy chain: G7H-G1T4 (sequence number: 368), light chain: G7L-LT0 (sequence number: 369)), a neutralizing antibody against human CXCL10, was used to design the ligand-fused light chain hCXCL10R75A.G4SGGGG.G7L-LT0 (sequence number: 371) by linking the human CXCL10 mutant hCXCL10R75A (sequence number: 370), which has been mutated to be protease-resistant, to the N-terminus of the light chain via a linker sequence consisting of a glycine-serine polymer. In addition, we designed a ligand-fused light chain, hCXCL10R75A.G7L.12aa0054-LT0 (sequence number: 372), in which a sequence (sequence number: 338) that is cleaved by urokinase (uPA) and matriptase (MT-SP1), which are expressed specifically in cancer, 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 sequence number: 368, ligand-fusion light chain sequence number: 371) and G7H-G1T4 / hCXCL10R75A.G7L.12aa0054-LT0 (heavy chain sequence number: 368, ligand-fusion light chain sequence number: 372). These were transiently expressed using Expi293 (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 of MabCXCL10_G7 are as follows: H-CDR1 (SFSIT, sequence number: 374), H-CDR2 (EITPMFGIANYAQKFQG, sequence number: 375), H-CDR3 (DGRFDVSDLLTDKPKVTINYNGMDV, sequence number: 376), L-CDR1 (SGSSSNIGSNTVN, sequence number: 377), L-CDR2 (NNDQRPS, sequence number: 378), L-CDR3 (ASWDDSLNGRV, sequence number: 379). We examined whether these fusion proteins could be cleaved by proteases. Human urokinase (huPA) (R&D Systems, 1310-SE-010) was used as the protease. Cleavage of the fusion proteins by proteases was assessed by reducing SDS-PAGE. 0.1 mg / ml of fusion protein was incubated with 30 nM huPA at 37°C for 1 hour, and then cleavage of the fusion proteins was assessed by reducing SDS-PAGE. G7H-G1T4 / hCXCL10R75A.G4SGGGG.G7L-LT0 was not cleaved by protease treatment, whereas G7H-G1T4 / hCXCL10R75A.G7L.12aa0054-LT0, which contained a protease cleavage sequence, generated a new band between 15 and 25 kDa upon protease treatment (Figure 15), confirming cleavage by protease treatment.

[0232] 8-2 Evaluation of the migration activity of anti-CXCL10 neutralizing antibody-CXCL10 fusion proteins containing a protease cleavage sequence following protease cleavage We evaluated whether an anti-CXCL10 neutralizing antibody-CXCL10 fusion protein, in which CXCL10 was fused with an anti-CXCL10 neutralizing antibody containing a protease cleavage sequence, could release CXCL10 by protease cleavage and induce cell migration. To compare the activity of hCXCL10R75A released from the fusion protein, hCXCL10R75A-His (SEQ ID NO: 373), which represents 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 had been mutated to be protease-resistant, to prepare the histidine-tagged human CXCL10 mutant hCXCL10R75A-His (SEQ ID NO: 373). hCXCL10R75A-His (SEQ ID NO: 373) was transiently expressed 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 by preparing Ba / F3 transfectant cells expressing mouse CXCR3 (mCXCR3) (hereafter referred to as BaF3 / mCXCR3) and then measuring the activity of these cells with HTS Transwell. TM -96 Permeable Support with 5.0 μm Pore Polycarbonate Membrane (Cat. 3387, Corning) was used for evaluation. The uPA(+) analyte was prepared in a 2.0 mL 96-well deep-well plate (Cat. P-DW-20-CS, Axygen) by adding recombinant huPA (Cat. 1310-SE, R&D Systems) to a final concentration of 30 nM to 0.15 μg / mL hCXCL10R75A-His, 1.5 μg / mL G7H-G1T4 / hCXCL10R75A.G4SGGGG.G7L-LT0 fusion protein lacking a protease cleavage sequence, or 1.5 μg / mL G7H-G1T4 / hCXCL10R75A.G7L.12aa0054-LT0 fusion protein containing a protease cleavage sequence. G7H-G1T4 / hCXCL10R75A.G4SGGGG.G7L-LT0 and G7H-G1T4 / hCXCL10R75A.G7L.12aa0054-LT0 (1.5 μg / mL) contain 0.15 μg / mL equivalent amounts of hCXCL10R75A. For uPA(-), we used 0.15 μg / mL hCXCL10R75A-His, 1.5 μg / mL G7H-G1T4 / hCXCL10R75A.G4SGGGG.G7L-LT0 (1.5 μg / mL), or 1.5 μg / mL G7H-G1T4 / hCXCL10R75A.G7L.12aa0054-LT0 (1.5 μg / mL) containing the protease cleavage sequence. 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. 5The cells were plated at 75 μL / well to achieve a total of 10 cells / well, and the reaction was carried out for 6 hours. The reaction was carried out under 5% carbon dioxide gas conditions at 37°C. After 6 hours of reaction, 100 μL of the solution in the lower chamber was transferred to an OptiPlate-96 (Cat. 6005299, PerkinElmer) and incubated with CellTiter-Glo. TM 100 μL of Luminescent Cell Viability Assay solution (Cat. G7571, Promega) was added. After 10 minutes of incubation at room temperature, luminescence intensity was measured using a SpectraMax M3 multimode microplate reader (Molecular Devices) to assess cell migration into the lower chamber. The results are shown in Figure 16. The luminescence intensity decreased with the addition of protease-untreated G7H-G1T4 / hCXCL10R75A.G7L.12aa0054-LT0 fusion protein compared to the addition of CXCL10R75A-His. Since the luminescence intensity reflects the amount of migrated cells, it was found that the bioactivity of CXCL10R75A in G7H-G1T4 / hCXCL10R75A.G7L.12aa0054-LT0 was neutralized. On the other hand, the addition of protease-treated G7H-G1T4 / hCXCL10R75A.G7L.12aa0054-LT0 restored luminescence intensity compared with untreated G7H-G1T4 / hCXCL10R75A.G7L.12aa0054-LT0, indicating that it induces cell migration equivalent to CXCL10R75A-His. The absence of the cleavage sequence in G7H-G1T4 / hCXCL10R75A.G4SGGGG.G7L-LT0 did not restore luminescence intensity even after protease treatment. These results suggest that the neutralizing activity of the antibody moiety in the fusion protein against CXCL10R75A is reduced upon protease cleavage.

[0233] Example 9: Preparation of anti-IL-12 neutralizing antibodies incorporating a protease cleavage sequence and a flexible linker sequence, and evaluation of IL-12 activation associated with protease cleavage 9-1. Construction of anti-IL-12 neutralizing antibodies incorporating protease cleavage sequences and flexible linker sequences IL-12 is a cytokine with immune stimulatory properties. IL-12 exerts antitumor effects by activating immune cells, but systemic exposure has also been reported to cause severe side effects (Nat Immunol. 2012 Jul 19;13(8):722-8.). A heavy chain variant of ustekinumab, 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 matriptase (MT-SP1), and a flexible linker consisting of a glycine-serine polymer, near the boundary between the variable and constant regions of the heavy chain of an anti-IL-12 antibody (UstkH-G1T4, heavy chain SEQ ID NO: 144), which has the same variable region as ustekinumab, a neutralizing antibody against human IL-12. This heavy chain variant, UstkH-G1T4CYTM1inP1 (SEQ ID NO: 146), was combined with the light chain of ustekinumab (UstkL-kT0, SEQ ID NO: 145) to prepare 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 those skilled in the art. This ustekinumab variant, UstkH-G1T4CYTM1inP1 / UstkL-kT0, was transiently expressed 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 variants 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), and L-CDR3 (QQYNIYPYT, SEQ ID NO: 391).

[0234] 9-2. Protease cleavage of anti-IL-12 neutralizing antibodies incorporating a protease cleavage sequence and a flexible linker sequence We examined whether the antibodies prepared in Section 9-1 above could be cleaved by proteases, including recombinant human matriptase / ST14 catalytic domain (human MT-SP1, hMT-SP1) (R&D Systems, 3946-SE-010), recombinant mouse matriptase / 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 its variant UstkH-G1T4CYTM1inP1 / UstkL-kT0 at final concentrations of 10.1, 16.9, or 9.17 μM, respectively, and incubating overnight at 37°C.

[0235] 9-3. Confirmation of cleavage of anti-IL-12 neutralizing antibodies incorporating cleaved protease cleavage sequences and flexible linker sequences and evaluation of IL-12 activation Cleavage of the antibody by proteases was evaluated by reducing SDS-PAGE. UstkH-G1T4 / UstkL-kT0 was not cleaved by each protease, whereas UstkH-G1T4CYTM1inP1 / UstkL-kT0, which contained a protease cleavage sequence and a flexible linker, produced a new band between 25 and 50 kDa upon protease treatment (Figure 17). Therefore, it was confirmed that the anti-IL-12 neutralizing antibody (UstkH-G1T4CYTM1inP1 / UstkL-kT0) containing the protease cleavage sequence and flexible linker sequence was cleaved by proteases. Next, we evaluated whether IL-12 is released from the antibody complex and exerts its physiological activity when the antibody is cleaved by protease. The physiological activity of IL-12 was evaluated based on the production of IFN-γ (interferon gamma, also referred to as IFN-g) by the NK92 human cell line. NK92 cells were cultured at 1 × 10 in a 96-well cell culture plate. 5Cells were seeded at 1000 cells / well. 10 ng / mL 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 was measured by ELISA 48 hours later. To evaluate the effect of antibodies on IL-12 activity, an experiment was also performed in which only protease-treated IL-12 was added without antibody (No Ab). Figure 18 shows the interferon gamma concentration. UstkH-G1T4 / UstkL-kT0 (without a protease cleavage sequence) treated with various proteases inhibited (neutralized) IL-12-induced interferon gamma production, reaching a similar level to that observed in the absence of IL-12 (No IL-12) at 0.8 μg / mL of antibody. On the other hand, UstkH-G1T4CYTM1inP1 / UstkL-kT0 (with a protease cleavage sequence) treated with various proteases produced more interferon gamma than UstkH-G1T4 / UstkL-kT0 without a protease cleavage sequence, regardless of antibody concentration. These results confirmed that UstkH-G1T4CYTM1inP1 / UstkL-kT0 allows IL-12 to act on cells by reducing its IL-12 neutralizing ability upon protease cleavage.

[0236] Example 10 Evaluation of antibodies obtained by introducing various protease cleavage sequences into anti-human CXCL10 neutralizing antibodies 10-1. Introduction of a protease cleavage sequence into anti-human CXCL10 neutralizing antibodies Expression vectors for MabCXCL10 (heavy chain: EEIVH (SEQ ID NO: 1), light chain: EEIVL (SEQ ID NO: 2)), an antibody that neutralizes CXCL10, and MabCXCL10_G7 (heavy chain: G7H-G1T4 (SEQ ID NO: 368), light chain: G7L-LT0 (SEQ ID NO: 369)) were prepared by methods known to those skilled in the art, and the antibodies were expressed and purified using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) by methods known to those skilled in the art. The cleavage sequences shown in Table 2 were inserted near the boundary between the variable and constant regions of the heavy chain of MabCXCL10 or MabCXCL10_G7 to prepare modified MabCXCL10 heavy chains. The sequences of the modified MabCXCL10 heavy chains with the protease cleavage sequences inserted are shown in Table 3.

[0237] [Table 2]

[0238] [Table 3]

[0239] The modified heavy chains and light chains in Table 3 were combined to produce the MabCXCL10 variants and MabCXCL10_G7 variants shown in Table 4. These variants were transiently expressed using FreeStyle293 cells (Invitrogen) or Expi293 cells (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.

[0240] [Table 4]

[0241] 10-2. Evaluation of protease cleavage of anti-human CXCL10 neutralizing antibodies containing multiple protease cleavage sequences in the heavy chain region We tested whether the antibodies prepared in 10-1 could be cleaved by proteases. Using recombinant human matriptase / ST14 catalytic domain (human MT-SP1, hMT-SP1) (R&D Systems, 3946-SE-010), the antibodies were incubated in PBS at 37°C for 20 hours with 10 nM protease and 50 μg / mL antibody for 20 hours. The results were then subjected to reducing SDS-PAGE. The results are shown in Figures 19A and 19B. For both the MabCXCL10 variants and the MabCXCL10_G7 variants shown in Table 4, a new band was generated at approximately 37 kDa upon treatment with hMT-SP1. This confirms that the protease cleavage sequences shown in Table 2 are cleaved by hMT-SP1. Furthermore, similar methods were used to confirm that the protease cleavage sequences shown in Table 2 are also cleaved by human uPA and mouse uPA.

[0242] Example 11: Preparation and evaluation of polypeptides incorporating cleavage sequences for various proteases 11-1 Construction of polypeptides incorporating recognition sequences for various proteases An expression vector 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, was prepared by a method known to those skilled in 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), and L-CDR3 (QQGNTLPYT, SEQ ID NO: 403). Table 5 shows peptide sequences known to be cleaved by MMP-2, MMP-7, and MMP-9, as well as peptide sequences containing a flexible linker consisting of a glycine-serine polymer adjacent to these sequences.

[0243] [Table 5]

[0244] These inserted sequences were inserted near the boundary between the heavy chain variable region and constant region of the MRA antibody to form 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. (heavy chain SEQ ID NO: 159) was designed, and expression vectors encoding these modified heavy chains were constructed by methods known to those skilled in the art. These modified heavy chains were combined with the MRA light chains to produce the MRA variants shown in Table 6. These variants were transiently expressed using FreeStyle293 cells (Invitrogen) or Expi293 cells (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.

[0245] [Table 6]

[0246] 11-2. Evaluation of protease cleavage of polypeptides incorporating various protease recognition sequences We investigated whether the modified MRA prepared in 11-1 could be cleaved by proteases. The proteases used were 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). 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. After incubation for 20 hours at 37°C using 50 nM, 100 nM, or 500 nM protease and 50 μg / mL antibody in 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 (hereafter referred to as Tris), protease cleavage was assessed by reducing SDS-PAGE. The results are shown in Figures 20A, 20B, and 21. The MRA modified antibodies were reacted with the proteases listed in Table 6. For MMP-2, cleavage occurred at MEIVHG4SMP2MP9G4S-MEIVHG4SMP2MP9G4SG1T4 / MRAL-k0, MEIVHG4SMP2.2G4S-MEIVHG4SMP2.2G4SG1T4 / MRAL-k0, MEIVHG4SMP2.4G4S-MEIVHG4SMP2.4G4SG1T4 / MRAL-k0, MEIVHMP2.1-MEIVHMP2.1G1T4 / MRAL-k0, and MEIVHMP2.3-MEIVHMP2.3G1T4 / MRAL-k0. For MMP-7, cleavage occurred at MEIVHMP7.2-MEIVHMP7.2G1T4 / MRAL-k0. For MMP-9, cleavage occurred at MEIVHG4SMP2MP9G4S-MEIVHG4SMP2MP9G4SG1T4 / MRAL-k0. Cleavage of MEIVHG4SMP9G4S-MEIVHG4SMP9G4SG1T4 / MRAL-k0 was observed.

[0247] Example 12 Evaluation of antibodies with protease cleavage sequences introduced at various positions in the heavy chain 12-1 Construction of antibodies with protease cleavage sequences 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 matriptase (MT-SP1), was inserted into different positions within the MRA heavy chain variable region (MRAH, SEQ ID NO: 161) to produce the MRA heavy chain variants shown in Table 7. These MRA heavy chain variable region variants were linked to the MRA heavy chain constant region (G1T4, SEQ ID NO: 162) to produce MRA heavy chain variants, 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 into different positions within the MRA heavy chain constant region (G1T4, SEQ ID NO: 162) to produce the MRA heavy chain constant region variants shown in Table 8. These MRA heavy chain constant region variants were linked to the MRA heavy chain variable region (MRAH, SEQ ID NO: 161) to produce MRA heavy chain variants, and expression vectors encoding the corresponding genes were prepared by methods known to those skilled in the art. The positions at which protease cleavage sequences were inserted in the prepared variants of the MRA heavy chain variable region and variants of the MRA heavy chain constant region are also shown in Tables 7 and 8. The insertion site in Table 7 refers to the position (Kabat numbering) in the antibody heavy chain variable region adjacent to the constant region, and the insertion site in Table 8 refers to the position (EU numbering) in the antibody heavy chain constant region adjacent to the variable region.

[0248] [Table 7] TIFF2025114603000019.tif135147

[0249] [Table 8]

[0250] The MRA heavy chain variants and MRA light chains prepared above were combined to produce the MRA variants shown in Table 9. These variants were transiently expressed using FreeStyle293 cells (Invitrogen) or Expi293 cells (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.

[0251] [Table 9] TIFF2025114603000022.tif229139

[0252] 12-2. Evaluation of protease cleavage of anti-human IL-6R neutralizing antibodies with protease cleavage sequences introduced into the antibody heavy chain We investigated whether the modified MRA prepared in 12-1 could be cleaved by proteases. Using recombinant human matriptase / ST14 catalytic domain (human MT-SP1, hMT-SP1) (R&D Systems, 3946-SE-010), the protease was 10 nM, and the antibody was 50 μg / mL in PBS at 37°C for 20 hours. The resulting mixture was then subjected to reducing SDS-PAGE. The results are shown in Figures 22A, 22B, 22C, 22D, 22E, 22F, 22G, 22H, and 22I, as well as Figures 23A, 23B, and 23C. After protease treatment, the heavy chain of the modified MRA was cleaved, and a heavy chain band appeared at a lower molecular weight than the heavy chain of the modified MRA that had not been treated with protease (a band appearing at approximately 50 kDa in the MT-SP1(-) lane in the figure). These results confirmed that the modified MRA prepared in 12-1 was cleaved by hMT-SP1.

[0253] Example 13 Evaluation of antibodies with protease cleavage sequences introduced at various positions in the light chain 13-1 Construction of antibodies with protease cleavage sequences introduced at various positions in the light chain Peptide sequence B (SEQ ID NO: 160), which has been reported to be cleaved by urokinase (uPA) and matriptase (MT-SP1), was inserted into different positions within the MRA light chain variable region (MRAL, SEQ ID NO: 230) to prepare the MRA light chain variants shown in Table 10. These MRA light chain variable region variants were each linked to the MRA light chain constant region (k0, SEQ ID NO: 231) to prepare MRA light chain variants, and expression vectors encoding the corresponding genes were prepared using methods known to those skilled in the art. Furthermore, peptide sequence B (SEQ ID NO: 160) was inserted into different positions within the MRA light chain constant region (k0, SEQ ID NO: 231) to prepare the MRA light chain constant region variants shown in Table 11. These MRA light chain constant region variants were each linked to the MRA light chain variable region (MRAL, SEQ ID NO: 230) to prepare MRA light chain variants, and expression vectors encoding the corresponding genes were prepared using methods known to those skilled in the art. The positions at which protease cleavage sequences are inserted in the prepared variants of the MRA light chain variable region and variants of the MRA light chain constant region are also shown in Tables 10 and 11. The insertion site in Table 10 refers to the position adjacent to the constant region of the indicated amino acid (Kabat numbering) in the antibody light chain variable region, and the insertion site in Table 11 refers to the position adjacent to the variable region of the indicated amino acid (EU numbering) in the antibody light chain constant region.

[0254] [Table 10] TIFF2025114603000024.tif74155

[0255] [Table 11]

[0256] The MRA light chain variants prepared above were combined with the MRA heavy chain to produce the MRA variants shown in Table 12. These were transiently expressed using FreeStyle293 cells (Invitrogen) or Expi293 cells (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.

[0257] [Table 12] TIFF2025114603000027.tif215125

[0258] 13-2. Evaluation of protease cleavage of anti-human IL-6R neutralizing antibodies containing a protease cleavage sequence in the antibody light chain variable region We investigated whether the modified MRA prepared in 13-1 could be cleaved by proteases. Recombinant human matriptase / ST14 catalytic domain (MT-SP1) (R&D Systems, 3946-SE-010) was used as the protease. After incubation for 20 hours at 37°C with 10 nM protease and 50 μg / mL antibody in PBS, the mixture was subjected to reducing SDS-PAGE. The results are shown in Figures 24A, 24B, 24C, 24D, and 24E, as well as Figures 25A and 25B. The modified MRA cleaved after protease treatment exhibited a light chain band at a lower molecular weight than the light chain of the untreated MRA (the band located around 25 kDa in the MT-SP1(-) lane in the figure).

[0259] Example 14: Preparation of anti-human PD-1 neutralizing antibodies incorporating a protease cleavage sequence and evaluation of binding to human PD-1 14-1. Introduction of a protease cleavage sequence into anti-human PD-1 neutralizing antibodies Neutralizing antibody 5C4H-G1T4 / 5C4L-KT0 against human PD-1 (heavy chain 5C4H-G1T4, SEQ ID NO: 297; heavy chain variable region 5C4H, SEQ ID NO: 300; heavy chain constant region G1T4, SEQ ID NO: 301; light chain 5C4L-KT0, SEQ ID NO: 298; light chain variable region 5C4L, SEQ ID NO: 302; light chain constant region KT0, SEQ ID NO: 303); Antibodies into which protease cleavage sequences have been introduced were prepared by inserting protease cleavage sequences into the heavy or light chains of H-CDR1 (NSGMH, SEQ ID NO: 392), H-CDR2 (VIWYDGSKRYYADSVKG, SEQ ID NO: 393), H-CDR3 (NDDY, SEQ ID NO: 394), L-CDR1 (RASQSVSSYLA, SEQ ID NO: 395), L-CDR2 (DASNRAT, SEQ ID NO: 396), and L-CDR3 (QQSSNWPRT, SEQ ID NO: 397). First, a peptide sequence (sequence number: 299) that has been reported to be cleaved by matriptase (MT-SP1), which is expressed specifically in cancer, was inserted into the heavy chain 5C4H-G1T4 or light chain 5C4L-KT0 of the aforementioned antibody to produce the heavy chain variants shown in Table 13 and the light chain variants shown in Table 14, which were then expressed by methods known to those skilled in the art.

[0260] [Table 13]

[0261] [Table 14]

[0262] An IgG1 antibody (Table 15) incorporating a protease cleavage sequence was prepared by combining a heavy chain variant in Table 13 with the light chain 5C4L-KT0, or a light chain variant in Table 14 with the heavy chain 5C4H-G1T4, and transiently expressed using Expi293 (Life Technologies) by a method known to those skilled in the art. Purification was carried out by a method known to those skilled in the art using Protein A. 5C4H-G1T4 / 5C4L-KT0 (heavy chain SEQ ID NO: 297, light chain SEQ ID NO: 298) was expressed and purified as a control antibody not containing a protease cleavage sequence.

[0263] [Table 15]

[0264] 14-2. Binding evaluation of anti-human PD-1 neutralizing antibodies containing protease cleavage sequences to human PD-1 14-2-1 Protease treatment For the protease-treated antibody, 10 μL of Recombinant Human Matriptase / ST14 Catalytic Domain (hMT-SP1, R&D systems 3946-SE-010) prepared at 1.8 μg / mL in PBS was added to the antibody prepared in 14-1 (final concentration 0.111 mg / mL). For the protease-untrea...

Claims

1. A polypeptide comprising a protease substrate, wherein the protease substrate comprises a sequence selected from the group consisting of SEQ ID NOs: 822, 823, 818-821, 824 and 830-836.

2. The polypeptide of claim 1, wherein the protease substrate is cleavable by human urokinase (huPA) or human matriptase (hMT-SP1).

3. 3. The polypeptide of claim 1 or 2, wherein the polypeptide comprises a biologically active moiety or is capable of binding to a biologically active moiety.

4. The polypeptide of claim 3, wherein the polypeptide comprises a transport moiety and a biological activity-inhibiting moiety.

5. The polypeptide of claim 4, wherein the transport moiety comprises an antibody constant region, or comprises an antibody constant region and a portion of the biological activity-inhibiting moiety.

6. The polypeptide of claim 4 or 5, wherein the biological activity-inhibiting portion comprises an antibody VH and an antibody VL.

7. 7. The polypeptide of claim 4, wherein the protease substrate is located between the biological activity-inhibiting moiety and the transport moiety.

8. A polypeptide described in any one of claims 4 to 7, wherein the biologically active portion is inhibited by binding to the uncleaved polypeptide and is activated or released by not binding to the cleaved polypeptide.

9. 9. The polypeptide of claim 4, wherein the binding of the cleaved polypeptide to the biologically active portion is weakened compared to the binding of the uncleaved polypeptide to the biologically active portion.

10. 10. The polypeptide of any one of claims 4 to 9, wherein the biologically active moiety is a cytokine or chemokine.

11. A polypeptide comprising: (a) a biologically active moiety or a binding moiety capable of binding to a biologically active moiety; (b) biological activity inhibiting moiety; (c) a transport moiety, the transport moiety comprising an antibody constant region or comprising a portion of the inhibitory moiety; (d) a protease substrate contained in the polypeptide of claim 1 or 2; wherein the protease substrate is positioned between the biological activity-inhibiting moiety and the delivery moiety.

12. The polypeptide of claim 11, wherein the biologically active moiety is a cytokine or a chemokine.

13. 13. The polypeptide of claim 11 or 12, wherein the biologically active portion is inhibited by binding to the uncleaved polypeptide and activated or released by not binding to the cleaved polypeptide.

14. 14. The polypeptide of claim 13, wherein the binding of the cleaved polypeptide to the biologically active portion is weakened compared to the binding of the uncleaved polypeptide to the biologically active portion.

15. A method for producing a polypeptide according to any one of claims 1 to 14, comprising the steps of culturing a host cell containing a polynucleotide encoding the polypeptide, and recovering the polypeptide from the cell.

16. 15. Use of a polypeptide according to any one of claims 1 to 14 to detect the presence of a protease which cleaves said protease substrate.

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