Ligand-binding molecules with tunable ligand-binding activity

Abstract

To provide ligand-binding molecules that selectively activate ligands such as cytokines or chemokines in target tissues; to pharmaceutical compositions comprising the ligand binding molecule; and to provide methods for producing the pharmaceutical composition.SOLUTION: Provided are: a ligand-binding molecule whose ligand-binding activity is attenuated by cleaving a cleavage site, and a method for producing the same; a complex formed by the ligand-binding molecule and a ligand; a fusion protein comprising the ligand-binding molecule and a ligand; and a pharmaceutical composition comprising the ligand-binding molecule or the fusion protein of the ligand-binding molecule and a ligand.SELECTED DRAWING: Figure 2

Description

[Technical field]

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

[0002] Antibodies are attracting attention as pharmaceuticals because they are highly stable in plasma and have few side effects. Many IgG-type antibody drugs are on the market, and many antibody drugs are currently being developed (Non-Patent Documents 1 and 2).

[0003] As cancer therapeutic drugs using antibody drugs, Rituxan, which targets the CD20 antigen, Cetuximab, which targets the EGFR antigen, and Herceptin, which targets the HER2 antigen, have been approved (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] In addition, a method is known in which a ligand having physiological activity such as a cytokine is delivered to a solid tumor by immunocytokines, which are antibodies that bind to cancer antigens highly expressed in cancer cells and are fused with the ligand. The cytokines delivered to solid tumors by immunocytokines activate the immune system and exert an antitumor effect. Since cytokines such as IL2, IL12, and TNF are highly toxic, it is expected that these cytokines can be delivered to the tumor site by antibodies to work locally in the tumor site, thereby reducing side effects and enhancing the effect (Non-Patent Documents 4, 5, 6). However, all of these have problems such as insufficient clinical efficacy when administered systemically, narrow therapeutic window, and high toxicity that prevents systemic administration, and have not yet been approved as a drug.

[0005] The main reason for this is that even if it is an immunocytokine, if it is administered systemically, it is exposed to the entire body, and therefore may act systemically and exert toxicity, or it can only be administered in extremely low doses to avoid toxicity. There is also a report that the antitumor effect was the same for an immunocytokine in which IL2 was fused to an antibody that binds to a cancer antigen and an immunocytokine in which IL2 was fused to an antibody that does not bind to a cancer antigen (Non-Patent Document 7).

[0006] As a method to avoid the above problems, a molecule has been reported in which a cytokine and a cytokine receptor are linked by a linker that is cleaved by a protease that is highly expressed in cancer. Cytokines are inhibited by the cytokine receptor linked by a linker, but when the linker is cleaved by a protease, the cytokine is released from the cytokine receptor and becomes active. For example, a molecule has been reported in which TNF-alpha and TNF-R are linked by a linker that is cleaved by uPA (Non-Patent Document 8), and a molecule has been reported in which IL2 and IL2R are linked by a linker that is cleaved by MMP2 (Non-Patent Document 9). However, in these molecules, the cytokines have activity even before the linker is cleaved, and cleavage of the linker only improves activity by about 10 times. In addition, a molecule has been reported in which an anti-IL2 scFv is linked to IL2 via a linker that is cleaved by MMP-2 instead of IL2R (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 view of the above circumstances, and one of its objects is to provide a ligand-binding molecule that selectively activates ligands such as cytokines or chemokines in target tissues, 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 intensive 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. The present inventors have also found that the ligand-binding molecule or a pharmaceutical composition containing the ligand-binding molecule is useful for treating a disease using the ligand, and that the ligand-binding molecule is useful for treating a disease including administering the ligand-binding molecule, and that the ligand-binding molecule is useful in the manufacture of a medicine for treating a disease. The present inventors have also 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 embodiments exemplified below. (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 of the molecule 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, when the cleavage site is cleaved, the ligand is released from the ligand-binding molecule. (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 comprising a sequence selected from the sequences set forth in SEQ ID NOs: 3, 34, 66, 70, 71, 72, 73, 35, 75, 76, and 345. (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 movable linker, or the protease cleavage sequence, the first movable linker, and the second movable 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 movable linker, or the protease cleavage sequence, the first movable linker, and the second movable 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 a 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 are 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), and 74 (Kabat numbering). The ligand-binding molecule according to (17), wherein the amino acid 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 movable linker, or the protease cleavage sequence, the first movable linker, and the second movable 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). (20) The ligand-binding molecule according to (13), wherein the cleavage site, or the protease cleavage sequence, or the protease cleavage sequence and a first movable linker, or the protease cleavage sequence, the first movable linker, and the second movable 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 movable linker, or the protease cleavage sequence, the first movable linker and the second movable 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 movable linker, or the protease cleavage sequence, the first movable linker, and the second movable 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 by 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, IL12, PD1, or IL6R. (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 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 VL 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 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) The 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), which 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, 356, and 367, or an antibody light chain selected from the sequences shown in SEQ ID NOs: 15 to 22. (30) The ligand is IL12, 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), which is an antibody comprising an antibody heavy chain shown in SEQ ID NO: 146. (32) The ligand is PD1, 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), which 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 having SEQ ID NO: 398, an H-CDR2 having SEQ ID NO: 399, and an H-CDR3 having SEQ ID NO: 400, and an antibody VL comprising an L-CDR1 having SEQ ID NO: 401, an L-CDR2 having SEQ ID NO: 402, and an L-CDR3 having 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), which 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), which 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 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 light chain 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) having 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 (42) or (43). (45) The ligand-binding molecule according to any one of (42) to (44), wherein the ligand is a variant of CXCL10 shown in SEQ ID NO: 370. (46) The ligand-binding molecule according to any one of (38) to (41), wherein the ligand is PD1, 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. (47) The ligand-binding molecule according to (46), wherein the cleavage site is contained in the antibody light chain or the antibody heavy chain. (48) The ligand-binding molecule according to (46) or (47), wherein the ligand is PD1, 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. (49) The ligand-binding molecule according to any one of (46) to (48), wherein the ligand is PD1 represented by SEQ ID NO: 320. (50) The ligand-binding molecule according to any one of (46) to (49), wherein the ligand is PD1, an antibody heavy chain contained in the ligand-binding molecule is fused to the ligand, and a series of polypeptides in which PD1 and the antibody heavy chain are fused include a sequence selected from the sequences shown in SEQ ID NOs: 323 and 324. (51) The ligand-binding molecule according to any one of (46) to (49), wherein the ligand is PD1, an antibody light chain contained in the ligand-binding molecule is fused to the ligand, and a series of polypeptides in which PD1 and the antibody light chain are fused include a sequence selected from the sequences shown in SEQ ID NOs: 325 to 334. (52) The ligand-binding molecule according to any one of (38) to (41), wherein the ligand is IL12, 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. (53) The ligand-binding molecule according to (52), wherein the cleavage site is contained in the antibody light chain or the antibody heavy chain. (54) The ligand-binding molecule according to (52) or (53), wherein the ligand is IL12, 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. (55) 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. (56) The ligand-binding molecule according to (55), wherein the cleavage site is contained in the antibody light chain or the antibody heavy chain. (57) The ligand-binding molecule according to (55) or (56), 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. (58) A complex formed by the ligand and the ligand-binding molecule according to any one of (1) to (36) that is bound to the ligand. (59) A fusion protein in which the ligand is fused to the ligand-binding molecule according to any one of (1) to (36). (60) The fusion protein according to (59), wherein the ligand-binding molecule does not bind to another ligand when fused to the ligand. (61) The fusion protein according to (59) or (60), wherein the ligand-binding molecule is fused to the ligand via a linker. (62) The fusion protein according to (61), wherein the linker does not contain a protease cleavage sequence. (63) The fusion protein according to (61) or (62), wherein the linker is a linker consisting of a glycine-serine polymer. (64) The fusion protein according to any one of (59) to (63), 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. (65) The fusion protein according to (64), wherein the cleavage site is contained in the antibody light chain or the antibody heavy chain of the ligand-binding molecule. (66) 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 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 light chain 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) having 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 (64) or (65). (67) The fusion protein according to any one of (64) to (66), wherein the ligand is a modified CXCL10 shown in SEQ ID NO: 370. (68) The fusion protein according to any one of (59) to (63), wherein the ligand is PD1, 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. (69) The ligand-binding molecule according to (68), wherein the cleavage site is contained in the antibody light chain or the antibody heavy chain. (70) The fusion protein according to (68) or (69), wherein the ligand is PD1, 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. (71) The fusion protein according to any one of (68) to (70), wherein the ligand is PD1 represented by SEQ ID NO: 320. (72) The fusion protein according to any one of (68) to (71), wherein the ligand is PD1, an antibody heavy chain contained in the ligand-binding molecule is fused to the ligand, and a series of polypeptides in which PD1 and the antibody heavy chain are fused include a sequence selected from the sequences shown in SEQ ID NOs: 323 and 324. (73) The fusion protein according to any one of (68) to (71), wherein the ligand is PD1, an antibody light chain contained in the ligand-binding molecule is fused to the ligand, and a series of polypeptides in which PD1 and the antibody light chain are fused include a sequence selected from the sequences shown in SEQ ID NOs: 325 to 334. (74) The fusion protein according to any one of (59) to (63), wherein the ligand is IL12, 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. (75) The fusion protein according to (74), wherein the cleavage site is contained in the antibody light chain or the antibody heavy chain. (76) The fusion protein according to (74) or (75), wherein the ligand is IL12, 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. (77) The fusion protein according to any one of (59) to (63), 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. (78) The fusion protein according to (77), wherein the cleavage site is contained in the antibody light chain or the antibody heavy chain. (79) The fusion protein according to (77) or (78), 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. (80) A pharmaceutical composition comprising the ligand-binding molecule according to any one of (1) to (57). (81) A pharmaceutical composition comprising a ligand-binding molecule according to any one of (1) to (37) and a ligand. (82) A pharmaceutical composition comprising the complex according to (58). (83) A pharmaceutical composition comprising the fusion protein according to any one of (59) to (79). (84) A method for producing a ligand-binding molecule according to any one of (1) to (57). (85) The method according to (84), which comprises introducing a protease cleavage sequence into a molecule capable of binding to a ligand. (86) A method for producing the fusion protein according to any one of (59) to (79), comprising fusing a ligand-binding molecule having a protease cleavage sequence to the ligand. (87) A polynucleotide encoding the ligand-binding molecule according to any one of (1) to (57). (88) A vector comprising the polynucleotide according to (87). (89) A host cell comprising the polynucleotide according to (87) or the vector according to (88). (90) A method for producing the ligand-binding molecule according to any one of (1) to (57), comprising the step of culturing the host cell according to (89). (91) A polynucleotide encoding the fusion protein according to any one of (59) to (79). (92) A vector comprising the polynucleotide according to (91). (93) A host cell comprising the polynucleotide according to (91) or the vector according to (92). (94) A method for producing the fusion protein according to any one of (59) to (79), comprising a step of culturing the host cell according to (93). The present invention also includes the embodiments described below. [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 of the molecule to the ligand is weakened when the molecule is cleaved at the at least one cleavage site. [2] The ligand-binding molecule of [1], wherein the cleavage site comprises a protease cleavage sequence. [3] The ligand-binding molecule according to [2], wherein the protease is a target tissue-specific protease. [4] The ligand-binding molecule according to any one of [1] to [3], wherein the ligand-binding molecule comprises an antibody VH, an antibody VL, and an antibody constant region. [5] The ligand-binding molecule according to [4], wherein the cleavage site or the protease cleavage sequence is located near the boundary between the antibody constant region and the antibody VH and / or near the boundary between the antibody constant region and the antibody VL. [6] The ligand-binding molecule according to [4] or [5], 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. [7] The ligand-binding molecule according to any one of [1] to [6], 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. [8] The ligand-binding molecule according to any one of [1] to [7], wherein the ligand is a cytokine or a chemokine. [9] The ligand-binding molecule of any one of [1] to [8], wherein the ligand-binding molecule is an IgG antibody.

[10] The ligand-binding molecule according to any one of [1] to [9], which is bound to the ligand.

[11] The ligand-binding molecule according to any one of [1] to [9], which is fused to the ligand.

[12] A pharmaceutical composition comprising the ligand-binding molecule according to any one of [1] to

[11] .

[13] A pharmaceutical composition comprising the ligand-binding molecule according to any one of [1] to

[10] and a ligand.

[14] A pharmaceutical composition comprising a fusion protein in which the ligand-binding molecule according to any one of [1] to [9] is fused to a ligand.

[15] A method for producing the ligand-binding molecule according to any one of [1] to

[11] . [Brief description of the drawings]

[0011] [Figure 1] Fig. 1 shows a fusion protein of an IgG antibody and a ligand, which comprises a VH molecule of a ligand-linker-anti-ligand antibody, that is specifically released in a target tissue, and one mode of its activation. The ligand and the anti-ligand antibody are linked by a linker. [Diagram 2] Figure 1 shows an IgG antibody that specifically releases a ligand in a target tissue, 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. [Diagram 3] This is a diagram showing an IgG antibody that specifically releases a ligand in a target tissue, 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 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]FIG. 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] FIG. 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 evaluating, using Biacore, 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 evaluating, using Biacore, 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), electrophoresed on reducing SDS-PAGE, and the degree of cleavage was evaluated by detection with Coomassie Brilliant Blue (CBB). Of the two new bands generated by protease treatment, the band at approximately 15 kDa is derived from VH, and the band appearing at 25-50 kDa is derived from the constant region. [Figure 7-2] (A) is a continuation of the figure, and (B) shows the results of evaluating the degree of cleavage by reducing SDS-PAGE after treating an antibody molecule created by inserting protease cleavage sequences into the variable and constant regions of the light chain of MabCXCL10 with a protease (MT-SP1). Two new bands derived from the light chain cleaved by the protease treatment are observed. [Figure 7-3] This is a continuation of (B). [Figure 8]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 at which 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 using Biacore to evaluate the interaction between 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 and human CXCL10. [Figure 10A] The antibody molecule was prepared 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. The antibody molecule was treated with proteases (uPA, MT-SP1), electrophoresed on reducing SDS-PAGE, and the degree of cleavage was evaluated by detection with CBB. Of the two new bands generated by protease treatment, the band at approximately 15 kDa is derived from VH, and the band appearing at 25-50 kDa is derived from the constant region. [Figure 10B] This is a continuation of Figure 10A. [Figure 11A] FIG. 1 shows the results of evaluating whether CXCL10 is released by treating the 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 a part of the amino acid sequence near the boundary between the variable and constant regions of MabCXCL10 with a protease cleavage sequence and a movable linker sequence, the site where the amino acid was inserted or modified, 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 are those that have been removed during insertion of the inserted sequence, i.e., replaced with the amino acid at the most C-terminus of the inserted sequence. [Figure 13] The antibody molecule was prepared by replacing a 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. The antibody molecule was treated with proteases (uPA, MT-SP1), electrophoresed on reducing SDS-PAGE, and the degree of cleavage was evaluated by detection with CBB. Of the two new bands generated by protease treatment, the band at approximately 15 kDa is derived from 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 cleavage of the CXCL10-anti-CXCL10 antibody fusion protein with protease. [Figure 16] FIG. 1 shows luciferase activity (luminescence value). [Figure 17] FIG. 13 is a diagram of reducing SDS-PAGE 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 is a graph showing 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 the addition of an 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 cleaving modified MRA with a protease. [Figure 22B] FIG. 1 shows the results of cleaving modified MRA with a protease. [Figure 22C] FIG. 1 shows the results of cleaving modified MRA with a protease. [Figure 22D] FIG. 1 shows the results of cleaving modified MRA with a protease. [Figure 22E] FIG. 1 shows the results of cleaving modified MRA with a protease. [Figure 22F] FIG. 1 shows the results of cleaving modified MRA with a protease. [Figure 22G] FIG. 1 shows the results of cleaving modified MRA with a protease. [Fig. 22H] FIG. 1 shows the results of cleaving modified MRA with a protease. [Figure 22I] FIG. 1 shows the results of cleaving modified MRA with a protease. [Figure 23A] FIG. 1 shows the results of cleaving modified MRA with a protease. [Figure 23B] FIG. 1 shows the results of cleaving modified MRA with a protease. [Figure 23C] FIG. 1 shows the results of cleaving modified MRA with a protease. [Figure 24A] FIG. 1 shows the results of cleaving modified MRA with a protease. [Figure 24B] FIG. 1 shows the results of cleaving modified MRA with a protease. [Figure 24C] FIG. 1 shows the results of cleaving modified MRA with a protease. [Figure 24D] FIG. 1 shows the results of cleaving modified MRA with a protease. [Figure 24E] FIG. 1 shows the results of cleaving modified MRA with a protease. [Figure 25A] FIG. 1 shows the results of cleaving modified MRA with a protease. [Figure 25B] FIG. 1 shows the results of cleaving modified MRA with a protease. [Figure 26] This figure compares real-time graphs showing the binding of PD1 to 5C4-bio in binding assessment samples containing protease-treated / protease-untreated antibodies and PD1. The thick black line is the binding assessment sample containing protease-treated antibody, and the thin gray line is the binding assessment sample containing 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 contained in the assessment sample, and in the None (antigen only) graph, only the 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 is a comparison of real-time graphs showing the binding of protease-treated and untreated antibodies to PD1. The thick black line shows the protease-treated antibody, and the thin gray line shows the protease-untreated antibody. The X-axis shows the measurement time (seconds), with the start of measurement set to 0 seconds. The Y-axis shows binding. The name of each graph indicates the antibody used, and 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 PD1 to 5C4-bio in a sample that was protease-treated in the presence of PD1 and a sample that was not protease-treated in the presence of PD1. The thick black line indicates the sample that was protease-treated, and the thin gray line indicates the sample that was not protease-treated. The X-axis indicates the measurement time (seconds), with the start of the measurement set to 0 seconds. The Y-axis indicates binding. The name of each graph indicates the antibody contained in the sample. In the "Antigen and protease" graph, the sample contained only PD1 and no antibody. [Diagram 30] A comparison of real-time graphs showing the binding of free PD1 to 5C4-bio in protease-treated fusion protein and protease-untreated protein solutions. The thick black line indicates the protease-treated sample, and the thin grey line indicates the non-protease-treated sample. The X-axis indicates the measurement time (seconds), with the start of the measurement at 0 seconds. The Y-axis indicates binding. The name of each graph indicates the fusion protein, and in the graph marked "None (antigen only)," no fusion protein was used as the evaluation sample, and only the antigen PD1 was used. In the graph marked "5C4H-G1T4 / 5C4L-KT0," no fusion protein was used, and only the 5C4H-G1T4 / 5C4L-KT0 antibody was used. [Diagram 31] Electrophoresis results of protease-treated antibody-PD1 fusion proteins. The protease (+) lane shows the protease-treated fusion protein, and the protease (-) lane shows the protease-untreated fusion protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The polypeptide of the present invention generally refers to a peptide or protein having a length of about 4 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, and may be, for example, a polypeptide derived from an 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.

[0013] As used herein, amino acids are represented by one-letter or three-letter codes, or both, e.g., Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, Val / V.

[0014] For modifying 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. In addition, as a method for modifying an amino acid by substituting an amino acid other than a natural amino acid, several known methods can also be used (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 a complementary amber suppressor tRNA of the UAG codon (amber codon), which is one of the stop codons, can be suitably used.

[0015] As used herein, the meaning of the term "and / or" used to describe the site of amino acid modification 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.

[0016] In the present specification, as an expression for an amino acid modification, an expression in which the one-letter code or three-letter code of the amino acid before and after the modification is written before and after a number representing a specific position can be appropriately used. For example, the modification F37V or Phe37Val used when adding a substitution of an amino acid contained in an antibody variable region represents a substitution of Phe at position 37 represented by Kabat numbering with Val. That is, the number represents the position of the amino acid represented by Kabat numbering, the one-letter code or three-letter code of the amino acid written before it represents the amino acid before the substitution, and the one-letter code or three-letter code of the amino acid written after it represents the amino acid after the substitution. Similarly, the modification P238A or Pro238Ala used when adding an amino acid substitution to the Fc region contained in an antibody constant region represents a substitution of Pro at position 238 represented by EU numbering with Ala. That is, the number represents the position of the amino acid represented by EU numbering, the one-letter code or three-letter code of the amino acid written before it represents the amino acid before the substitution, and the one-letter code or three-letter code of the amino acid written after it represents the amino acid after the substitution.

[0017] 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, and refers to a molecule capable of binding to a ligand.

[0018] The ligand-binding molecule of the present invention is a molecule capable of binding to a ligand, particularly a molecule capable of binding to a ligand in an uncleaved state. The term "binding" as used herein generally refers to binding by interactions mainly based on non-covalent bonds such as electrostatic forces, van der Waals forces, and hydrogen bonds. Suitable examples of the ligand-binding mode of the ligand-binding molecule of the present invention include, but are not limited to, antigen-antibody reactions in which an antigen-binding region, an antigen-binding molecule, an antibody, and an antibody fragment bind to an antigen.

[0019] In addition, being able to bind to a ligand means that the ligand-binding molecule can bind to the ligand even if the ligand-binding molecule and the ligand are different molecules, and does not mean that the ligand-binding molecule and the ligand are connected by a covalent bond. For example, being able to bind to a ligand is not said just because a ligand and a ligand-binding molecule are covalently bonded via a linker. Furthermore, being attenuated in binding to a ligand means that the ability to bind is attenuated. For example, when a ligand and a ligand-binding molecule are covalently bonded via a linker, cleavage of the linker cannot be considered to be attenuating the binding to the ligand. 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 able to bind to the ligand.

[0020] 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 may be used as long as it is capable of binding 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 (sdAbs), modules called A domains of about 35 amino acids contained in Avimers, which are cell membrane proteins present in living organisms (International Publication Nos. WO2004 / 044011 and WO2005 / 040229), Adnectins containing the 10Fn3 domain, which is a domain that binds to proteins in fibronectin, a glycoprotein expressed in cell membranes (International Publication No. WO2002 / 032925), Affibodies using as a scaffold an IgG-binding domain that constitutes a bundle of three helices consisting of 58 amino acids of Protein A (International Publication No. WO1995 / 001937), ankyrin repeats having a structure in which a turn containing 33 amino acid residues and two antiparallel helices and loop subunits are repeatedly stacked, and the like. 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 WO2002 / 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 WO2003 / 029462); and a concave region of a parallel sheet structure inside a horseshoe-shaped structure in which leucine-rich-repeat (LRR) modules are repeatedly stacked in the variable lymphocyte receptor (VLR) that does not have an immunoglobulin structure and serves as the adaptive immune system of jawless fish such as lampreys and hagfish (International Publication WO2008 / 016854).

[0021] 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.

[0022] 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 appropriately produced according to the following examples.

[0023] Anti-IL-6R antibodies can be obtained as polyclonal or monoclonal antibodies using known means. As anti-IL-6R antibodies, monoclonal antibodies derived from mammals can be preferably produced. Mammalian-derived monoclonal antibodies include those produced by hybridomas and those produced by host cells transformed with an expression vector containing an antibody gene by genetic engineering techniques. Antibodies referred to in the present application include "humanized antibodies" and "chimeric antibodies".

[0024] 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. Hybridomas producing anti-IL-6R antibodies can then be selected by screening for monoclonal antibody-producing cells using a conventional screening method.

[0025] Specifically, monoclonal antibodies are produced, for example, as follows. First, an IL-6R protein to be used as a sensitizing antigen for obtaining antibodies can be obtained by expressing the IL-6R gene. That is, 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, a soluble IL-6R is expressed as described by Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968). Purified natural IL-6R protein can also be used as a sensitizing antigen.

[0026] The purified IL-6R protein can be used as a sensitizing antigen for immunizing a mammal. A partial peptide of IL-6R can also be used as a sensitizing antigen. In this case, the partial peptide can be obtained by chemical synthesis from the amino acid sequence of human IL-6R. It can also be obtained by incorporating a part of the IL-6R gene into an expression vector and expressing it. It can also be obtained by decomposing 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 to be 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.

[0027] In addition, a fusion protein in which a desired partial polypeptide or peptide of the IL-6R protein is fused 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 produced by fusing genes encoding two or more desired polypeptide fragments in frame and inserting the fusion gene into an expression vector as described above. A method for producing a fusion protein is described in Molecular Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58 (1989) Cold Spring Harbor Lab. press). A method for obtaining IL-6R used as a sensitizing antigen and a method for immunization using the same are also specifically described in WO2003 / 000883, WO2004 / 022754, WO2006 / 006693, etc.

[0028] 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. In general, rodents such as mice, rats, hamsters, rabbits, monkeys, etc. are preferably used.

[0029] The above-mentioned animals are immunized with the sensitizing antigen according to a known method. For example, as a general method, immunization is performed by administering the sensitizing antigen intraperitoneally or subcutaneously to a mammal. Specifically, the sensitizing antigen diluted at an appropriate dilution ratio with PBS (Phosphate-Buffered Saline) or physiological saline, etc., is mixed with a conventional adjuvant, such as Freund's complete adjuvant, if desired, and emulsified, and then the sensitizing antigen is administered to the mammal several times every 4 to 21 days. In addition, a suitable carrier may 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.

[0030] 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 body of the immunized animal, thereby providing immune stimulation. Compared to general immunization methods in which a protein antigen is administered to an immunized animal, DNA immunization is expected to have the following advantages: -It can maintain the structure of membrane proteins such as IL-6R and provide immune stimulation. -No need to purify the immunogen

[0031] To obtain the monoclonal antibody of the present invention by DNA immunization, first, DNA expressing 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. As the expression vector, for example, a commercially available expression vector such as pcDNA3.1 can be suitably used. As a method for administering a vector to a living body, a commonly used method can be used. For example, DNA immunization is performed by introducing gold particles to which an expression vector is adsorbed into the cells of an individual animal to be immunized using a gene gun. Furthermore, an antibody that recognizes IL-6R can also be produced using the method described in International Publication WO 2003 / 104453.

[0032] After a mammal is immunized in this manner and an increase in the titer of an antibody that binds to IL-6R in the serum is confirmed, immune cells are collected from the mammal and subjected to cell fusion. As preferred immune cells, splenocytes in particular can be used.

[0033] Mammalian myeloma cells are used as the cells to be fused with the immune cells. The myeloma cells are preferably provided with an appropriate selection marker for screening. The selection marker refers to a trait that allows (or does not allow) a cell to survive 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 with HGPRT or TK deficiency have hypoxanthine-aminopterin-thymidine sensitivity (hereinafter abbreviated as HAT sensitivity). HAT-sensitive cells cannot synthesize DNA in HAT selection medium and die, but when fused with normal cells, they can continue to synthesize DNA by utilizing the salvage circuit of normal cells, and therefore grow even in HAT selection medium.

[0034] 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 that cannot incorporate these pyrimidine analogs can survive in the selective medium. Another selection marker called G418 resistance confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) via the neomycin resistance gene. Various myeloma cells suitable for cell fusion are known.

[0035] 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.

[0036] Basically, cell fusion between the immune cells and myeloma cells is carried out according to known methods, for example, 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 normal nutrient culture medium in the presence of a cell fusion promoter, such as polyethylene glycol (PEG) or Sendai virus (HVJ), and if desired, an auxiliary agent such as dimethyl sulfoxide can be added to enhance the fusion efficiency.

[0037] The ratio of immune cells to myeloma cells may be set arbitrarily. For example, it is preferable to use 1 to 10 times more immune cells than myeloma cells. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium suitable for the growth of the myeloma cell line, MEM culture medium, or other usual culture medium used for this type of cell culture may be used, and serum supplements such as fetal calf serum (FCS) may be suitably added.

[0038] For cell fusion, a predetermined amount of the immune cells and myeloma cells are thoroughly mixed in the culture medium, and a PEG solution (e.g., average molecular weight of about 1000 to 6000) pre-warmed to about 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, the appropriate culture medium listed above is successively added, and the procedure of centrifuging and removing the supernatant is repeated to remove cell fusion agents and the like that are undesirable for hybridoma growth.

[0039] The hybridomas thus obtained can be selected by culturing them in a conventional selection medium, such as HAT medium (a medium containing hypoxanthine, aminopterin and thymidine). The culture can be continued using the above HAT medium for a sufficient time (usually several days to several weeks) for cells other than the desired hybridoma (non-fused cells) to die. Then, screening and single cloning of hybridomas producing the desired antibody are performed by a conventional limiting dilution method.

[0040] The hybridomas thus obtained can be selected by using a selection medium according to the selection marker possessed by the myeloma used in the cell fusion. For example, cells lacking HGPRT or TK can be selected by culturing in HAT culture medium (a culture medium containing hypoxanthine, aminopterin and thymidine). That is, when HAT-sensitive myeloma cells are used in the cell fusion, cells that have successfully fused with normal cells can selectively grow in the HAT culture medium. The culture is continued using the above-mentioned HAT culture medium for a sufficient time 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. Next, screening and single cloning of hybridomas that produce the desired antibody can be performed by the usual limiting dilution method.

[0041] Screening and monocloning of the desired antibody can be suitably carried out by a screening method based on a known antigen-antibody reaction. For example, a monoclonal antibody that binds to IL-6R can bind to IL-6R expressed on the cell surface. Such a monoclonal antibody can be screened, for example, by FACS (fluorescence activated cell sorting). FACS is a system that allows the binding of an antibody to a cell surface to be measured by analyzing cells contacted with a fluorescent antibody with laser light and measuring the fluorescence emitted by individual cells.

[0042] To screen for hybridomas producing the monoclonal antibody of the present invention by FACS, cells expressing IL-6R are first prepared. The preferred cells for screening are mammalian cells in which IL-6R is forcibly expressed. By using non-transformed mammalian cells used as host cells as a control, the binding activity of the antibody to IL-6R on the cell surface can be selectively detected. That is, by selecting hybridomas producing antibodies that do not bind to host cells but bind to IL-6R-forcibly expressing cells, hybridomas producing IL-6R monoclonal antibodies can be obtained.

[0043] Alternatively, the binding activity of an antibody to immobilized IL-6R-expressing cells can be evaluated based on the principle of ELISA. For example, IL-6R-expressing cells are immobilized in the wells of an ELISA plate. The culture supernatant of a hybridoma is contacted with the immobilized cells in the wells, and antibodies that bind to the immobilized cells are detected. When the monoclonal antibody is derived from a mouse, the antibody that binds to the cells can be detected by an anti-mouse immunoglobulin antibody. Hybridomas that produce the desired antibody that has the ability to bind to the antigen and are selected by these screenings can be cloned by limiting dilution or the like.

[0044] The hybridomas producing the monoclonal antibodies thus prepared can be subcultured in a normal culture medium, and can be stored for a long period of time in liquid nitrogen.

[0045] The hybridoma is cultured according to a conventional method, and the desired monoclonal antibody can be obtained from the culture supernatant. Alternatively, the hybridoma can be administered to a compatible mammal to proliferate, and the monoclonal antibody can be obtained from the ascites. The former method is suitable for obtaining highly pure antibodies.

[0046] Antibodies encoded by antibody genes cloned from antibody-producing cells such as hybridomas can also be suitably used. The cloned antibody gene is incorporated into an appropriate vector and introduced into a host, whereby the antibody encoded by the gene is expressed. Methods for isolating antibody genes, introducing them into vectors, and transforming host cells have already been established, for example, by Vandamme et al. (Eur. J. Biochem. (1990) 192 (3), 767-775). Methods for producing recombinant antibodies are also known, as described below.

[0047] For example, cDNA encoding the variable region (V region) of an anti-IL-6R antibody is obtained from a hybridoma cell that produces the anti-IL-6R antibody. To do this, generally, total RNA is first extracted from the hybridoma. For example, the following method 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)

[0048] The extracted mRNA can be purified using an mRNA Purification Kit (GE Healthcare Biosciences) or the like. Alternatively, kits for directly extracting total mRNA from cells, such as QuickPrep mRNA Purification Kit (GE Healthcare Biosciences), are also commercially available. Using such a kit, mRNA can be obtained from a hybridoma. cDNA encoding an antibody V region can be synthesized from the obtained mRNA using reverse transcriptase. cDNA can be synthesized using an AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Kogyo Co., Ltd.) or the like. In addition, for the synthesis and amplification of cDNA, a 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 appropriately used. Furthermore, during the synthesis of such cDNA, appropriate restriction enzyme sites (described below) can be introduced at both ends of the cDNA.

[0049] The desired cDNA fragment is purified from the obtained PCR product and then ligated to a vector DNA. The recombinant vector is thus prepared, introduced into E. coli or the like, and colonies are selected, after which the desired recombinant vector can be prepared from the E. coli that formed the colonies. Then, whether or not the recombinant vector has the base sequence of the desired cDNA is confirmed by a known method, for example, the dideoxynucleotide chain termination method.

[0050] To obtain genes encoding variable regions, it is easy to use the 5'-RACE method using primers for amplifying variable region genes. First, cDNA is synthesized using RNA extracted from hybridoma cells as a template to obtain a 5'-RACE cDNA library. A commercially available kit such as the SMART RACE cDNA Amplification Kit can be used to synthesize the 5'-RACE cDNA library.

[0051] The resulting 5'-RACE cDNA library is used as a template to amplify antibody genes by PCR. 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 desirable to determine the subclass in advance using a commercially available kit such as the Iso Strip Mouse Monoclonal Antibody Isotyping Kit (Roche Diagnostics).

[0052] Specifically, for example, when the objective is to obtain a gene encoding mouse IgG, primers capable of amplifying genes encoding γ1, γ2a, γ2b, and γ3 as heavy chains and κ and λ chains as light chains can be used. To amplify the variable region genes of IgG, a primer that anneals to a portion corresponding to the constant region close to the variable region is generally used as the 3' primer. On the other hand, a primer included in the 5' RACE cDNA library construction kit is used as the 5' primer.

[0053] Using the PCR product thus amplified, an immunoglobulin consisting of a combination of heavy and light chains can be reconstituted. The desired antibody can be screened using the binding activity of the reconstituted immunoglobulin to IL-6R as an index. For example, when the aim is to obtain an antibody against IL-6R, it is more preferable that the binding of the antibody to IL-6R is specific. 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) Selecting an antibody that binds to IL-6R-expressing cells.

[0054] Methods for detecting the binding between an antibody and IL-6R-expressing cells are known. Specifically, the binding between an antibody and IL-6R-expressing cells can be detected by techniques such as FACS described above. Fixed specimens of IL-6R-expressing cells can be appropriately used to evaluate the binding activity of an antibody.

[0055] As a method for screening antibodies using binding activity as an index, a panning method using a phage vector is also preferably used. When antibody genes are obtained as a library of heavy and light chain subclasses from a polyclonal antibody-expressing cell group, a screening method using a phage vector is advantageous. Genes encoding the variable regions of the heavy and light chains can be linked with an appropriate linker sequence to form a single chain Fv (scFv). By inserting a gene encoding an scFv into a phage vector, a phage expressing scFv on its surface can be obtained. After contacting this phage with a desired antigen, the phage bound to the antigen can be recovered to recover DNA encoding an scFv having the desired binding activity. By repeating this operation as necessary, scFv having the desired binding activity can be concentrated.

[0056] After obtaining a cDNA encoding the V region of the desired anti-IL-6R antibody, the cDNA is digested with a restriction enzyme that recognizes the restriction enzyme sites inserted at both ends of the cDNA. A preferred restriction enzyme recognizes and digests a base sequence that appears infrequently in the base sequence constituting the antibody gene. Furthermore, in order to insert one copy of the digested fragment into a vector in the correct direction, it is preferable to insert a restriction enzyme that gives a sticky end. 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. At this time, if a gene encoding an antibody constant region (C region) and a gene encoding the V region are fused in frame, a chimeric antibody can be obtained. Here, a chimeric antibody refers to an antibody whose 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 antibody of the present invention. A chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector that already has a constant region. Specifically, for example, a restriction enzyme recognition sequence for a restriction enzyme that digests the V region gene can be appropriately positioned on the 5' side of an expression vector carrying DNA encoding a desired antibody constant region (C region). A chimeric antibody expression vector is constructed by fusing the two genes digested with the same combination of restriction enzymes in frame.

[0057] To produce an anti-IL-6R monoclonal antibody, an antibody gene is incorporated into an expression vector so that it is expressed under the control of an expression control region. Expression control regions for expressing an antibody include, for example, enhancers and promoters. In addition, a suitable signal sequence can be added to the amino terminus so that the expressed antibody is secreted outside the cell. For example, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 536) can be used as a signal sequence, but other suitable signal sequences can also be added. The expressed polypeptide is cleaved at the carboxyl terminal portion of the above sequence, and the cleaved polypeptide can be secreted outside the cell as a mature polypeptide. Then, a suitable host cell is transformed with this expression vector to obtain a recombinant cell expressing DNA encoding an anti-IL-6R antibody.

[0058] "Antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen to which the complete 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.

[0059] 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.

[0060] The term "variable region" or "variable domain" refers to the domain of an antibody's heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of an antibody's heavy and light chains (VH and VL, respectively) usually 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.

[0061] The term "complementarity determining region" or "CDR" as used herein refers to the regions of the variable domain of an antibody that are hypervariable in sequence and / or form structurally defined loops ("hypervariable loops") and / or antigen contact residues ("antigen contacts"). Typically, 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) combinations 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.

[0062] "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 the VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0063] The term "constant region" or "constant domain" as used herein refers to a portion of an antibody other than the variable region. For example, an IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded, with each heavy chain having, from N-terminus to C-terminus, a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by a heavy chain constant region (CH) that includes a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain. Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chain of a native antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0064] 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), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0065] The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that includes 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, with the exception that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) 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.

[0066] The ligand-binding molecule of the present invention is a polypeptide that includes 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 at any position in the polypeptide, as long as the cleavage site can attenuate the binding of the ligand-binding molecule to the ligand. The polypeptide can include one or more cleavage sites.

[0067] Furthermore, the ligand-binding molecule of the present invention has weaker (i.e., attenuated) ligand binding in the cleaved state compared to the uncleaved state. In an embodiment in which the binding between the ligand-binding molecule and the ligand is an antigen-antibody reaction, the attenuation of ligand binding can be evaluated by the ligand binding activity of the ligand-binding molecule.

[0068] The binding activity of the ligand-binding molecule and the ligand can be evaluated by well-known methods such as FACS, ELISA format, ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), the BIACORE method using the surface plasmon resonance (SPR) phenomenon, and the BLI (Bio-Layer Interferometry) method (Octet) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010). The ALPHA screen is carried out by ALPHA technology using two beads, a donor and an acceptor, based on the following principle: A luminescence 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 in the donor bead excited by a laser converts the surrounding oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and, when it reaches the nearby acceptor bead, triggers a chemiluminescence reaction in the bead, which ultimately emits light. If there is no interaction between the molecules bound to the donor bead and the acceptor bead, the singlet oxygen produced by the donor bead does not reach the acceptor bead, and no chemiluminescence reaction occurs.

[0069] For example, a biotin-labeled ligand-binding molecule is bound to a donor bead, and a ligand tagged with glutathione S-transferase (GST) is bound to an acceptor bead. In the absence of competing untagged ligand-binding molecules, the ligand-binding molecule and the ligand interact to generate a signal at 520-620 nm. The untagged ligand-binding molecule competes with the interaction between the tagged ligand-binding molecule and the ligand. Relative binding affinity can be determined by quantifying the decrease in fluorescence resulting from the competition. It is known to biotinylate a ligand-binding molecule such as an antibody using Sulfo-NHS-biotin or the like. As a method for tagging a ligand with GST, a method of expressing a GST-fused ligand in a cell or the like that contains a vector capable of expressing a fusion gene in which a polynucleotide encoding a ligand and a polynucleotide encoding GST are fused in frame, and purifying the GST-fused ligand using a glutathione column, or the like, can be appropriately adopted. The resulting signals are suitably analyzed by fitting to a one-site competition model utilizing non-linear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

[0070] One of the substances (ligand) for which interaction is to be observed is fixed on the gold thin film of a sensor chip, and light is applied from the back of the sensor chip so that it is totally reflected at the interface between the gold thin film and the glass. When the other substance (analyte) for which interaction is to be observed is poured onto the surface of the sensor chip and the ligand and analyte bind, the mass of the immobilized ligand molecule increases, and the refractive index of the solvent on the surface of the sensor chip changes. This change in refractive index shifts the position of the SPR signal (conversely, when the bond is dissociated, the position of the signal returns). The Biacore system takes the amount of shift mentioned above, that is, the change in mass on the sensor chip surface, as the vertical axis, and displays the change in mass over time as measurement data (sensorgram). The kinetics: binding rate constant (ka) and dissociation rate constant (kd) can be calculated from the curve of the sensorgram, and the dissociation constant (KD) can be calculated from the ratio of these constants. Inhibition measurement methods and equilibrium value analysis methods are also preferably used in 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.

[0071] The term "the function of a ligand-binding molecule to bind to a ligand is attenuated" means, for example, that 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, 15% or less, particularly preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, compared to the control ligand-binding molecule, based on the above-mentioned measurement method. Any suitable index of binding activity may be used, for example, dissociation constant (KD). When dissociation constant (KD) is used as an evaluation index of binding activity, a larger dissociation constant (KD) of the test ligand-binding molecule to the ligand compared to the dissociation constant (KD) of the control ligand-binding molecule to the ligand indicates that the binding activity of the test ligand-binding molecule to the ligand is weaker than that of the control ligand-binding molecule. The function of binding to a ligand is weakened, for example, when 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 can be, for example, an uncleaved form of the ligand-binding molecule.

[0072] In one embodiment of the present invention, the cleavage site of the ligand-binding molecule of the present invention is cleaved, thereby releasing the ligand from the ligand-binding molecule. 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 the portion of the ligand-binding molecule via the linker (see, for example, FIG. 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 molecule.

[0073] A method for detecting the release of a ligand from a ligand-binding molecule due to cleavage at the cleavage site includes a method for detecting the ligand using a ligand-detecting antibody or the like 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. Detection of the ligand 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 the surface plasmon resonance (SPR) phenomenon, and BLI (Bio-Layer Interferometry) method (Octet) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010). For example, when detecting the release of a ligand using Octet, a ligand-detecting antibody that recognizes the ligand is biotinylated, contacted with a biosensor, and then the binding to the ligand in the sample is measured to detect the release of the ligand. Specifically, the amount of the ligand is measured using a ligand detection antibody for a sample containing a ligand-binding molecule and a ligand before or after protease treatment, and the amount of the ligand detected in the sample before and after protease treatment is compared to detect the release of the ligand. In addition, the amount of the ligand is measured using a ligand detection antibody for 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, and the amount of the ligand detected in the sample with and without protease is compared to detect the release of the ligand. More specifically, the release of the ligand can be detected by the method 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 the ligand is measured using a ligand detection antibody for a sample containing a fusion protein before or after protease treatment, and the amount of the ligand detected in the sample before and after protease treatment is compared to detect the release of the ligand. In addition, the amount of ligand is measured using a ligand detection antibody for a sample containing a protease and a fusion protein and a sample containing a fusion protein without a protease, and the amount of ligand detected in the samples with and without the protease is 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.

[0074] In an embodiment in which the physiological activity of the ligand is inhibited when it is bound to the ligand-binding molecule, the release of the ligand can be detected by measuring the physiological activity of the ligand in the sample as a method for detecting the release from the ligand-binding molecule. Specifically, the release of the ligand can be detected by measuring and comparing the physiological activity of the ligand for a sample containing the ligand-binding molecule and the ligand before or after protease treatment. The release of the ligand can also be detected by measuring and comparing the physiological activity of the ligand for a sample containing the protease, the ligand-binding molecule, and the ligand, and for a sample containing the ligand-binding molecule and the ligand without the protease. When the ligand-binding molecule is fused with the ligand to form a fusion protein, the release of the ligand can be detected by measuring and comparing the physiological activity of the ligand for a sample containing the fusion protein before or after the protease treatment. The release of the ligand can also be detected by measuring and comparing the physiological activity of the ligand for a sample containing the protease and the fusion protein, and for a sample containing the fusion protein without the protease.

[0075] In one embodiment of the invention, the cleavage site comprises a protease cleavage sequence and is cleaved by a protease.

[0076] 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 the type is not particularly limited. In some embodiments, a target tissue-specific protease is used. The target tissue-specific protease is, 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.

[0077] As used herein, the term "target tissue" refers to a tissue that contains at least one target cell. In some embodiments of the invention, the target tissue is cancerous tissue. In some embodiments of the invention, the target tissue is inflamed tissue.

[0078] The term "cancer tissue" refers to tissue that contains 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 that cancer tissue contains 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.

[0079] 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 skin conditions -Spinal nerve damage in herniated discs and chronic lower back pain

[0080] Proteases that are specifically expressed or specifically activated in several types of target tissues or that are thought to be associated with disease states of target tissues (target tissue-specific proteases) are known. For example, International Publication WO2013 / 128194, International Publication WO2010 / 081173, International Publication WO2009 / 025846, etc. disclose proteases that are specifically expressed in cancer tissues. In addition, 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.

[0081] In addition to proteases that are specifically expressed in target tissues, there are also proteases that are specifically activated in target tissues. For example, proteases may be expressed in an inactive form and then become active. 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 may escape inhibition and become specifically activated. Active proteases can be measured using an antibody that recognizes active proteases (PNAS 2013 Jan 2; 110(1): 93-98.) or a method in which a peptide recognized by a protease is fluorescently labeled and quenched before cleavage but emits light after cleavage (Nat Rev Drug Discov. 2010 Sep;9(9):690-701. doi: 10.1038 / nrd3053.).

[0082] From one perspective, the term "target tissue specific protease" means: (i) a protease that is expressed at a higher level in a target tissue than in normal tissue; (ii) a protease that has higher activity in the target tissue than in normal tissue; (iii) a protease that is expressed at a higher level in the target cell than in normal cells; (iv) a protease that has a higher activity in target cells than in normal cells; It can refer to either:

[0083] 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), A disintegrin and metalloproteases (ADAMs), metalloproteases with A disintegrin or thrombospondin motifs (ADAMTS), meprins (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-LikeProteases, AMSH, gamma secretase, A-antiplasmin cleaving enzyme (APCE), decysin 1, N-Acetylated Alpha-Linked Acidic Dipeptidase-Like 1 (NAALADL1), furin, etc.

[0084] From another perspective, the target tissue-specific protease can refer to a cancer tissue-specific protease or an inflamed tissue-specific protease.

[0085] Examples of cancer tissue-specific proteases include proteases that are specifically expressed in cancer tissues, such as those disclosed in International Publication WO2013 / 128194, International Publication WO2010 / 081173, and International Publication WO2009 / 025846.

[0086] The type of cancer tissue-specific protease has a higher specificity of expression in the cancer tissue of the treatment target, and the more effective it is in reducing side effects. The concentration of the cancer tissue-specific protease in the cancer tissue is preferably 5 times higher than that in the normal tissue, more preferably 10 times higher, even more preferably 100 times higher, particularly preferably 500 times higher, and most preferably 1000 times higher. In addition, the activity of the cancer tissue-specific protease in the cancer tissue is preferably 2 times higher than that in the normal tissue, more preferably 3 times higher, 4 times higher, 5 times higher, or 10 times higher, more preferably 100 times higher, particularly preferably 500 times higher, and most preferably 1000 times higher. In addition, 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 outside the cell. When the cancer tissue-specific protease is not bound to the cell membrane of the cancer cell, in order for the cytotoxicity by the immune cell to be specific to the cancer cell, it is preferable that the cancer tissue-specific protease is present inside or near the cancer tissue. In this specification, "near the cancer tissue" means within a range in which the cancer tissue-specific protease cleavage sequence is cleaved to exert the effect of reducing the ligand binding activity. However, it is preferable that the range is one in which normal cells are not damaged as much as possible. From another perspective, cancer tissue-specific proteases are (i) a protease that is expressed at a higher level in cancer tissue than in normal tissue; (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 one type alone or two or more types in combination. The number of types of cancer tissue-specific proteases can be appropriately determined by those skilled in the art in consideration of the type of cancer to be treated.

[0087] 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, MMP2 and MMP9 are even more preferred.

[0088] The more specific the type of inflammatory tissue-specific protease is expressed in the inflammatory tissue of the treatment target, the more effective it is in reducing side effects. The concentration of the inflammatory tissue-specific protease in the inflammatory tissue is preferably 5 times higher than that in the normal tissue, more preferably 10 times higher, even more preferably 100 times higher, particularly preferably 500 times higher, and most preferably 1000 times higher. In addition, the activity of the inflammatory tissue-specific protease in the inflammatory tissue is preferably 2 times higher than that in the normal tissue, more preferably 3 times higher, 4 times higher, 5 times higher, or 10 times higher, more preferably 100 times higher, particularly preferably 500 times higher, and most preferably 1000 times higher. In addition, the inflammatory tissue-specific protease may be one that is bound to the cell membrane of an inflammatory cell, or one that is not bound to the cell membrane and is secreted outside the cell. When the inflammatory tissue-specific protease is not bound to the cell membrane of an inflammatory cell, in order for the cytotoxicity by immune cells to be specific to the inflammatory cell, it is preferable that the inflammatory tissue-specific protease is present inside or near the inflammatory tissue. In this specification, "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 the ligand binding activity. However, it is preferable that the range is one in which normal cells are not damaged as much as possible. From another perspective, inflammatory tissue-specific proteases are (i) a protease that is expressed at a higher level in inflamed tissue than in normal tissue; (ii) proteases that have higher activity in inflamed tissues than in normal tissues; (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 one type alone or two or more types in combination. The number of types of inflammatory tissue-specific proteases can be appropriately determined by those skilled in the art, taking into consideration the pathology of the subject to be treated.

[0089] From the above viewpoints, as the inflammatory tissue-specific protease, among the above-mentioned proteases, metalloproteases are preferable, and among the metalloproteases, ADAMTS5, MMP2, MMP7, MMP9 and MMP13 are more preferable.

[0090] A protease cleavage sequence is a specific amino acid sequence that is specifically recognized by a target tissue-specific protease when a polypeptide is hydrolyzed by 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 in the target tissue / cells to be treated 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 above-mentioned cancer tissue-specific proteases and inflammatory tissue-specific proteases disclosed in International Publication WO2013 / 128194, International Publication WO2010 / 081173, International Publication WO2009 / 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. In addition, 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 protease cleavage sequence in a protein that changes its molecular shape upon cleavage by a protease, such as TGF-β being converted to its latent form upon cleavage by a protease, may be used.

[0091] 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. More preferably, the protease cleavage sequence is an amino acid sequence that is specifically hydrolyzed by a suitable target tissue-specific protease, as described above. 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 MMP2 and MMP9) VPLSLTMG (SEQ ID NO:35, cleavable by MMP7) 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 MMP1) GAGIPVSLRSGAG ​​(SEQ ID NO: 70, cleavable by MMP2) GPLGIAGQ (SEQ ID NO:71, cleavable by MMP2) GGPLGMLSQS (SEQ ID NO: 72, cleavable by MMP2) PLGLWA (SEQ ID NO:73, cleavable by MMP2) GAGRPFSMIMGAG (SEQ ID NO:74, cleavable by MMP3) GAGVPLSLTMGAG (SEQ ID NO: 75, cleavable by MMP7) GAGVPLSLYSGAG (SEQ ID NO:76, cleavable by MMP9) AANLRN (SEQ ID NO:77, cleavable by MMP11) AQAYVK (SEQ ID NO:78, cleavable by MMP11) AANYMR (SEQ ID NO:79, cleavable by MMP11) AAALTR (SEQ ID NO:80, cleavable by MMP11) AQNLMR (SEQ ID NO:81, cleavable by MMP11) AANYTK (SEQ ID NO:82, cleavable by MMP11) GAGPQGLAGQRGIVAG (SEQ ID NO: 83, cleavable by MMP13) PRFKIIGG (SEQ ID NO:84, cleavable by pro-urokinase) PRFRIIGG (SEQ ID NO:85, cleavable by pro-urokinase) 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) TSTSGRSANPRG (SEQ ID NO: 345)

[0092] In one embodiment of the invention, the protease cleavage sequence further comprises a flexible linker at 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 the 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) The flexible linker in this embodiment is preferably a peptide linker. The first flexible linker and the second flexible linker are each independently and optionally present, and are the same or different flexible linkers that contain at least one flexible amino acid (such as Gly). For example, a sufficient number of residues (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, especially Gly, etc.) are included so that the protease cleavage sequence has the desired protease accessibility.

[0093] Flexible linkers suitable for use on either end of the protease cleavage sequence are typically those that improve the accessibility of the protease to the protease cleavage sequence and increase the cleavage efficiency of the protease. Suitable flexible linkers can be readily selected and are of different lengths, such as 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 invention, the flexible linker is a peptide linker of 1 to 7 amino acids.

[0094] 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 thus more likely 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 (SGGGGGG, SEQ ID NO: 54) (Gly·Gly·Gly·Gly·Ser (GGGGS, SEQ ID NO: 41)) (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.

[0095] 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')2, complete antibodies, etc.

[0096] 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 one of the amino acid sequences shown in SEQ ID NOs: 55, 56, 57, and 58, or an Fc region mutant obtained by modifying these Fc regions, can be used. In some embodiments of the present invention, the ligand-binding molecule comprises an antibody constant region.

[0097] In some more specific embodiments of the present invention, the ligand-binding molecule is an antibody. When an antibody is used as the ligand-binding molecule, binding to the ligand is achieved by the variable region. In some more specific embodiments, the ligand-binding molecule is an IgG antibody. When an IgG antibody is used as the ligand-binding molecule, the type is not limited, and IgG1, IgG2, IgG3, IgG4, etc. can be used. Even when an IgG antibody is used as the ligand-binding molecule, binding to the ligand is achieved by the variable region, and binding to the ligand can be achieved by one or both of the two variable regions of the IgG antibody.

[0098] In some embodiments of the present invention, cleavage of a cleavage site / protease cleavage sequence in a ligand-binding molecule disrupts a domain having ligand-binding activity in the ligand-binding molecule, thereby weakening binding to a ligand. For example, when an IgG antibody is used as a ligand-binding molecule, an embodiment includes a cleavage site / protease cleavage sequence provided in the antibody variable region, in which the antibody variable region cannot be formed in its entirety in the cleaved state, thereby weakening binding to a ligand.

[0099] In the present specification, the term "association" can be rephrased as, for example, a state in which two or more polypeptide regions interact with each other. In general, hydrophobic bonds, hydrogen bonds, ionic bonds, etc. are formed between the target polypeptide regions to form an association. As a common 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 pairing structure through non-covalent bonds between them.

[0100] In some embodiments of the present invention, the VH and VL contained in the ligand-binding molecule are associated. The association between the antibody VH and the antibody 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 dissolution of all or part of the interaction state between two or more polypeptide regions. Dissolution of the association between VH and VL may mean dissolution of 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 dissolved by cleavage of the cleavage site or by cleavage of the protease cleavage sequence with a protease.

[0101] In some embodiments of the present invention, the 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 the antibody VL in the ligand-binding molecule are associated, and cleavage of the cleavage site / protease cleavage sequence cancels the association of the antibody VH and the 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 it can attenuate the binding of the ligand-binding molecule to a ligand upon cleavage.

[0102] In some further embodiments of the invention, the ligand binding molecule comprises an antibody VH, an antibody VL and an antibody constant region. It is known that VH and VL, and CH and CL of an antibody interact with each other through many amino acid side chains between the domains, as described by Rothlisberger et al. (J Mol Biol. 2005 Apr 8;347(4):773-89.). It is known that VH-CH1 and VL-CL can form a stable structure as a Fab domain, but as reported, the amino acid side chains between VH and VL are generally about 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 the VL domain are present, the proportion of them that form an associated state is low.

[0103] 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, prior to cleavage, all of the heavy chain-light chain interactions are present between the two peptides in the Fab structure, whereas upon cleavage of the cleavage site / protease cleavage sequence, the interaction between a peptide comprising VH (or a portion of VH) and a peptide comprising VL (or a portion of VL) is weakened and the association between VH and VL is dissolved.

[0104] 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 on the variable region side of amino acid 140 (EU numbering) in the antibody heavy chain constant region, preferably on the variable region side of amino acid 122 (EU numbering) in the antibody heavy chain constant region. In some specific embodiments, the cleavage site / protease cleavage sequence is 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. 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 inserted 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.

[0105] In one embodiment of the invention, the cleavage site / protease cleavage sequence is located in the antibody VH or in the antibody 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 inserted at the position of a residue forming a loop structure or a residue close to the loop structure in the antibody VH or antibody VL. The loop structure in the antibody VH or antibody VL refers to a portion of the antibody VH or antibody VL that does not form a secondary structure such as an α-helix or a β-seed. The positions of the residues forming the loop structure or the 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), from amino acid 95 (Kabat numbering), and from amino acid 100 (Kabat numbering). It can refer to the range from amino acid number 7 (Kabat numbering) to amino acid number 99 (Kabat numbering), amino acid number 101 (Kabat numbering) to amino acid number 113 (Kabat numbering), antibody VL amino acid number 7 (Kabat numbering) to amino acid number 19 (Kabat numbering), amino acid number 39 (Kabat numbering) to amino acid number 46 (Kabat numbering), amino acid number 49 (Kabat numbering) to amino acid number 62 (Kabat numbering), and amino acid number 96 (Kabat numbering) to amino acid number 107 (Kabat numbering). In some more specific embodiments, the cleavage site / protease cleavage sequence is inserted at any position in the antibody VH sequence from amino acid number 7 (Kabat numbering) to amino acid number 16 (Kabat numbering), from amino acid number 40 (Kabat numbering) to amino acid number 47 (Kabat numbering), from amino acid number 55 (Kabat numbering) to amino acid number 69 (Kabat numbering), from amino acid number 73 (Kabat numbering) to amino acid number 79 (Kabat numbering), from amino acid number 83 (Kabat numbering) to amino acid number 89 (Kabat numbering), from amino acid number 95 (Kabat numbering) to amino acid number 99 (Kabat numbering), or from amino acid number 101 (Kabat numbering) to amino acid number 113 (Kabat numbering). In some more specific embodiments, the cleavage site / protease cleavage sequence is inserted 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), from amino acid 96 (Kabat numbering) to amino acid 107 (Kabat numbering).

[0106] 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 can refer 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 between amino acid 109 (Kabat numbering) of the antibody VH and amino acid 122 (EU numbering) of the antibody heavy chain constant region, or 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 number 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 number 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 number 112) of the antibody light chain constant region.

[0107] In one embodiment, the cleavage site / protease cleavage sequence is located near the interface between the antibody VL and the antibody constant region. Near the interface 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 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 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, and preferably refers to 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 can refer to 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.

[0108] A plurality of cleavage sites / protease cleavage sequences can be provided in a ligand molecule, for example, at a plurality of 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, a person skilled in the art who has come into contact with the present invention can change the shape of a molecule containing the antibody VH, antibody VL, and antibody constant region, such as by swapping the antibody VH and antibody VL, and such molecular shape does not depart from the scope of the present invention.

[0109] 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 a biological signaling pathway that typically is involved in the cell that bears the receptor.

[0110] In the present specification, the term "ligand" includes a desired molecule that exhibits biological activity by interacting with a biomolecule. For example, the term "ligand" does not only mean a molecule that interacts with a receptor, but also includes a molecule that exhibits biological activity by interacting with the molecule, and for example, a receptor that interacts with the molecule and a binding fragment thereof are also included in the ligand. For example, a protein that contains a ligand-binding site of a protein known as a receptor or a site where the receptor interacts with another molecule is included in the ligand in the present invention. Specifically, a soluble receptor, a soluble fragment of a receptor, an extracellular domain of a transmembrane receptor, and a polypeptide containing them are included in the ligand in the present invention.

[0111] The ligands of the 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, such as 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 less than or equal to 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.

[0112] Examples of molecules with biological activity 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, a chemokine, a cell growth factor, a TGF-β family, a myokine, an adipokines, or a neurotrophic factor. In more particular 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, or MIP-1b.

[0113] Chemokines are a family of homogeneous serum proteins between 7 and 16 kDa, originally characterized by their ability to induce leukocyte migration. Most chemokines have four characteristic cysteines (Cys) and are classified into CXC, or alpha, CC, or beta, C, or gamma, and CX3C, or 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. In general, disulfide bridges are 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).

[0114] Interferon-inducible protein-10 (IP-10 or CXCL10) is induced by interferon-γ and TNF-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-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking," J Immunol., 168:3195-204, 2002). In addition, 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).

[0115] 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 anti-tumor immunity (Nomura et al., Int. J. Cancer 91:597-606, 2001; Mach and Dranoff, Curr. Opin. Immunol. 12:571-75, 2000). For example, the biological activity of chemokines has been discussed in reports known to those skilled in the art (Bruce, L. et al., "Radiolabeled Chemokine binding assays," Methods in Molecular Biology (2000) vol. 138, pp129-134; Raphaele, B. et al. "Calcium Mobilization," Methods in Molecular Biology (2000) vol. 138, pp143-148; Paul D. Ponath et al., "Transwell Chemotaxis," Methods in Molecular Biology (2000) vol. 138, pp113-120 Humana Press. Totowa, New Jersey).

[0116] 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 by measuring the cell migration activity of CXCL10, by Reporter assay using a CXCR3 stable expressing cell line (see PLoS One. 2010 Sep 13;5(9):e12700), and by PathHunter assay using B-Arrestin recruitment induced in the early stage of GPCR signaling. TM Examples include β-Arrestin recruitment assay.

[0117] 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 a variety of 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.

[0118] Interleukin-12 binds to IL-12 receptors expressed on the plasma membrane of cells (e.g., T cells, NK cells) and can thereby alter (e.g., initiate, block) biological processes. For example, binding of IL-12 to the IL-12 receptor stimulates the proliferation of preactivated T cells and NK cells, enhances the cytolytic activity of cytotoxic T cells (CTLs), NK cells and LAK (lymphokine-activated killer) 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, CTLs) and for setting up 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).

[0119] Methods for measuring the physiological activity of IL12 include measuring the cell proliferation activity of IL12, STAT4 reporter assay, cell activation by IL12 (cell surface marker expression, cytokine production, etc.), and promotion of cell differentiation by IL12.

[0120] The protein Programmed Death 1 (PD-1) is an inhibitory member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells (Okazaki et al. (2002) Curr. Opin. Immunol. 14:391779-82; Bennett et al. (2003) J Immunol 170:711-8). The first members of the family, CD28 and ICOS, were discovered by their functional effects on increasing T cell proliferation after 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.

[0121] The PD-1 gene is a 55 kDa type I transmembrane protein that is part of the Ig gene superfamily. PD-1 contains a membrane-proximal immunoreceptor tyrosine 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) that is important for B7-1 and B7-2 binding. Two ligands for PD-1, PD-L1 and PD-L2, have been identified and have been shown to negatively regulate T cell activation when bound to PD-1 (Freeman et al. (2000) J Exp Med 192:1027-34; Latchman et al. (2001) Nat Immunol 2:261-8; Carter et al. (2002) Eur J Immunol 32:634-43). Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1 but not to other CD28 family members. PD-L1, one of the ligands for PD-1, is abundant in a variety of human cancers (Dong et al. (2002) Nat. Med. 8:787-9). The interaction of PD-1 with PD-L1 results in a reduction in 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). Immunosuppression can be restored 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).

[0122] 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 with arthritis and nephritis (Nishimura et al. (1999) Immunity 11:141-51, Nishimura et al. (2001) Science 291:319-22). In addition, PD-1 has been found to play an important role in autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease (GVHD), type I 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 it is essential for inhibiting the flux of ATP and the tyrosine phosphorylation of downstream effector molecules (Okazaki et al. (2001) PNAS 98:13866-71).

[0123] 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, which are secreted by glial cells of the nervous system and by many cells of the immune system. Cytokines can be classified as proteins, peptides or glycoproteins, and encompass a large and diverse family of regulators. Cytokines bind to cell surface receptors and induce intracellular signaling, which can result in the regulation of enzyme activity, up- or down-regulation of some genes and their transcription factors, or feedback inhibition, etc. In some embodiments, the cytokines of the present invention include immune regulators 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.

[0124] In some embodiments of the present invention, the ligand is a chemokine. Chemokines generally act as chemoattractants to recruit immune effector cells to the site of chemokine expression. It may be beneficial to express certain chemokine genes, for example together 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.

[0125] In addition, 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.

[0126] In some embodiments of the present invention, the ligand is selected from CXCL10, PD1, IL12, and IL6R. The CXCL10, PD1, IL12, and IL6R may have the same sequence as naturally occurring CXCL10, PD1, IL12, and IL6R, or may be a variant having a different sequence from naturally occurring CXCL10, PD1, IL12, and IL6R but retaining the physiological activity of the corresponding natural ligand. To obtain a variant of the ligand, the ligand sequence may be artificially added for various purposes, and preferably, a variant of the ligand is obtained by adding a modification that is not susceptible to protease cleavage (protease resistant).

[0127] 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 an uncleaved ligand-binding molecule to a ligand substantially or significantly interferes with or competes with the binding of the ligand to its binding partner. When an antibody or 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 binding to the ligand exerting its neutralizing activity.

[0128] In one embodiment of the present invention, it is preferable that the uncleaved ligand-binding molecule can sufficiently neutralize the biological activity of the ligand by binding to the ligand. That is, it is preferable that the biological activity of the ligand bound to the uncleaved ligand-binding molecule is lower than the biological activity of the ligand not bound to the uncleaved ligand-binding molecule. Although not limited thereto, for example, the biological activity of the ligand bound to the uncleaved ligand-binding molecule may be 90% or less, preferably 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, particularly preferably 20% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less compared to the biological activity of the ligand not bound to the uncleaved ligand-binding molecule. By sufficiently neutralizing the biological activity of the ligand, it is expected that when the ligand-binding molecule is administered, the ligand will not exert its biological activity before reaching the target tissue.

[0129] 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 in vivo natural binding partner (e.g., natural receptor for the ligand) to the ligand. For example, 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 compared to the binding amount of the natural binding partner to the ligand in the body (per unit binding partner), but is not limited thereto. As an index of binding activity, an appropriate index may be used, for example, the dissociation constant (KD). When the dissociation constant (KD) is used as an evaluation index of binding activity, a dissociation constant (KD) of the cleaved ligand-binding molecule for the ligand that is larger than the dissociation constant (KD) of the 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 dissociation constant (KD) of the cleaved ligand-binding molecule for the ligand is, for example, 1.1 times or more, preferably 1.5 times or more, 2 times or more, 5 times or more, 10 times or more, and particularly preferably 100 times or more, compared to the dissociation constant (KD) of the natural binding partner in vivo for the ligand. Since the cleaved ligand-binding molecule has only low binding activity to the ligand or has almost no binding activity to the ligand, it is expected that the ligand-binding molecule will be guaranteed to release the ligand after cleavage and will be prevented from rebinding to another ligand molecule.

[0130] 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, so that the function of the ligand-binding molecule to inhibit the biological activity of the ligand is also attenuated. Those skilled in the art can confirm the biological activity of the ligand by known methods, for example, by detecting the binding of the ligand to its binding partner.

[0131] 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 a non-covalent bond between the ligand-binding molecule and the ligand, such as an antigen-antibody bond.

[0132] In some embodiments of the present invention, the 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. Even if 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 an embodiment 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 an embodiment in which the ligand-binding molecule and the ligand are not fused. When the ligand-binding molecule is cleaved, the non-covalent bond is weakened. That is, 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 when fusing 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), and the like, 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. For example, but not limited to, in the case of a peptide linker: Ser Gly·Ser(GS) Ser·Gly(SG) Gly Gly Ser (GGS) Gly·Ser·Gly (GSG) Ser Gly Gly (SGG) Gly·Ser·Ser (GSS) Ser·Ser·Gly (SSG) Ser Gly Ser (SGS) Gly Gly Gly Ser (GGGS, 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 (SGGGGGG, SEQ ID NO: 54) (Gly·Gly·Gly·Gly·Ser (GGGGS, SEQ ID NO: 41)) (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.

[0133] The synthetic chemical linkers (chemical crosslinkers) are crosslinkers 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, and these crosslinkers are commercially available.

[0134] The present invention also relates to a pharmaceutical composition (drug) comprising the ligand-binding molecule of the present invention and a pharma- ceutical acceptable carrier, a pharmaceutical composition (drug) comprising the ligand-binding molecule of the present invention, a ligand, and a pharma- ceutical acceptable carrier, and a pharmaceutical composition (drug) comprising a fusion protein in which the ligand-binding molecule of the present invention and a ligand are fused, and a pharma- ceutical composition (drug) comprising a pharma- ceutical acceptable carrier.

[0135] As used herein, "treatment" (and its grammatical derivatives, such as "treat", "treating", etc.) refers to a clinical intervention intended to modify the natural course of the individual being treated, and may be performed for prophylaxis or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, relief 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 control the biological activity of the ligand and are used to delay the onset of the disease or slow the progression of the disease.

[0136] In the present invention, the pharmaceutical composition generally refers to a drug for treating or preventing a disease, or for testing or diagnosing a disease. 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 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."

[0137] 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 still bound to the ligand. The ligand-binding molecule transported to the target tissue is cleaved in the target tissue, weakening the binding to the ligand and releasing the ligand bound to the target tissue. The released ligand exerts biological activity in the target tissue and can treat a disease caused by the target tissue. In an embodiment 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 is not exerted during transport, but is only exerted when cleaved in the target tissue, allowing the disease to be treated and systemic side effects to be suppressed.

[0138] 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. A composition containing both a ligand-binding molecule and a ligand can also 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 ligand administered to the individual and is transported in the body while still bound to the ligand. The ligand-binding molecule transported to the target tissue is cleaved in the target tissue, weakening its binding to the ligand and allowing the ligand bound to the target tissue to be released. The released ligand exerts its biological activity in the target tissue and can treat a disease caused by the target tissue. In an embodiment 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 to the target tissue, the biological activity of the ligand is not exerted during transport, but is only exerted when cleaved in the target tissue, allowing the disease to be treated and systemic side effects to be suppressed. A ligand-binding molecule administered to an individual can bind not only to the ligand administered to the individual, but also to ligands that are originally present in the individual, and can transport the ligand originally present in the individual or the ligand administered to the individual in the bound state within the body.

[0139] In some embodiments of the present invention, a fusion protein in which a ligand-binding molecule and a ligand are fused 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 in which a ligand-binding molecule and a ligand are fused is administered to an individual, the fusion protein is delivered in the body, and the ligand-binding molecule portion in the fusion protein is cleaved in the target tissue, thereby weakening the non-covalent bond of the ligand-binding molecule portion to 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 can exert the biological activity of the ligand in the target tissue, and a disease caused by the target tissue can be treated. In an embodiment 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 during delivery is not exerted, but the biological activity of the ligand can be exerted only after cleavage in the target tissue, and the disease can be treated, and systemic side effects can be suppressed.

[0140] The pharmaceutical composition of the present invention can be formulated using a method known to those skilled in the art. For example, it can be used parenterally in the form of a sterile solution or suspension injection with water or other pharma- ceutically acceptable liquid. For example, it can be formulated by appropriately combining with a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc., and mixing in a unit dose form required for generally accepted pharmaceutical practice. The amount of active ingredient in these preparations is set so that an appropriate volume within the indicated range is obtained.

[0141] Sterile compositions for injection can be formulated according to common pharmaceutical practice using a vehicle such as distilled water for injection. Aqueous solutions for injection include, for example, isotonic solutions containing physiological saline, glucose, and other auxiliary agents (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). Appropriate solubilizing agents such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80(TM), HCO-50, etc.) can be used in combination.

[0142] The oily liquid may include sesame oil and soybean oil, and may also be used in combination with benzyl benzoate and / or benzyl alcohol as a solubilizing agent. It may also be combined with a buffer (e.g., phosphate buffer and sodium acetate buffer), a soothing agent (e.g., procaine hydrochloride), a stabilizer (e.g., benzyl alcohol and phenol), and an antioxidant. The prepared injection solution is usually filled into a suitable ampule.

[0143] The pharmaceutical composition of the present invention is preferably administered parenterally. For example, the composition is administered in the form of an injection, a nasal administration, a pulmonary administration, or a transdermal administration. For example, the composition can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.

[0144] The administration method may be appropriately selected depending on the age and symptoms of the patient. The dosage of the pharmaceutical composition containing the ligand-binding molecule may be set, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight per administration. Alternatively, for example, the dosage may be set to 0.001 to 100,000 mg per patient, but 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 a person skilled in the art can set an appropriate dosage and administration method taking these conditions into consideration.

[0145] 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 of the present invention, there is provided 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.

[0146] 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, and a method in which a part of the amino acid sequence of a polypeptide capable of binding to a ligand is replaced with a protease cleavage sequence.

[0147] An example of a method for obtaining a molecule capable of binding to a ligand is a method for obtaining a ligand-binding domain having the ability to bind to a ligand, which 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 it is in the ligand-binding region, or only the Fv region of the obtained antibody may be used, or when the Fv region is a single chain (also called "sc") and capable of recognizing an antigen, only the single chain may be used. Also, a Fab region containing the Fv region may be used.

[0148] Specific methods for producing antibodies are well known to those skilled in the art. For example, monoclonal antibodies may be produced by the hybridoma method (Kohler and Milstein, Nature 256:495 (1975)) or recombinant method (U.S. Patent No. 4,816,567). They may also be isolated from a phage antibody library (Clackson et al., Nature 352:624-628 (1991); Marks et al., J.Mol.Biol. 222:581-597 (1991)). They may also be isolated from a single B cell clone (N. Biotechnol. 28(5): 253-457 (2011)).

[0149] A humanized antibody is also called a reshaped human antibody. Specifically, humanized antibodies in which the CDRs of an antibody from a non-human animal, such as a mouse antibody, are grafted onto a human antibody are known. General gene recombination techniques for obtaining humanized antibodies are also known. Specifically, overlap extension PCR is known as a method for grafting the CDRs of a mouse antibody onto human FRs.

[0150] A vector for expressing a humanized antibody can be prepared by inserting a DNA encoding an antibody variable region in which three CDRs and four FRs are linked with a 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 a recombinant cell, the recombinant cell is cultured to express the DNA encoding the humanized antibody, and the humanized antibody is produced in the culture of the cultured cell (see European Patent Application Publication No. 239400 and International Publication No. WO 1996 / 002576).

[0151] 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 for grafting mouse CDRs to human FRs.

[0152] 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, WO 1996 / 033735) are used as immunized animals, and desired human antibodies can be obtained by DNA immunization.

[0153] Furthermore, a technique for obtaining a human antibody 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. A phage expressing an scFv that binds to an antigen can be selected. By analyzing the gene of the selected phage, the DNA sequence encoding the Fv region of a human antibody that binds to an antigen can be determined. After determining the DNA sequence of the scFv that binds to an 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 as 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 and WO 1995 / 015388).

[0154] A molecule in which a protease cleavage sequence has been introduced into a molecule capable of binding to a ligand is the ligand-binding molecule of the present invention. Optionally, it can be confirmed whether the ligand-binding molecule is cleaved by treatment with a protease corresponding to the protease cleavage sequence. For example, it can be confirmed whether the protease cleavage sequence has been cleaved by contacting a molecule in which a protease cleavage sequence has been introduced into a molecule capable of binding to a ligand with a protease and confirming the molecular weight of the product after protease treatment by electrophoresis such as SDS-PAGE.

[0155] The present invention also relates to polynucleotides encoding ligand-binding molecules whose binding to a ligand is attenuated in a cleaved state, or polynucleotides encoding fusion proteins in which the ligand-binding molecule is fused with a ligand.

[0156] The polynucleotide of the present invention is usually carried (inserted) in a suitable vector and introduced into a host cell. The vector is not particularly limited as long as it stably retains the inserted nucleic acid. For example, if E. coli is used as the host, a cloning vector such as pBluescript vector (Stratagene) is preferred, but various commercially available vectors can be used. When a vector is used for the purpose of producing the ligand-binding molecule or fusion protein of the present invention, an expression vector is particularly useful. The expression vector is not particularly limited as long as it expresses the ligand-binding molecule in a test tube, in E. coli, in cultured cells, or in an individual organism. For example, the pBEST vector (Promega) for in vitro expression, the pET vector (Invitrogen) for E. 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 are preferred. The DNA of the present invention can be inserted into a vector by standard methods, for example, 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).

[0157] The host cell is not particularly limited, and various host cells are 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. Vector introduction into a host cell can be performed by known methods such as calcium phosphate precipitation, electric pulse perforation (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), lipofectamine method (GIBCO-BRL), and microinjection.

[0158] 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 a host cell into the lumen of the endoplasmic reticulum, into the periplasmic space, or into the extracellular environment. These signals can be endogenous to the ligand binding molecule or fusion protein of interest or they can be heterologous signals.

[0159] In the above production methods, the ligand-binding molecule or fusion protein is recovered by collecting the medium when the polypeptide of the present invention is secreted into the medium, or by lysing the cells first and then recovering the ligand-binding molecule or fusion protein when the ligand-binding molecule or fusion protein is produced intracellularly.

[0160] Ligand binding molecules or fusion proteins of the invention can be recovered and purified from recombinant cell cultures 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.

[0161] It is naturally understood by those skilled in the art that any combination of one or more of the embodiments described herein is included in the present invention, so long as there is no technical inconsistency based on the technical common knowledge of those skilled in the art. In addition, an invention that excludes any combination of one or more of the embodiments described herein from the present invention is also to be considered as an invention contemplated and described in the present specification, so long as there is no technical inconsistency based on the technical common knowledge of those skilled in the art. EXAMPLES

[0162] The following are examples of the methods and compositions of the present invention. In light of the general description above, it will be understood that various other embodiments may be practiced.

[0163] 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 end of the targeting IgG or scFv (Expert Opin Investig Drugs. 2009 Jul;18(7):991-1000., Curr Opin Immunol. 2016 Jun;40:96-102.). Cytokines such as IL2, IL12, and TNF are highly toxic, so it is expected that these cytokines can be delivered to the cancer local area by antibodies to work at the cancer local area while reducing side effects and enhancing their effectiveness (Non-Patent Documents 4, 5, 6). However, all of these have problems such as not showing sufficient clinical effects when administered systemically, having a narrow therapeutic window, and being highly toxic and not being able to be administered systemically. The main reason for this is that even if they are immunocytokines, cytokines administered systemically are exposed to the entire body, so they may act systemically and exhibit toxicity, or they can only be administered at extremely low doses to avoid toxicity. In addition, immunocytokines that bind to cancer antigens are internalized by cancer cells in tumors and disappear, 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 IL2 is fused to an antibody that binds to a cancer antigen is the same as that of an immunocytokine in which IL2 is fused to an antibody that does not bind to a cancer antigen (Non-Patent Document 7).

[0164] As a method for reducing the systemic effect, which is a major issue for immunocytokines, molecules have been reported in which the cytokine and cytokine receptor are linked by a linker that is cleaved by a protease that is 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. For example, 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 IL2 and IL2R are linked by a linker that is cleaved by MMP2 (Non-Patent Document 9) have been reported. However, in these molecules, the cytokines have biological activity even before the linker is cleaved, and cleavage of the linker only increases activity by about 10 times. This is due to two reasons: first, the affinity between the cytokine and the cytokine receptor is not strong, so that the cytokine retains a certain degree of activity even before protease cleavage; and second, the cytokine receptor can bind to the cytokine even after the linker is cleaved by the protease, thereby inhibiting the biological activity of the cytokine.

[0165] A molecule has been reported in which anti-IL2 scFv, instead of IL2R, is bound to IL2 via a linker that is cleaved by MMP-2 (Non-Patent Document 9). Considering that this molecule in which IL2 and anti-IL2 scFv are bound via a protease-cleavable linker also liberates IL2 upon linker cleavage, just like a molecule in which a cytokine is bound to a cytokine receptor, it is natural to use an anti-IL2 scFv that does not have a strong affinity for IL2. In addition, unlike the above-mentioned IgG-IL2 fusion, these reported protease-activated cytokines do not have an Fc region, so they are expected to have 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.

[0166] 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, and have the activity of 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 cause the migration of leukocytes from within blood vessels into inflamed tissues. Since chemokines can control the migration of white blood cells, they may be useful in cancer immunotherapy. If T cells, antigen-presenting cells, M1 macrophages, etc. can be induced to migrate to the local area of ​​solid cancer, it is believed that an antitumor 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 using systemic administration of chemokines (chemokine therapy) is not considered realistic.

[0167] Example 3: Concept of a ligand-binding molecule capable of releasing a target tissue-specific ligand 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, side effects occur because cytokines act throughout the body, or they can only be administered in low doses to avoid side effects, meaning that high exposure cannot be achieved in the tumor. 2. In the case of protease-activated cytokines, where a linker that can be cleaved by a protease is used to connect the cytokine receptor (or antibody) and the cytokine, neutralization of cytokine activity is insufficient and the cytokine retains a certain degree of 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, thus inhibiting the biological activity of the cytokine. 4. In the case of cytokines that are activated by proteases, the half-life of the inactive cytokines is short and their retention time in the blood is short, so that a large dose is required.

[0168] In order 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 by ligand-binding molecules (biological activity is minimized). 2. The biological activity of the ligand is restored by cleavage with a protease (it becomes an active ligand). 3. Ligand-binding molecules lose their ligand-binding activity upon 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.

[0169] As a pharmaceutical composition that satisfies the above conditions, we have devised 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 produce a ligand-binding molecule.

[0170] Example 4: Examples of anti-ligand antibodies incorporating a protease cleavage sequence 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 inserted 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 inserted. It is confirmed that the ligand dissociates when cleaved by a protease while bound to the anti-ligand neutralizing antibody. It is confirmed that the dissociated ligand exerts biological activity. In FIG. 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 inserted near the boundary between VH and CH1. If the affinity of the anti-ligand antibody to the ligand is strong enough, the biological activity of the ligand is sufficiently inhibited. Even if this ligand-anti-ligand antibody fusion is administered systemically, the ligand is neutralized and does not exhibit its biological activity, and the ligand-anti-ligand antibody fusion has a long half-life because it has an Fc region. When the protease cleavage sequence near the boundary between VH and CH1 of the systemically administered ligand-anti-ligand antibody fusion is cleaved by a protease highly expressed in tumor tissue, the VH molecule of the ligand-linker-anti-ligand antibody is released. Since VH or VL alone cannot bind to the ligand (both VH and VL are required to bind to the ligand), the neutralization of the ligand is released and it is possible to exert a biological effect in the tumor tissue. Furthermore, since the VH molecule of this released ligand-linker-anti-ligand 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, and the ligand is administered in a mixture with an anti-ligand antibody with a protease cleavage sequence inserted near the boundary between VH and CH1. If the affinity of the anti-ligand antibody to the ligand is strong enough and there is enough 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, and the ligand-anti-ligand antibody complex has a long half-life because it has an Fc region. When the protease cleavage sequence near the boundary between VH and CH1 of the systemically administered ligand-anti-ligand antibody complex is cleaved by a protease highly expressed in tumor tissue, the VH molecule of the anti-ligand antibody is released. Since VH or VL alone cannot bind to the ligand (both VH and VL are required to bind to the ligand), the neutralization of the ligand is released and it is possible to exert a biological effect in the 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, thereby minimizing systemic side effects caused by the ligand. In Figure 3, an anti-ligand antibody with a protease cleavage sequence inserted near the boundary between VH and CH1 is administered systemically. The administered antibody binds to the ligand originally present in the body, and the subsequent steps are the same as those described above in Figure 2. In this way, by using an anti-ligand antibody in which a protease cleavage sequence has been inserted near the boundary between VH and CH1, it is possible to selectively release the ligand in tissues where the protease is expressed, and to allow the ligand to exert its biological effect. If the ligand is a cytokine, the cytokine can be made to act selectively 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, and the chemokine concentration in peripheral blood is low, allowing cells expressing the chemokine receptor to migrate to tissues where the protease is expressed.

[0171] Example 5: Production and evaluation of CXCL10-releasing antibodies 5-1. Introduction of a protease cleavage sequence into anti-CXCL10 neutralizing antibodies CXCL10 is one of the chemokines that has a migratory effect on effector T cells. An expression vector for MabCXCL10 (heavy chain: EEIVH (SEQ ID NO: 1), light chain: EEIVL (SEQ ID NO: 2)), a neutralizing antibody against human CXCL10, was prepared by a method known to those skilled in the art, and expressed and purified by a method known to those skilled in the art using FreeStyle 293 (Life Technology). 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 the amine coupling method using NHS·EDC, and 1.563 nM human CXCL10 was run as an analyte in a running buffer of 20 mM ACES, 0.05% Tween20, 200 mM NaCl, pH 7.4, after the antibody was captured. The binding of the antibody to the antigen at 37°C was evaluated. The sensorgram showing the amount of binding over time, which is the difference from the blank in which only the running buffer was used as the analyte, is shown in Figure 4. The time when the analyte started to flow is taken as the starting point on the horizontal axis. The vertical axis represents the response (amount of binding) at each time point, with the response at the time when the analyte started to flow being 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. We designed the heavy and light chains shown in Figure 5 in which peptide sequence A (SEQ ID NO: 3), a sequence that has been reported to be cleaved by urokinase (uPA) and matriptase (MT-SP1), which are expressed in a cancer-specific manner, was inserted at seven sites near the boundary between the variable and constant regions of the heavy or light chain. We also designed a variant in which glycosylation would not occur due to the insertion of the cleavage sequence. Expression vectors encoding the heavy chain variants EEIVHA (SEQ ID NO: 4), EEIVHB (SEQ ID NO: 5), EEIVHC (SEQ ID NO: 6), EEIVHD (SEQ ID NO: 7), EEIVHE (SEQ ID NO: 8), EEIVHF (SEQ ID NO: 9), EEIVHG (SEQ ID NO: 10), EEIVHBG (SEQ ID NO: 11), EEIVHCG (SEQ ID NO: 12), EEIVHDG (SEQ ID NO: 13), EEIVHEG (SEQ ID NO: 14), and the light chain variants EEIVLA (SEQ ID NO: 15), EEIVLB (SEQ ID NO: 16), EEIVLC (SEQ ID NO: 17), EEIVLD (SEQ ID NO: 18), EEIVLE (SEQ ID NO: 19), EEIVLF (SEQ ID NO: 20), EEIVLG (SEQ ID NO: 21), and EEIVLEG (SEQ ID NO: 22) were prepared by methods known to those skilled in the art. The following IgG1 antibodies were synthesized by combining these heavy chain variants with native light chains, or by combining native heavy chains with light chain variants and inserting a protease cleavage sequence near the boundary between the heavy chain variable region and the constant region: 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 expressed by transient expression 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.

[0172] 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 the amine coupling method using NHS·EDC, and 3.125, 1.563, and 0.781 nM human CXCL10 were run as an analyte in a running buffer of 20 mM ACES, 0.05% Tween20, 150 mM NaCl, pH 7.4, and the antibody was captured. The binding of the antibody to the antigen at 25°C was evaluated. The sensorgram showing the amount of binding over time, calculated as the difference from the blank value using only the running buffer as the analyte, is shown in Figure 6. The horizontal axis indicates the starting point of the time when the analyte flow started. The vertical axis indicates the response (amount of binding) at each time, with the response at the time when the analyte flow started being set to 0. As shown in the sensorgrams in Figure 6, all of the antibodies bound to human CXCL10. In other words, it was possible to insert a protease cleavage sequence near the boundary between the antibody variable region and constant region without losing the binding activity to the antigen.

[0173] 5-3. Evaluation of protease cleavage of anti-CXCL10 neutralizing antibodies incorporating a protease cleavage sequence We examined whether the antibodies prepared in 5-1 were cleaved by proteases. We used recombinant human matriptase / ST14 catalytic domain (MT-SP1) (R&D Systems, 3946-SE-010) as the protease, and reacted the antibodies for 20 hours under the conditions of 20 nM protease, 60 or 100 μg / mL antibody, PBS, and 37°C. After that, we evaluated the cleavage by proteases by reducing SDS-PAGE. The results are shown in Figure 7. As a result, new bands between 25 kDa and 50 kDa were generated by protease treatment for EEIVHA / EEIVL, EEIVHE / EEIVL, EEIVHF / EEIVL, EEIVHG / EEIVL, EEIVHEG / EEIVL, and EEIVHBG / EEIVL. Furthermore, in EEIVH / EEIVLEG, EEIVH / EEIVLF, and EEIVH / EEIVLG, bands of 25 kDa or less were generated by protease treatment, confirming that the antibodies were cleaved by proteases in EEIVHA / EEIVL, EEIVHE / EEIVL, EEIVHF / EEIVL, EEIVHG / EEIVL, EEIVHEG / EEIVL, EEIVHBG / EEIVL, EEIVH / EEIVLEG, EEIVH / EEIVLF, and EEIVH / EEIVLG.

[0174] 5-4. Introduction of a flexible linker sequence near the protease cleavage sequence of an anti-CXCL10 neutralizing antibody In 5-3, an investigation was carried out into inserting a sequence containing a linker made of a glycine-serine polymer near the protease cleavage sequence of EEIVHC / EEIVL that was not cleaved by recombinant human matriptase / ST14 (MT-SP1) catalytic domain (R&D Systems, 3946-SE-010). Five types of heavy chains were designed, as shown in FIG. 8. Expression vectors encoding the heavy chain variants EEIVHC002 (SEQ ID NO: 23), EEIVHC003 (SEQ ID NO: 24), EEIVHC004 (SEQ ID NO: 25), EEIVHC005 (SEQ ID NO: 26), and EEIVHC006 (SEQ ID NO: 27) were prepared by a method 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 region and constant region 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 expressed by transient expression 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.

[0175] 5-5. Evaluation of 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, and 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, and 6.25, 3.125, 1.563, and 0.781 nM human CXCL10 was run as an analyte in a running buffer of 20 mM ACES, 0.05% Tween20, 300 mM NaCl, pH 7.4, and the antibody was captured. The binding of the antibody to the antigen at 25°C was evaluated. The sensorgram showing the amount of binding over time, calculated as the difference from the blank in which only the running buffer was used as the analyte, is shown in Figure 9. The horizontal axis indicates the starting point of the time when the analyte started to flow. The vertical axis indicates the response (amount of binding) at each time when the response at the time when the analyte started to flow was set to 0. As shown in the sensorgram in Figure 9, all of the antibodies bound to human CXCL10. In other words, it was possible to insert a protease cleavage sequence and a flexible linker sequence near the boundary between the antibody variable region and constant region without losing the binding activity to the antigen.

[0176] 5-6. Evaluation of protease cleavage of anti-CXCL10 neutralizing antibodies incorporating a protease cleavage sequence and a flexible linker sequence We examined whether the antibody prepared in 5-5 was 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, and the antibody was reacted for 2 and 20 hours under the conditions of 12.5 nM protease, 133 μg / mL antibody, PBS, and 37°C. The cleavage by the proteases was evaluated by reducing SDS-PAGE. The results are shown in Figure 10. As a result, in EEIVHC002 / EEIVL, EEIVHC003 / EEIVL, EEIVHC004 / EEIVL, EEIVHC005 / EEIVL, and EEIVHEC006 / EEIVL, new bands were generated between 25 kDa and 50 kDa upon protease treatment, confirming that the antibodies were cleaved by proteases in EEIVHC002 / EEIVL, EEIVHC003 / EEIVL, EEIVHC004 / EEIVL, EEIVHC005 / EEIVL, and EEIVHEC006 / EEIVL. These results demonstrated that even if an antibody is not cleaved by a protease when only a protease cleavage site is introduced near the boundary between the variable and constant regions, as in the case of EEIVHC / EEIVL, a molecule that can be cleaved by a protease can be created by introducing a flexible linker sequence near the cleavage sequence. Therefore, it was demonstrated that an antibody that can be cleaved by a protease can be created by arbitrarily combining a protease cleavage sequence and a flexible linker.

[0177] 5-7. CXCL10 - Ligand activation by protease cleavage of anti-CXCL10 neutralizing antibodies Next, whether human CXCL10 bound to the antibody prepared in 5-5 is released by protease treatment was evaluated using Biacore. Specifically, using the antibody EEIVHC006a / EEIVL (heavy chain sequence number: 33, light chain sequence number: 2) prepared in 5-5, analytes with / with antigen and with protease, without antigen and with protease, and with antigen and without protease were prepared. The analyte with / with antigen and 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 analyte without antigen / with protease was prepared by treating only the antibody with 20 nM recombinant human matriptase / ST14 catalytic domain (MT-SP1) (R&D Systems, 3946-SE-010) for 20 hours. The analyte with antigen / without protease was prepared by binding the antibody to human CXCL10. In addition, CXCL10 was prepared as an antigen-only analyte as a system control to confirm that the response was due to CXCL10 binding. Anti-CXCL10 antibody was immobilized on a CM5 sensor chip (BR100530, GE Healthcare) by a method known to those skilled in the art. Four types of analyte were run using 20 mM ACES, 0.05% Tween20, pH 7.4 as a running buffer: antigen with / with protease, antigen without / with protease, antigen with / without protease, and antigen only, to evaluate the binding of anti-CXCL10 antibody on the sensor chip to human CXCL10 at 25°C. In addition, using MabCXCL10a (heavy chain: EEIVHa (sequence number: 65), light chain: EEIVL (sequence number: 2)), which has a Fab region similar to that of antibody MabCXCL10 that does not have a protease cleavage sequence, analytes with antigen / with protease, without antigen / with protease, and with antigen / without protease were prepared in the same manner as antibody EEIVHC006a / EEIVL. Similarly, anti-CXCL10 antibody was immobilized on a CM5 sensor chip (BR100530, GE Healthcare) by a method known to those skilled in the art, and four types of analyte were run using 20 mM ACES, 0.05% Tween20, pH 7.4 as the running buffer: analyte with antigen / with protease, analyte without antigen / with protease, analyte with antigen / without protease, and analyte only with antigen (CXCL10), to evaluate the binding of the anti-CXCL10 antibody on the sensor chip to human CXCL10 at 25°C. Figure 11 shows a sensorgram showing the amount of binding over time, calculated as the difference from the amount of binding in a flow cell on which anti-CXCL10 antibody was not immobilized. The time when the analyte started to flow is set as the starting point on the vertical axis. The vertical axis shows the response at each time, with the response at the start of the analyte flow set as 100. As a result, as shown in Figure 11(A), protease treatment of MabCXCL10a, which does not have a cleavage sequence inserted, did not release CXCL10, but as shown in Figure 11(B), protease treatment of EEIVHC006a / EEIVL released CXCL10.

[0178] 5-8. Preparation of anti-CXCL10 neutralizing antibodies in which part of the amino acid sequence near the boundary between the antibody variable and constant regions is replaced with a protease cleavage sequence and part of the flexible linker sequence, and evaluation of cleavage by proteases An investigation was carried out into 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 sequence of a flexible linker. A heavy chain shown in FIG. 12 was designed in which a portion of the amino acids in the heavy chain was replaced with peptide sequence A (SEQ ID NO: 3), a sequence that has been 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 prepared by 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 expressed by transient expression 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 examined whether EESVHA009 / EEIVL and EESVHA012 / EEIVL are 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, and the samples were reacted for 20 hours under the conditions of 12.5 nM protease, 100 μg / mL antibody, PBS, and 37°C. The results of the evaluation of protease cleavage by the proteases using reducing SDS-PAGE are shown in Figure 13. As a result, new bands between 25 kDa and 50 kDa were generated in EESVHA009 / EEIVL and EESVHA012 / EEIVL upon protease treatment. Therefore, it was confirmed that the antibodies in EESVHA009 / EEIVL and EESVHA012 / EEIVL were cleaved by proteases.

[0179] Example 6 Consideration of permissible sites for inserting cleavage sequences to eliminate antigen-binding ability by protease cleavage There is 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 the in vitro evaluation of its function (International Publication WO2004 / 021861A2). Although no experimental data is given, it is claimed that the antigen is released from the antigen-antibody complex when this antibody is mixed with an antigen and then treated with a medium containing the corresponding protease. The 216th amino acid in the heavy chain of the human IgG1 antibody, in which the report claims that a protease cleavage sequence has been inserted, is not an aspartic acid in any of the numbering systems, Kabat numbering, EU numbering, or OU numbering, described in Kabat, E. et al. Sequences of Proteins of Immunological Interest 5th edition. On the other hand, referring to a different document, the 216th amino acid in the heavy chain of the human IgG1 antibody is considered to be an aspartic acid immediately following the cysteine ​​that forms a disulfide bond between the heavy chain and the light chain (Nature 344, 667-670 (12 April 1990), Kabat, E. et al. Sequences of Proteins of Immunological Interest 4th edition). When a protease cleavage sequence is inserted immediately before this 216th aspartic acid, it is considered that an Fab region similar to that formed when the hinge region of the antibody is cleaved by papain is formed from the antibody cleaved by the protease. It is generally recognized that cleavage of the hinge region of an antibody by papain is unlikely to result in loss of antigen-binding ability. Therefore, even if an antibody having a protease cleavage sequence inserted immediately before aspartic acid 216 is cleaved with the corresponding protease, it is unlikely to lose its antigen-binding ability. Let us also consider the case where a protease cleavage sequence is inserted just before the 216th amino acid (Kabat numbering) of the heavy chain of a human IgG1 antibody in the Kabat numbering described in Kabat, E. et al. Sequences of Proteins of Immunological Interest 5th edition. This site is located a few amino acids N-terminal to the 220th cysteine ​​(Kabat numbering) where a disulfide bond is formed between the heavy chain and the light chain. Therefore, it is assumed that the effect of the cleavage of the heavy chain by a protease occurring at this site is similar to the effect caused by the loss of the disulfide bond formed between the heavy chain and the light chain. Based on previous literature, it is unlikely that antigen binding will be lost even in the Fab region where disulfide bonds cannot be formed between the heavy chain and the light chain (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 considered that the antigen-binding ability is not lost by protease cleavage.

[0180] 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 having a protease cleavage sequence prepared in Example 5 liberates CXCL10 through protease cleavage, and whether CXCL10 exerts cell migration activity. The cell migration activity of CXCL10 was evaluated by preparing Ba / F3 transfectant cells expressing mouse CXCR3 (mCXCR3) (hereafter referred to as BaF3 / mCXCR3) and then measuring the cell migration activity of CXCL10 using HTS Transwell. TMThe assay was performed using -96 Permeable Support with 5.0μm Pore Polycarbonate Membrane (Cat. 3387, Corning). Five types of analytes were prepared: CXCL10+protease, EEIVHC006a / EEIVL+CXCL10, EEIVHC006a / EEIVL+CXCL10+protease, EEIVHC006a / EEIVL+protease, and MabCXCL10+CXCL10+protease. In a 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 further added after the above reaction to a final concentration of 12.5 nM. 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 so that the cells were 100 cells / well, and the reaction was allowed to proceed for 6 hours. The reaction was carried out under conditions of 5% carbon dioxide and 37°C. After the 6-hour reaction, 100 μL of the solution in the lower chamber was transferred to a fluorescent luminescence 96-well plate (Cat. 3912, Corning) and analyzed by CellTiter-Glo. TM 100 μL of Luminescent Cell Viability Assay solution (Cat. G7571, Promega) was added. After reacting at room temperature for 10 minutes, the luminescence value was measured using a SpectraMax M3 multimode microplate reader (Molecular Devices) to evaluate the degree of cell migration to the lower chamber. The results are shown in Figure 14. The luminescence intensity decreased when EEIVHC006a / EEIVL+CXCL10 analyte was added compared to CXCL10+protease analyte. The luminescence intensity reflected the amount of migrated cells, indicating that EEIVHC006a / EEIVL formed a complex with CXCL10 and neutralized the action of CXCL10. On the other hand, the luminescence intensity was restored when EEIVHC006a / EEIVL+CXCL10+protease analyte was added compared to EEIVHC006a / EEIVL+CXCL10 analyte, indicating that it induces cell migration in the same way as when CXCL10+protease analyte was added. When MabCXCL10 antibody without a cleavage sequence was used and MabCXCL10+CXCL10+protease analyte was added, the luminescence intensity did not recover. These results demonstrated that the neutralizing ability of EEIVHC006a / EEIVL to CXCL10 decreased following antibody cleavage by proteases.

[0181] Example 8 Evaluation of migration activity following protease cleavage of anti-CXCL10 neutralizing antibody-CXCL10 fusion protein incorporating a protease cleavage sequence 8-1 Preparation of anti-CXCL10 neutralizing antibody-CXCL10 fusion protein and protease cleavage evaluation 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 fusion 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 resistant to proteases, to the N-terminus of the light chain via a linker sequence consisting of a glycine-serine polymer. The ligand fusion light chain and MabCXCL10_G7 heavy chain G7H-G1T4 were combined to produce a fusion protein: G7H-G1T4 / hCXCL10R75A.G4SGGGG.G7L-LT0 (heavy chain sequence number: 368, ligand fusion light chain sequence number: 371) which was expressed by transient expression 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 this fusion protein was cleaved by proteases. Human urokinase (human uPA, huPA) (R&D Systems, 1310-SE-010) was used as the protease. Cleavage of the fusion protein by proteases was evaluated by reducing SDS-PAGE. 0.1 mg / ml of the fusion protein was reacted with 30 nM huPA at 37°C for 1 hour, and then cleavage of the fusion protein was evaluated by reducing SDS-PAGE. As a result, G7H-G1T4 / hCXCL10R75A.G4SGGGG.G7L-LT0 was not cleaved by protease treatment (Figure 15).

[0182] 8-2. Evaluation of migration activity following protease cleavage of anti-CXCL10 neutralizing antibody-CXCL10 fusion protein We evaluated whether an anti-CXCL10 neutralizing antibody-CXCL10 fusion protein, in which CXCL10 was fused to an anti-CXCL10 neutralizing antibody containing a protease cleavage sequence, could release CXCL10 by protease cleavage and induce cell migration. As a control for comparing 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 human CXCL10 mutant hCXCL10R75A (SEQ ID NO: 370), which had been mutated to be resistant to proteases, to prepare 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. The 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 cell migration activity using HTS Transwell. TM -96 Permeable Support with 5.0μm Pore Polycarbonate Membrane (Cat. 3387, Corning) was used for evaluation. As an analyte for uPA(+), recombinant huPA (Cat. 1310-SE, R&D systems) was added to 0.15 μg / mL hCXCL10R75A-His and 1.5 μg / mL G7H-G1T4 / hCXCL10R75A.G4SGGGG.G7L-LT0, a fusion protein lacking a protease cleavage sequence, in a 2.0 mL 96-well deep well plate (Cat. P-DW-20-CS, Axygen) to a final concentration of 30 nM. G7H-G1T4 / hCXCL10R75A.G4SGGGG.G7L-LT0 1.5 μg / mL contains 0.15 μg / mL equivalent amount of hCXCL10R75A. The uPA(-) analytes used were hCXCL10R75A-His at 0.15 μg / mL and the fusion protein G7H-G1T4 / hCXCL10R75A.G4SGGGG.G7L-LT0 at 1.5 μg / mL, which does not have a 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. 5 The cells were seeded at 75 μL / well so that the cells were 100 cells / well, and the reaction was allowed to proceed for 6 hours. The reaction was carried out under conditions of 5% carbon dioxide and 37°C. After the 6-hour reaction, 100 μL of the solution in the lower chamber was transferred to an OptiPlate-96 (Cat. 6005299, PerkinElmer) and analyzed by CellTiter-Glo. TM 100 μL of Luminescent Cell Viability Assay solution (Cat. G7571, Promega) was added. After 10 minutes of reaction at room temperature, the luminescence intensity was measured using a SpectraMax M3 multimode microplate reader (Molecular Devices) to evaluate the degree of cell migration to the lower chamber. The results are shown in Figure 16. Compared to the case where CXCL10R75A-His was added, G7H-G1T4 / hCXCL10R75A.G4SGGGG.G7L-LT0, which does not have a cleavage sequence, did not recover in luminescence intensity even after protease treatment.

[0183] 8-3 Construction of anti-CXCL10 neutralizing antibody-CXCL10 fusion protein incorporating a protease cleavage sequence and evaluation of migration activity following protease cleavage The amino acid sequence is designed to include a protease cleavage sequence near the boundary between the variable and constant regions of the ligand fusion light chain hCXCL10R75A.G4SGGGG.G7L-LT0 (SEQ ID NO: 371) constructed in 8-1. The ligand fusion light chain into which the protease cleavage sequence has been introduced is combined with G7H-G1T4 of the MabCXCL10_G7 heavy chain, and the fusion protein is expressed by transient expression 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. We will verify whether these fusion proteins are cleaved by proteases. Human urokinase (human uPA, huPA) (R&D Systems, 1310-SE-010) is used as the protease. Cleavage of the fusion proteins by proteases is evaluated by reducing SDS-PAGE. After reacting 0.1 mg / ml of the fusion proteins with 30 nM huPA at 37°C for 1 hour, cleavage of the fusion proteins is evaluated by reducing SDS-PAGE. We will evaluate whether the anti-CXCL10 neutralizing antibody-CXCL10 fusion protein, which is a fusion protein of CXCL10 and an anti-CXCL10 neutralizing antibody with a protease cleavage sequence, releases CXCL10 by protease cleavage and induces cell migration. We prepared Ba / F3 transfectant cells expressing mouse CXCR3 (mCXCR3) (hereinafter referred to as BaF3 / mCXCR3) and used these cells in HTS Transwell. TM -96 Permeable Support with 5.0μm Pore Polycarbonate Membrane (Cat. 3387, Corning) is used for evaluation. For the uPA(+) analyte, add recombinant huPA (Cat. 1310-SE, R&D systems) to a final concentration of 30 nM in a 2.0 mL 96-well deep well plate (Cat. P-DW-20-CS, Axygen) to 0.15 μg / mL hCXCL10R75A-His or 1.5 μg / mL fusion protein with a protease cleavage sequence prepared in 8-2. The 1.5 μg / mL fusion protein with a protease cleavage sequence contains 0.15 μg / mL of hCXCL10R75A. For the uPA(-) analyte, use 0.15 μg / mL hCXCL10R75A-His or 1.5 μg / mL fusion protein with a 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. 5 The cells were seeded at 75 μL / well so that the cells were 100 cells / well, and incubated for 6 hours. The incubation was carried out under 5% carbon dioxide gas at 37°C. After 6 hours of incubation, 100 μL of the solution in the lower chamber was transferred to an OptiPlate-96 (Cat. 6005299, PerkinElmer) and incubated with CellTiter-Glo. TM Add 100 μL of Luminescent Cell Viability Assay solution (Cat. G7571, Promega). After 10 minutes of incubation at room temperature, measure the luminescence intensity using a SpectraMax M3 multimode microplate reader (Molecular Devices) to evaluate the degree of cell migration to the lower chamber. Migration activity can be evaluated based on the luminescence intensity.

[0184] 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 one of the cytokines that activates the immune system. IL-12 exerts an antitumor effect by activating immune cells, but it has also been reported that systemic exposure to IL-12 can cause severe side effects (Nat Immunol. 2012 Jul 19;13(8):722-8.). A peptide sequence A (SEQ ID NO: 3), which has been reported to be cleaved by urokinase (uPA) and matriptase (MT-SP1), and a sequence including a movable linker consisting of a glycine-serine polymer were inserted near the boundary between the variable and constant regions of the heavy chain (UstkH-G1T4, heavy chain SEQ ID NO: 144) of an anti-IL12 antibody having the same variable region as Ustekinumab, a neutralizing antibody against human IL-12, to design a modified Ustekinumab heavy chain UstkH-G1T4CYTM1inP1 (SEQ ID NO: 146). This 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) by a method known to those skilled in the art. The Ustekinumab variant UstkH-G1T4CYTM1inP1 / UstkL-kT0 was expressed by transient expression using FreeStyle 293 (Life Technologies) by a method known to those skilled in the art, and purified by a method known to those skilled in the art using protein A. The CDR sequences contained in the anti-IL12 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).

[0185] 9-2. Protease cleavage of anti-IL-12 neutralizing antibodies incorporating a protease cleavage sequence and a flexible linker sequence We examined whether the antibody prepared in 9-1 above was cleaved by proteases. The proteases used were 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 the ustekinumab variant UstkH-G1T4CYTM1inP1 / UstkL-kT0 to final concentrations of 10.1, 16.9, or 9.17 μM, respectively, and incubating overnight at 37°C.

[0186] 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. As a result, UstkH-G1T4 / UstkL-kT0 was not cleaved by each protease, whereas UstkH-G1T4CYTM1inP1 / UstkL-kT0, which had a protease cleavage sequence and a flexible linker inserted therein, produced a new band between 25 kDa and 50 kDa upon treatment with each protease (Figure 17). Therefore, it was confirmed that the anti-IL-12 neutralizing antibody (UstkH-G1T4CYTM1inP1 / UstkL-kT0) with the protease cleavage sequence and flexible linker sequence introduced therein was cleaved by proteases. Next, we evaluated whether IL-12 is released from the antibody complex and exerts its biological activity when the antibody is cleaved by protease. The biological activity of IL-12 was evaluated based on the production of IFN-γ (interferon gamma, also written as IFN-g) by the NK92 human cell line. NK92 cells were cultured at 1 × 10 5The cells 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 the amount of IFN-γ produced after 48 hours was measured by ELISA. To evaluate the effect of the antibody on IL12 activity, an experiment was also performed in which only protease-treated IL12 was added without the addition of antibody (No Ab). Figure 18 shows the results of measuring the concentration of interferon gamma. UstkH-G1T4 / UstkL-kT0 (without a protease cleavage sequence) treated with various proteases inhibited (called neutralization) the production of interferon gamma by IL-12, and was at the same level as when no IL-12 was added (No IL-12) when the antibody was 0.8 μg / mL. On the other hand, UstkH-G1T4CYTM1inP1 / UstkL-kT0 (with a protease cleavage sequence) treated with various proteases produced interferon gamma at all antibody concentrations, compared to when UstkH-G1T4 / UstkL-kT0 without a protease cleavage sequence was added. These results confirmed that UstkH-G1T4CYTM1inP1 / UstkL-kT0 allows IL-12 to act on cells by reducing its neutralizing ability against IL-12 due to cleavage by proteases.

[0187] Example 10: Evaluation of an anti-human CXCL10 neutralizing antibody with a protease cleavage sequence introduced therein 10-1. Introduction of a protease cleavage sequence into anti-human CXCL10 neutralizing antibody Expression vectors for MabCXCL10 (heavy chain: EEIVH (sequence number: 1), light chain: EEIVL (sequence number: 2)) and MabCXCL10_G7 (heavy chain: G7H-G1T4 (sequence number: 368), light chain: G7L-LT0 (sequence number: 369)), which are antibodies that neutralize CXCL10, were prepared by methods known to those skilled in the art, and expressed and purified using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life technologies) by methods known to those skilled in the art. The cleavage sequence shown in sequence number 345 was inserted near the boundary between the variable and constant regions of the heavy chain of MabCXCL10 or MabCXCL10_G7, respectively, to produce the MabCXCL10 modified heavy chain EldHA0003-G1T4 (sequence number: 356) and the MabCXCL10_G7 modified heavy chain G7H.12aa-G1T4 (sequence number: 367).

[0188] By combining the above two types of modified heavy chains and light chains, MabCXCL10 variant EldHA0003 (heavy chain sequence number: 356, light chain sequence number: 2) and MabCXCL10_G7 variant G7H.12aa (heavy chain sequence number: 367, light chain sequence number: 369) were expressed by transient expression using FreeStyle293 cells (Invitrogen) or Expi293 cells (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.

[0189] 10-2. Evaluation of protease cleavage of anti-human CXCL10 neutralizing antibodies with a protease cleavage sequence introduced into the heavy chain region We verified whether the antibody prepared in 10-1 was cleaved by protease. Recombinant human matriptase / ST14 catalytic domain (human MT-SP1, hMT-SP1) (R&D Systems, 3946-SE-010) was used as the protease, and the antibody was reacted for 20 hours under the conditions of 10 nM protease, 50 μg / mL antibody, PBS, and 37°C, and then subjected to reducing SDS-PAGE. The results are shown in Figures 19A and 19B. In both the MabCXCL10 variant EldHA0003 and the MabCXCL10_G7 variant G7H.12aa, a new band was generated around 37 kDa by treatment with hMT-SP1. In other words, it was confirmed that the protease cleavage sequence shown in SEQ ID NO: 345 was cleaved by hMT-SP1. Furthermore, by a similar method, it was confirmed that the protease cleavage sequence shown in SEQ ID NO: 345 was also cleaved by human uPA and mouse uPA.

[0190] Example 11: Preparation and evaluation of polypeptides incorporating cleavage sequences for various proteases 11-1 Preparation 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 IL6R, 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), L-CDR3 (QQGNTLPYT, SEQ ID NO: 403). Table 4 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.

[0191] [Table 1]

[0192] These insertion sequences were inserted near the boundary between the heavy chain variable region and constant region of the MRA antibody to give 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), MEIVHMP7.2-MEIVHMP7.2G1T4 (heavy chain SEQ ID NO: 159) were designed, and expression vectors encoding these modified heavy chains were constructed by methods known to those skilled in the art. These modified heavy chains and MRA light chains were combined to produce the MRA modifications shown in Table 5, which were expressed by transient expression 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.

[0193] [Table 2]

[0194] 11-2. Evaluation of protease cleavage of polypeptides containing recognition sequences for various proteases We examined whether the MRA modified form 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. The assay was performed with 50 nM, 100 nM, or 500 nM protease, 50 μg / mL antibody, and assay buffer (MMP Activity Assay Kit (Fluorometric - Green) (ab112146), Component C: Assay Buffer). )) or 20 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, pH 7.2 (hereinafter referred to as Tris) at 37°C for 20 hours, and the results of evaluation of protease cleavage by reducing SDS-PAGE are shown in Figures 20A, 20B, and 21. The MRA modified antibodies were reacted with the proteases shown in Table 5. In MMP-2, the cleavage was MEIVHG4SMP2MP9G4S-MEIVHG4SMP2MP9G4SG1T4 / MRAL-k0, MEIVHG4SMP2.2G4S-MEIVHG4SMP2.2G4SG1T4 / MRAL-k0, MEIVHG4SMP2.4G4S-MEIVHG4SMP2.4G4SG1T4 / MRAL-k0, MEIVHMP2.1-MEIVHMP2.1G1T4 / MRAL-k0, MEIVHMP2.3-MEIVHMP23G1T4 / MRAL-k0. In MMP-7, the cleavage was MEIVHMP7.2-MEIVHMP7.2G1T4 / MRAL-k0. In MMP-9, the cleavage was MEIVHG4SMP2MP9G4S-MEIVHG4SMP2MP9G4SG1T4 / MRAL-k0, Cleavage of MEIVHG4SMP9G4S-MEIVHG4SMP9G4SG1T4 / MRAL-k0 was observed.

[0195] Example 12 Evaluation of antibodies with protease cleavage sequences introduced at various positions in the heavy chain 12-1 Preparation 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 in the MRA heavy chain variable region (MRAH, SEQ ID NO: 161) to prepare variants of the MRA heavy chain variable region shown in Table 6. These variants of the MRA heavy chain variable region were linked to the MRA heavy chain constant region (G1T4, SEQ ID NO: 162) to prepare variants of the MRA heavy chain, and expression vectors encoding the corresponding genes were prepared by a method known to those skilled in the art. In addition, peptide sequence B (SEQ ID NO: 160) was inserted into different positions in the MRA heavy chain constant region (G1T4, SEQ ID NO: 162) to prepare variants of the MRA heavy chain constant region shown in Table 7. These variants of the MRA heavy chain constant region were linked to the MRA heavy chain variable region (MRAH, SEQ ID NO: 161) to prepare variants of the MRA heavy chain, and expression vectors encoding the corresponding genes were prepared by a method known to those skilled in the art. The positions at which the protease cleavage sequences are 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 6 and 7. The insertion site in Table 6 refers to the position (Kabat numbering) in the antibody heavy chain variable region adjacent to the constant region, and the insertion site in Table 7 refers to the position (EU numbering) in the antibody heavy chain constant region adjacent to the variable region.

[0196] [Table 3] TIFF2025041812000005.tif135147

[0197] [Table 4]

[0198] The MRA heavy chain variants and MRA light chains prepared above were combined to produce the MRA variants shown in Table 8, which were expressed by transient expression 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.

[0199] [Table 5] TIFF2025041812000008.tif229139

[0200] 12-2. Evaluation of protease cleavage of anti-human IL6R neutralizing antibodies with protease cleavage sequences introduced into the antibody heavy chain We verified whether the modified MRA prepared in 12-1 could be cleaved by proteases. Recombinant human matriptase / ST14 catalytic domain (human MT-SP1, hMT-SP1) (R&D Systems, 3946-SE-010) was used as the protease, and the mixture was reacted for 20 hours under the conditions of 10 nM protease, 50 μg / mL antibody, PBS, and 37°C, and then subjected to reducing SDS-PAGE. The results are shown in Figures 22A, 22B, 22C, 22D, 22E, 22F, 22G, 22H, 22I, and Figures 23A, 23B, and 23C. In the modified MRA after protease treatment, the heavy chain was cleaved, and a heavy chain band appeared at a position with a smaller molecular weight compared to the heavy chain of the modified MRA that was not treated with protease (a band that appears at about 50 kDa in the MT-SP1(-) lane in the figure). From these results, it was confirmed that the modified MRA prepared in 12-1 was cleaved by hMT-SP1.

[0201] Example 13 Evaluation of antibodies with protease cleavage sequences introduced at various positions in the light chain 13-1 Preparation of antibodies with protease cleavage sequences 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 in the MRA light chain variable region (MRAL, SEQ ID NO: 230) to prepare the variants of the MRA light chain variable region shown in Table 9. These variants of the MRA light chain variable region were linked to the MRA light chain constant region (k0, SEQ ID NO: 231) to prepare the MRA light chain variants, and expression vectors encoding the corresponding genes were prepared by a method known to those skilled in the art. In addition, peptide sequence B (SEQ ID NO: 160) was inserted into different positions in the MRA light chain constant region (k0, SEQ ID NO: 231) to prepare the variants of the MRA light chain constant region shown in Table 10. These variants of the MRA light chain constant region were linked to the MRA light chain variable region (MRAL, SEQ ID NO: 230) to prepare the MRA light chain variants, and expression vectors encoding the corresponding genes were prepared by a method known to those skilled in the art. The positions at which the protease cleavage sequences were 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 9 and 10. The insertion site in Table 9 refers to the position adjacent to the constant region side of the indicated amino acid (Kabat numbering) in the antibody light chain variable region, and the insertion site in Table 10 refers to the position adjacent to the variable region side of the indicated amino acid (EU numbering) in the antibody light chain constant region.

[0202] [Table 6] TIFF2025041812000010.tif74155

[0203] [Table 7]

[0204] The MRA light chain variants and MRA heavy chains prepared above were combined to produce the MRA variants shown in Table 11, which were expressed by transient expression 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.

[0205] [Table 8] TIFF2025041812000013.tif215125

[0206] 13-2. Evaluation of protease cleavage of anti-human IL6R neutralizing antibodies with protease cleavage sequences introduced into the antibody light chain variable region We verified whether the modified MRA prepared in 13-1 was cleaved by protease. Recombinant human matriptase / ST14 catalytic domain (MT-SP1) (R&D Systems, 3946-SE-010) was used as the protease, and the mixture was reacted for 20 hours under the conditions of 10 nM protease, 50 μg / mL antibody, PBS, and 37°C, and then subjected to reducing SDS-PAGE. The results are shown in Figures 24A, 24B, 24C, 24D, and 24E, and Figures 25A and 25B. The modified MRA cleaved after protease treatment has a light chain band at a position with a smaller molecular weight than the light chain of the modified MRA that was not treated with protease (the band applied at about 25 kDa of the MT-SP1(-) lane in the figure).

[0207] Example 14: Preparation of anti-human PD1 neutralizing antibodies incorporating a protease cleavage sequence and evaluation of binding to human PD1 14-1. Introduction of a protease cleavage sequence into anti-human PD1 neutralizing antibodies Neutralizing antibody 5C4H-G1T4 / 5C4L-KT0 against human PD1 (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; A protease cleavage sequence was inserted into the heavy or light chain 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), or L-CDR3 (QQSSNWPRT, SEQ ID NO: 397) to prepare an antibody into which a protease cleavage sequence was introduced. First, a peptide sequence (sequence number: 299) that has been reported to be cleaved by matriptase (MT-SP1) that 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 12 and the light chain variants shown in Table 13, which were then expressed by methods known to those skilled in the art.

[0208] [Table 9]

[0209] [Table 10]

[0210] An IgG1 antibody (Table 14) into which a protease cleavage sequence has been introduced was produced by combining a heavy chain variant in Table 12 with a light chain 5C4L-KT0, or a light chain variant in Table 13 with a heavy chain 5C4H-G1T4, and expressed by transient expression using Expi293 (Life Technologies) by a method known to those skilled in the art, and purified by a method known to those skilled in the art using Protein A. As a control antibody not containing a protease cleavage sequence, 5C4H-G1T4 / 5C4L-KT0 (heavy chain SEQ ID NO: 297, light chain SEQ ID NO: 298) was expressed and purified.

[0211] [Table 11]

[0212] 14-2. Binding evaluation of anti-human PD1 neutralizing antibodies with protease cleavage sequences to human PD1 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-untreated antibody, 10 μL of PBS alone was added to the antibody prepared in 14-1 (final concentration 0.111 mg / mL). The sample volume at the time of reaction was 90 μL, and the final concentration of protease was 0.2 μg / mL. Each sample was incubated at 37°C for 12 hours.

[0213] 14-2-2 Preparation of biotinylated anti-human PD1 neutralizing antibody A biotinylated anti-human PD1 neutralizing antibody having the same variable region sequence as 5C4H-G1T4 / 5C4L-KT0 was prepared. Specifically, a gene fragment encoding 5C4VH-G1dGSBAP (SEQ ID NO: 317) was prepared by adding the heavy chain constant region of the antibody and biotin (AviTag sequence, SEQ ID NO: 316) to the heavy chain variable region 5C4H (SEQ ID NO: 300), and was introduced into an animal cell expression vector by a method known to those skilled in the art. The constructed expression vector and a vector expressing the light chain 5C4L-KT0B (SEQ ID NO: 298) were introduced into FreeStyle293 cells (Invitrogen) using 293fectin (Invitrogen). At this time, a gene expressing EBNA1 (SEQ ID NO: 318) and a gene expressing biotin ligase (BirA, SEQ ID NO: 319) were simultaneously introduced, and biotin was added for the purpose of biotin labeling. The cells into which the gene was introduced were cultured at 37°C and 8% CO2, and the target biotinylated anti-human PD1 neutralizing antibody (5C4-bio) was secreted into the culture supernatant. 5C4-bio was purified from the culture supernatant by a method known to those skilled in the art.

[0214] 14-2-3 Binding evaluation of each antibody to human PD1 before and after protease treatment Human PD1 was added to 80 μL of the protease-treated antibody / protease-untreated antibody prepared in 14-2-1 to a final concentration of 0.67 μM, and allowed to bind at room temperature for 30 minutes to prepare a sample for binding evaluation. The amount of PD1 that did not bind to the antibody was evaluated to evaluate the binding of the antibody to PD1 when protease-treated / untreated. Specifically, the amount of PD1 that did not bind to the antibody was evaluated by biolayer interferometry (BLI) using the biotinylated anti-human PD1 neutralizing antibody (5C4-bio) prepared in Example 14-2-2. The binding evaluation sample, 5C4-bio, and PBS were each dispensed into different wells of tilted bottom (TW384) Micro plates (ForteBio, 18-5076). Streptavidin biosensors (ForteBio, 18-0009) were hydrated with PBS and measurements were performed with Octet RED 384 at 30°C. A baseline measurement was performed for 30 seconds in the wells containing PBS, after which 5C4-bio was allowed to bind to the Streptavidin sensor for 200 seconds. A baseline measurement was performed again for 30 seconds in the wells containing PBS, after which binding was measured for 180 seconds in the wells containing the binding evaluation sample, and dissociation was measured for 180 seconds in the wells containing PBS. A real-time binding graph showing the binding process is shown in Figure 26. As shown in Figure 26, in the case of antibodies into which a protease cleavage sequence had been introduced, the amount of human PD1 bound to 5C4-bio was greater when measured using binding assessment samples containing protease-treated antibodies than when measured using binding assessment samples containing protease-untreated antibodies. That is, the binding activity of each antibody into which a protease cleavage sequence had been introduced to PD1 was reduced by protease treatment, and PD1 was released and bound to 5C4-bio.

[0215] 14-2-4 Confirmation of antibody protease cleavage (SDS-PAGE) We confirmed by SDS-PAGE whether the antibodies used in 14-2-3 were cleaved by protease treatment. 10 μL of the protease-cleaved antibodies / protease-untreated antibodies prepared in 14-2-3 were mixed with 3.3 μL of sample buffer and incubated at 95°C for 5 minutes. Next, electrophoresis was performed using Mini-PROTEAN TGX gel (4-20% 15well) (Bio-Rad #456-1096), and the proteins were stained with Sample Blue Safe Stain (novex, LC6065). The results are shown in Figure 27. As shown in Figure 27, each antibody with a protease cleavage sequence introduced was cleaved by protease treatment.

[0216] 14-2-5 Evaluation of antibody PD1 binding before and after protease treatment The binding activity of each antibody into which a protease cleavage sequence had been introduced to PD1 before and after protease treatment was also measured by a separate method. 10 μL of the protease-treated or protease-untreated antibody prepared in 14-2-1 was mixed with 70 μL of PBS to prepare a sample for PD1 binding measurement. PD1 binding of the sample was evaluated by biolayer interferometry (BLI). The protease-treated antibody / protease-untreated antibody prepared in 14-2-1 and human PD1 (250 nM) were each dispensed into different wells of tilted bottom (TW384) Microplates (ForteBio, 18-5076). Protein G sensor (ForteBio, 18-0022) was hydrated with PBS, and measurements were performed with Octet RED 384 at 30°C. A baseline measurement was performed for 30 seconds in the wells containing PBS, and then the antibody was allowed to bind to the Protein G sensor for 200 seconds. Again, a baseline measurement was performed for 30 seconds in wells containing PBS, followed by binding measurement for 180 seconds in wells containing human PD1, and dissociation measurement for 180 seconds in wells containing PBS. A real-time binding graph showing the state of binding is shown in Figure 28. As shown in Figure 28, when each antibody containing a protease cleavage sequence was used, the amount of human PD1 binding of the protease-treated antibody was reduced compared to the protease-untreated antibody.

[0217] 14-3. Evaluation of protease-induced ligand release from the complex of anti-human PD1 neutralizing antibody containing a protease cleavage sequence and ligand (human PD1) 14-3-1 Protease treatment in the presence of ligand 10μL of human PD1 adjusted to 6.67μM in PBS was added to the antibody (final concentration 0.100mg / mL) prepared in 14-1 to prepare an antibody-PD1 complex solution. For the protease-treated sample, 10μL of Recombinant Human Matriptase / ST14 Catalytic Domain (hMT-SP1, R&D systems 3946-SE-010) adjusted to 5.28μg / mL in PBS was added to the antibody-PD1 complex solution, and for the non-protease-treated sample, 10μL of PBS alone was added. The final concentration of protease during the reaction was 0.528μg / mL. Each sample was incubated at 37℃ for 12 hours.

[0218] 14-3-2 Evaluation of PD1 release after protease treatment The amount of PD1 not complexed with the antibody was evaluated by biolayer interferometry (BLI) using the biotinylated anti-human PD1 neutralizing antibody (5C4-bio) prepared in Example 14-2-2. Each sample prepared in 14-3-1, 5C4-bio, and PBS were dispensed into different wells of tilted bottom (TW384) Micro plates (ForteBio, 18-5076). Streptavidin biosensors (ForteBio, 18-0009) were hydrated with PBS and measurements were performed with Octet RED 384 at 30°C. A baseline measurement was performed in the wells containing PBS for 30 seconds, and then 5C4-bio was allowed to bind to the Streptavidin sensor for 200 seconds. A baseline measurement was performed again in the wells containing PBS for 30 seconds, and binding was measured for 180 seconds in wells containing protease-treated or protease-untreated samples, and dissociation was measured for 180 seconds in wells containing PBS. A real-time binding graph showing the binding is shown in Figure 29. As shown in Figure 29, for each antibody into which a protease cleavage sequence had been introduced, the amount of human PD1 bound to 5C4-bio was increased in the protease-treated sample compared to the protease-untreated sample. In other words, the binding activity of each antibody to PD1 was weakened by protease treatment, and PD1 was released from the antibody-PD1 complex.

[0219] Example 15: Preparation and evaluation of a fusion protein of anti-human PD1 neutralizing antibody and human PD1 (anti-PD1 neutralizing antibody-PD1 fusion protein) incorporating a protease cleavage sequence 15-1. Preparation of fusion protein of anti-human PD1 neutralizing antibody and human PD1 A human PD1 sequence (SEQ ID NO: 320) was linked to the N-terminus of the antibody heavy chain or heavy chain variant prepared in Example 14-1 via a flexible linker consisting of a glycine-serine polymer (SEQ ID NO: 321) to prepare a PD1 fusion heavy chain (Table 15).

[0220] [Table 12]

[0221] In addition, a human PD1 sequence (sequence number: 320) was linked to the N-terminus of the antibody light chain or modified light chain prepared in Example 14-1 via a flexible linker consisting of a glycine-serine polymer (sequence number: 321) to prepare a PD1-fused light chain (Table 16).

[0222] [Table 13]

[0223] The following anti-PD1 neutralizing antibody-PD1 fusion proteins combine the PD1 fusion heavy chain and light chain 5C4L-KT0 from Table 15, or the PD1 fusion light chain and heavy chain 5C4H-G1T4 from Table 16: hPD15C4HA12aa-G1T4 / 5C4L-KT0 (PD1 fusion heavy chain SEQ ID NO: 323, light chain SEQ ID NO: 298) hPD15C4HE12aa-G1T4E / 5C4L-KT0 (PD1 fusion heavy chain SEQ ID NO: 324, light chain SEQ ID NO: 298) 5C4H-G1T4 / hPD15C4LH12aa-KT0 (heavy chain SEQ ID NO: 297, light chain SEQ ID NO: 325) 5C4H-G1T4 / hPD15C4LI12aa-KT0 (heavy chain SEQ ID NO: 297, PD1 fusion light chain SEQ ID NO: 326) 5C4H-G1T4 / hPD15C4LC12aa-KT0 (heavy chain SEQ ID NO: 297, PD1 fusion light chain SEQ ID NO: 327) 5C4H-G1T4 / hPD15C4LD12aa-KT0 (heavy chain SEQ ID NO: 297, PD1 fusion light chain SEQ ID NO: 328) 5C4H-G1T4 / hPD15C4LE12aa-KT0E (heavy chain SEQ ID NO: 297, PD1 fusion light chain SEQ ID NO: 329) 5C4H-G1T4 / hPD15C4LB12aa-KT0B (heavy chain SEQ ID NO: 297, PD1 fusion light chain SEQ ID NO: 330) 5C4H-G1T4 / hPD15C4LF12aa-KT0F (heavy chain SEQ ID NO: 297, PD1 fusion light chain SEQ ID NO: 331) 5C4H-G1T4 / hPD15C4LG12aa-KT0G (heavy chain SEQ ID NO: 297, PD1 fusion light chain SEQ ID NO: 332) 5C4H-G1T4 / hPD15C4LJ12aa-KT0J (heavy chain SEQ ID NO: 297, PD1 fusion light chain SEQ ID NO: 333) 5C4H-G1T4 / hPD15C4LK12aa-KT0K (heavy chain SEQ ID NO: 297, PD1 fused light chain SEQ ID NO: 334) was expressed by transient expression using Expi293 (Life Technologies) by a method known to those skilled in the art, and purified by a method known to those skilled in the art using protein A. As a control antibody not containing a protease cleavage sequence, 5C4H-G1T4 / 5C4L-KT0 (heavy chain SEQ ID NO: 297, light chain SEQ ID NO: 298) was expressed and purified in the same manner.

[0224] 15-2. Evaluation of protease cleavage of anti-PD1 neutralizing antibody-PD1 fusion protein 15-2-1 Protease treatment For the protease-treated fusion protein, 30 μg of the fusion protein prepared in 15-1 was added with 4.9 μL of Recombinant Human Matriptase / ST14 Catalytic Domain (hMT-SP1, R&D systems 3946-SE-010) adjusted to 16.7 μg / mL in PBS. For the protease-untreated fusion protein, 30 μg of the fusion protein prepared in 15-1 was added with 4.9 μL of PBS alone. The protease-treated fusion protein or the protease-untreated fusion protein was incubated at 37°C for 12 hours.

[0225] 15-2-2 Evaluation of PD1 release by protease treatment PD1 release by protease treatment was evaluated by biolayer interferometry (BLI) using the biotinylated anti-human PD1 neutralizing antibody (5C4-bio) prepared in Example 14-2-2. The protease-treated fusion protein, protease-untreated fusion protein, 5C4-bio, and PBS prepared in 15-2-1 were dispensed into different wells of tilted bottom (TW384) Micro plates (ForteBio, 18-5076). Streptavidin biosensors (ForteBio, 18-0009) were hydrated with PBS and measurements were performed with Octet RED 384 at 30°C. A baseline measurement was performed for 30 seconds in the wells containing PBS, and then 5C4-bio was allowed to bind to the Streptavidin sensor for 200 seconds. A baseline measurement was performed again for 30 seconds in the wells containing PBS, and then binding was measured for 180 seconds in the wells containing protease-treated or protease-untreated fusion protein, and dissociation was measured for 180 seconds in the wells containing PBS. A real-time binding graph showing the binding is shown in Figure 30. As shown in Figure 30, in the case of antibody-PD1 fusion proteins containing antibodies with protease cleavage sequences introduced therein, the amount of human PD1 bound to 5C4-bio was increased in the protease-treated samples compared to the protease-untreated samples. In other words, the binding activity of the antibody in the fusion protein to PD1 was reduced by protease treatment, and PD1 was released from the fusion protein.

[0226] 15-2-3 Confirmation of cleavage of anti-PD1 neutralizing antibody-PD1 fusion protein (SDS-PAGE) The protease-treated fusion protein prepared in 15-2-1 was confirmed by SDS-PAGE to be cleaved by protease treatment. 10 μL of the protease-treated fusion protein prepared in 15-2-1 or the protease-untreated fusion protein was mixed with 3.3 μL of sample buffer and incubated at 95°C for 5 minutes. Next, electrophoresis was performed using Mini-PROTEAN TGX gel (4-20% 15well) (Bio-Rad #456-1096), and the protein was stained with Sample Blue Safe Stain (novex, LC6065). The results are shown in Figure 31. As shown in Figure 31, the fusion protein containing the antibody into which the protease cleavage sequence was introduced was cleaved by protease treatment.

[0227] The foregoing invention has been described in detail by way of illustration and example for purposes of facilitating a clear understanding, but the descriptions and illustrations herein should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein by reference in their entireties. [Industrial Applicability]

[0228] The ligand-binding molecule of the present invention is transported in vivo with the ligand still bound thereto, and is cleaved in diseased tissue to weaken the binding to the ligand, thereby specifically releasing the ligand in the diseased tissue, thereby specifically exposing the diseased tissue to the ligand. Furthermore, since the ligand-binding molecule suppresses the biological activity of the ligand during transport, the risk of the ligand acting systemically is reduced, making it extremely useful for the treatment of diseases.

Claims

1. A molecule capable of binding to a ligand, the molecule being a polypeptide having at least one cleavage site, and the binding of the molecule to the ligand is weakened when the molecule is cleaved at the at least one cleavage site, and the ligand is (a) cytokine or chemokine (b) a ligand selected from interleukins, interferons, hematopoietic factors, the TNF superfamily, chemokines, cell growth factors, and the TGF-β family; or (c) CXCL10, IL12, PD1, or IL6R; Ligand-binding molecules.

2. A ligand-binding molecule as described in claim 1, wherein the cleavage site comprises a protease cleavage sequence.

3. A ligand-binding molecule as described in claim 2, wherein the protease cleavage sequence is cleaved by a target tissue-specific protease.

4. The ligand-binding molecule described in claim 1 or 2, wherein the ligand-binding molecule comprises an antibody VH, an antibody VL, and an antibody constant region.

5. The ligand-binding molecule described in claim 4, wherein the cleavage site or the protease cleavage sequence is located near the boundary between the antibody constant region and the antibody VH, and / or near the boundary between the antibody constant region and the antibody VL.

6. The ligand-binding molecule described in claim 4, wherein the antibody VL and the antibody VH are associated, and the association is dissolved by cleavage of the cleavage site or by cleavage of the protease cleavage sequence by a protease.

7. A ligand-binding molecule as described in claim 1, 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.

8. The ligand-binding molecule described in claim 1, wherein the ligand-binding molecule is an IgG antibody.

9. A ligand-binding molecule described in claim 1, bound to the ligand.

10. A ligand-binding molecule described in claim 1, fused to the ligand.

11. A pharmaceutical composition comprising the ligand-binding molecule of claim 1.

12. A pharmaceutical composition comprising the ligand-binding molecule of claim 1 and a ligand.

13. A pharmaceutical composition comprising a fusion protein in which the ligand-binding molecule of claim 1 is fused with a ligand.

14. The ligand-binding molecule of claim 2, (a) a first flexible linker is attached to one end of the protease cleavage sequence, and the first flexible linker may be a flexible linker consisting of a glycine-serine polymer; or (b) a first flexible linker is attached to one end of the protease cleavage sequence, and a second flexible linker is attached to the other end of the protease cleavage sequence, and the second flexible linker may be a flexible linker consisting of a glycine-serine polymer; Ligand-binding molecules.

15. The ligand-binding molecule of claim 2, (a) the protease is a cancer tissue-specific protease or an inflamed tissue-specific protease, or at least one protease selected from matriptase, urokinase (uPA), and a metalloprotease; or (b) the protease cleavage sequence contained in the ligand-binding molecule comprises a sequence selected from the sequences set forth in SEQ ID NOs: 3, 34, 66, 70, 71, 72, 73, 35, 75, 76, and 345; Ligand-binding molecules.

16. The ligand-binding molecule of claim 14, comprising a first movable linker and a second movable linker; (a) 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; (b) 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; (c) 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; (d) 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; (e) 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 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) of an antibody VH; (f) 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; (g) 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; (h) 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; or (i) 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); Ligand-binding molecules.

17. A ligand-binding molecule as described in claim 10, wherein the ligand-binding molecule is fused to the ligand via a linker, which may comprise a glycine-serine polymer, and which may not include a protease cleavage sequence.

18. The ligand-binding molecule of claim 17, comprising an antibody light chain and an antibody heavy chain, wherein the antibody light chain or the antibody heavy chain is fused to the ligand, and the cleavage site may be contained in the antibody light chain or the antibody heavy chain.

19. A method for producing a fusion protein of a ligand-binding molecule and its ligand according to claim 1, which has a protease cleavage sequence, comprising fusing the ligand to the ligand.

20. The ligand-binding molecule described in claim 1, wherein the ligand is IL12.

21. A ligand-binding molecule according to claim 16, wherein the protease cleavage sequence, or the protease cleavage sequence and the first movable linker, or the protease cleavage sequence, the first movable linker and the second movable linker, is inserted at any position in the sequence from amino acid 109 (Kabat numbering) of antibody VH to amino acid 122 (EU numbering) of the antibody heavy chain constant region.

22. A ligand-binding molecule according to claim 1, wherein the ligand is IL12, at least one of the cleavage sites comprises a protease cleavage sequence, a first movable linker is attached to one end of the protease cleavage sequence and a second movable linker is attached to the other end of the protease cleavage sequence, and the protease cleavage sequence, or the protease cleavage sequence and the first movable linker, or the protease cleavage sequence, the first movable linker and the second movable linker are inserted at any position in the sequence from amino acid 109 (Kabat numbering) of antibody VH to amino acid 122 (EU numbering) of the antibody heavy chain constant region, and the second movable linker may be composed of a glycine-serine polymer.

23. The ligand-binding molecule of claim 20, wherein the protease is matriptase, and the protease cleavage sequence contained in the ligand-binding molecule comprises the sequence set forth in SEQ ID NO: 345.

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