Ligand binding fusion protein

Abstract

To provide a fusion protein capable of selectively activating its ligand moiety such as a cytokine or chemokine in a target tissue.SOLUTION: Fusion proteins are provided comprising a ligand moiety connected via a peptide linker to a ligand binding moiety which binds to the ligand moiety, such as a cytokine or chemokine, but which is capable of releasing the ligand moiety in the presence of a protease. Also provided are methods for their production, their use and pharmaceutical compositions containing such fusion proteins. It also relates to improved variants of such fusion proteins. In addition, the in vitro activity of the variants was evaluated for several cytokines.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] The present invention relates to fusion proteins in which a ligand moiety is connected via a peptide linker to a ligand-binding moiety that binds to said ligand moiety. The invention also relates to methods for their preparation, their use, and pharmaceutical compositions comprising such fusion proteins. [Background technology]

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

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

[0004] A method for delivering a ligand to solid tumors using immunocytokines containing a physiologically active ligand, such as a cytokine, fused to an antibody molecule that binds to a cancer antigen highly expressed on cancer cells is also known. Cytokines delivered to solid tumors by immunocytokines activate the immune system, thereby exerting antitumor effects. Because cytokines, including IL-2, IL-12, and TNF, are highly toxic, it is expected that local delivery of these cytokines to tumors using antibodies will enhance their local effects while reducing side effects (Non-Patent Documents 4, 5, and 6). However, none of these cytokines have yet been approved as pharmaceuticals due to problems such as insufficient clinical efficacy when administered systemically; a narrow therapeutic window; and severe toxicity that precludes systemic administration.

[0005] This is mainly because cytokines, including immunocytokines, are exposed to the entire body when administered systemically and therefore may exert toxicity due to systemic effects, or because cytokines can only be administered at very low doses to avoid toxicity. It has also been reported that the antitumor effect was not different between an immunocytokine containing IL-2 fused to an antibody that binds to a cancer antigen and an immunocytokine containing IL-2 fused to an antibody that does not bind to a cancer antigen (Non-Patent Document 7).

[0006] As a way to avoid the above problems, molecules containing a cytokine and a cytokine receptor connected via a linker cleavable by a protease highly expressed in cancer have been reported. The cytokine is inhibited by the cytokine receptor connected via the linker, but is released from the cytokine receptor by protease cleavage of the linker, thereby becoming active. For example, a molecule containing TNF-α and TNF-R connected via a linker cleavable by uPA (Non-Patent Document 8) has been reported, and a molecule containing IL-2 and IL-2R connected via a linker cleavable by MMP-2 (Non-Patent Document 9) has been reported. However, the cytokine in these molecules retains activity even before cleavage of the linker, and cleavage of the linker improves activity only approximately 10-fold. On the other hand, molecules containing a cytokine connected to an anti-cytokine scFv instead of the cytokine receptor via a linker cleavable by MMP (Non-Patent Documents 9 and 10) have also been reported. Several other documents (e.g., Patent Documents 1 to 6) also relate to fusion polypeptides containing cleavable moieties. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO 2009 / 025846 [Patent Document 2] WO 2011 / 123683

Patent document 3

Patent document 4

Patent document 5

Patent document 6

Non-licensed literature

[0008] [Non-licensed 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-licensed Document 2

Non-licensed Document 4

Direct Environment 5

Outdoor Configuration6

Direct Environment 7

Outdoor Track 8

Outdoor Tools9

[0009] The present invention has been made in view of these circumstances, and aims to provide a fusion protein capable of selectively activating its ligand moiety, such as a cytokine or a chemokine, in a target tissue, a method for producing the same, a use thereof, and a pharmaceutical composition containing the fusion protein. [Means for solving the problem]

[0010] The present inventors have conducted extensive research to achieve this goal and have consequently developed a fusion protein in which a ligand moiety (e.g., a cytokine or chemokine) is connected to a ligand-binding moiety via a peptide linker, and the ligand-binding moiety binds to the ligand moiety but can release the ligand moiety in the presence of a protease. The present inventors have also found that such a fusion protein or a pharmaceutical composition containing the fusion protein is useful for treating a disease using a ligand, and that the fusion protein or pharmaceutical composition is useful for treating a disease by administering the fusion protein; and that the fusion protein is useful for producing a medicament for treating a disease. The present inventors have also developed a method for producing the fusion protein, thereby completing the present invention. The molecular format of the fusion protein of the present invention is advantageous over other molecular formats known in the prior art in that it may enable higher expression levels and higher activity.

[0011] The present invention is based on these findings and specifically includes the following exemplary embodiments. [1] (a) a ligand-binding portion comprising a ligand-binding domain and at least one first protease cleavage site; (b) at least one ligand moiety; and (c) at least one peptide linker connecting at least one ligand moiety to the C-terminal region of the ligand-binding moiety; A fusion protein comprising: the ligand-binding domain is capable of binding to the at least one ligand moiety and releasing the at least one ligand moiety in the presence of a protease; Fusion proteins. [2] The fusion protein of [1], wherein the at least one peptide linker does not contain any protease cleavage site. [3] The fusion protein of [1], wherein the at least one peptide linker comprises a second protease cleavage site. [4] The fusion protein according to any one of [1] to [3], wherein the ligand-binding domain comprises an antibody variable region. [5] The fusion protein of [4], wherein the ligand-binding domain comprises a VH region and a VL region that associate with each other. [6] The fusion protein of [4] or [5], wherein the ligand-binding portion further comprises a CH1 domain and a CL domain. [7] The fusion protein of [6], wherein at least one of the at least one first protease cleavage site is located near the boundary between the VH or VL domain and the CH1 or CL domain. [8] The fusion protein according to any one of [4] to [7], wherein the ligand-binding portion further comprises an Fc region. [8-1] A fusion protein according to [8], comprising a constant region including a linker. [8-2] A fusion protein of [8-1], comprising a constant region comprising the sequence of SEQ ID NO: 901 (C1). [8-3] A fusion protein of [8-1], comprising a constant region comprising the sequence of SEQ ID NO: 905 (C2) or SEQ ID NO: 932 (C5). [8-4] A fusion protein according to [8-1], comprising a heavy chain and a light chain, wherein the linker is positioned in the hinge region so as to promote disulfide bond formation between Cys at position 220 (C220) (EU numbering) of the heavy chain and Cys at position 214 (C214) (EU numbering) of the light chain. [8-5] The fusion protein of [8-4], wherein the hinge region comprises the following amino acid sequence from position 216 (EU numbering): EPKSCGGGGSGGGGSDKTHTCPPCP (sequence number: 935). [8-6] The fusion protein of [8-1], wherein the ligand-binding portion comprises a heavy chain and a light chain, and amino acid residues in the heavy chain and the light chain have been modified so that no disulfide bond is formed between position 220 (EU numbering) of the heavy chain and position 214 (EU numbering) of the light chain. [8-7] The fusion protein of [8-6], wherein the light chain contains a C214S (EU numbering) modification and the heavy chain contains a C220S (EU numbering) modification. [8-8] A fusion protein of [8-1], comprising a constant region comprising the sequence of SEQ ID NO: 908 (C3). [8-9] The fusion protein of [8-1], wherein the ligand-binding portion comprises a heavy chain and a light chain, and the heavy chain is modified to enable disulfide bond formation between position 131 (EU numbering) of the heavy chain and position 214 (EU numbering) of the light chain. [8-10] The fusion protein of [8-9], wherein the heavy chain contains the modifications S131C (EU numbering) and C220S (EU numbering). [8-11] A fusion protein of [8-1], comprising a constant region comprising the sequence of SEQ ID NO: 910 (C4). [9] The fusion protein of [8], wherein the ligand-binding portion comprises a full-length antibody.

[10] The fusion protein of [9], wherein the full-length antibody is an IgG antibody.

[11] The fusion protein of [9] or

[10] , wherein the at least one ligand moiety comprises two ligand moieties and the at least one peptide linker comprises two peptide linkers, each of which is connected to the C-terminal region of the ligand-binding moiety via a respective peptide linker.

[12] Any one of the fusion proteins [8] to

[11] , wherein the at least one ligand moiety is connected to an amino acid residue exposed on the surface of the CH3 region of the Fc region via the at least one peptide linker.

[13] The fusion protein of any one of [1] to

[12] , wherein the at least one ligand moiety is connected to the C-terminal amino acid residue of the ligand-binding moiety via the at least one peptide linker.

[14] The fusion protein of any one of [1] to

[13] , further comprising a cleavable linker comprising a third protease cleavage site, wherein the at least one ligand moiety is connected to the N-terminal region of the ligand-binding moiety via the cleavable linker.

[15] The fusion protein of any one of [8] to

[13] , further comprising a cleavable linker comprising a third protease cleavage site, wherein the at least one ligand moiety is connected via the cleavable linker to one of amino acid residues 1 to 230 from the N-terminus of the ligand-binding moiety.

[16] The fusion protein of

[14] or

[15] , wherein the at least one ligand moiety is connected to the N-terminal amino acid residue of the ligand-binding moiety via the cleavable linker.

[17] The fusion protein of any one of [1] to

[16] , wherein the at least one ligand moiety has biological activity, and binding of the ligand-binding domain to the ligand moiety inhibits the biological activity of the ligand moiety.

[18] The fusion protein of

[17] , wherein the at least one ligand moiety comprises a protein or polypeptide having biological activity.

[19] The fusion protein of

[18] , wherein the at least one ligand moiety comprises a cytokine or a chemokine.

[20] The fusion protein of

[18] , wherein the at least one ligand moiety comprises a ligand protein or polypeptide selected from the group consisting of CXCL10, IL-2, IL-12, IL-22, PD-1, or IL-6R.

[21] The fusion protein of any one of [1] to

[20] , wherein the at least one ligand moiety comprises IL-12, and the fusion protein comprises an antibody heavy chain and a light chain selected from the group consisting of: (a) a light chain comprising the amino acid sequence of SEQ ID NO: 874, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 875; (b) a light chain comprising the amino acid sequence of SEQ ID NO: 876, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 880; (c) a light chain comprising the amino acid sequence of SEQ ID NO: 876, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 881; (d) a light chain comprising the amino acid sequence of SEQ ID NO: 874, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 884; (e) a light chain comprising the amino acid sequence of SEQ ID NO: 876, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 885; (f) a light chain comprising the amino acid sequence of SEQ ID NO: 876, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 886; (g) a light chain comprising the amino acid sequence of SEQ ID NO: 887, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 888; (h) a light chain comprising the amino acid sequence of SEQ ID NO: 890, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 891; (i) a light chain comprising the amino acid sequence of SEQ ID NO: 876, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 904; (j) a light chain comprising the amino acid sequence of SEQ ID NO: 906, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 907; (k) a light chain comprising the amino acid sequence of SEQ ID NO: 876, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 909; and (l) An antibody heavy chain and a light chain that compete with the antibody heavy chain and the antibody light chain described in (a) to (k). [21-a] Any one of the fusion proteins of [1] to

[20] , wherein the at least one ligand moiety comprises IL-12, and the ligand-binding moiety comprises an antibody variable region comprising any one of the combinations of H chain CDR1, CDR2, and CDR3 and L chain CDR1, CDR2, and CDR3 selected from the following (a) to (l), or an antibody variable region comprising any one of the combinations of H chain CDR1, CDR2, and CDR3 and L chain CDR1, CDR2, and CDR3 of an antibody variable region functionally equivalent thereto: (a) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 875; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 874; (b) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 880; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 876; (c) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 881; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 876; (d) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 884; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 874; (e) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 885; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 876; (f) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 886; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 876; (g) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 888; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 887; (h) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 891; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 890; (i) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 904; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 876; (j) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 907; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 906; (k) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 909; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 876; and (l) H-chain and L-chain CDR1, CDR2, and CDR3 contained in antibody variable regions that compete with the antibody heavy chain variable region and antibody light chain variable region described in (a) to (k). [21-b] The fusion protein of any one of [1] to

[20] , wherein the at least one ligand moiety comprises IL-12, and the ligand-binding moiety comprises any one of the combinations of a heavy chain variable region (VH) and a light chain variable region (VL) selected from the following (a) to (l): (a) VH comprised in SEQ ID NO: 875; and VL comprised in SEQ ID NO: 874; (b) VH comprised in SEQ ID NO: 880; and VL comprised in SEQ ID NO: 876; (c) VH comprised in SEQ ID NO: 881; and VL comprised in SEQ ID NO: 876; (d) VH comprised in SEQ ID NO: 884; and VL comprised in SEQ ID NO: 874; (e) VH comprised in SEQ ID NO: 885; and VL comprised in SEQ ID NO: 876; (f) VH comprised in SEQ ID NO: 886; and VL comprised in SEQ ID NO: 876; (g) VH comprised in SEQ ID NO: 888; and VL comprised in SEQ ID NO: 887; (h) VH comprised in SEQ ID NO: 891; and VL comprised in SEQ ID NO: 890; (i) VH comprised in SEQ ID NO: 904; and VL comprised in SEQ ID NO: 876; (j) VH comprised in SEQ ID NO: 907; and VL comprised in SEQ ID NO: 906; (k) VH comprised in SEQ ID NO: 909; and VL comprised in SEQ ID NO: 876; and (l) VH and VL that compete with the VH and VL described in (a) to (k). [21-c] The fusion protein of any one of [1] to

[20] , wherein the at least one ligand moiety comprises IL-12, and the fusion protein comprises any one of the combinations selected from the following (a) to (d): (a) a first light chain comprising the sequence of SEQ ID NO: 876, a first heavy chain comprising the sequence of SEQ ID NO: 881, a second light chain comprising the sequence of SEQ ID NO: 882, and a second heavy chain comprising the sequence of SEQ ID NO: 883; (b) a first light chain comprising the sequence of SEQ ID NO: 876, a first heavy chain comprising the sequence of SEQ ID NO: 886, a second light chain comprising the sequence of SEQ ID NO: 882, and a second heavy chain comprising the sequence of SEQ ID NO: 883; (c) a first light chain comprising the sequence of SEQ ID NO: 887, a first heavy chain comprising the sequence of SEQ ID NO: 888, a second light chain comprising the sequence of SEQ ID NO: 882, and a second heavy chain comprising the sequence of SEQ ID NO: 883; and (d) a first light chain comprising the sequence of SEQ ID NO: 890, a first heavy chain comprising the sequence of SEQ ID NO: 891, a second light chain comprising the sequence of SEQ ID NO: 882, and a second heavy chain comprising the sequence of SEQ ID NO: 883. [21-2] The fusion protein of any one of [1] to

[20] , wherein the at least one ligand moiety comprises IL-22, and the fusion protein comprises an antibody heavy chain and a light chain selected from the group consisting of: (a) a light chain comprising the amino acid sequence of SEQ ID NO: 912, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 913; (b) a light chain comprising the amino acid sequence of SEQ ID NO: 915, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 916; (c) a light chain comprising the amino acid sequence of SEQ ID NO: 912, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 929; (d) a light chain comprising the amino acid sequence of SEQ ID NO: 912, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 930; and (e) An antibody heavy chain and a light chain that compete with the antibody heavy chain and the antibody light chain described in (a) to (d). [21-2a] Any one of the fusion proteins of [1] to

[20] , wherein the at least one ligand moiety comprises IL-22, and the ligand-binding moiety comprises an antibody variable region comprising any one of the combinations of H chain CDR1, CDR2, and CDR3 and L chain CDR1, CDR2, and CDR3 selected from the following (a) to (e), or an antibody variable region comprising any one of the combinations of H chain CDR1, CDR2, and CDR3 and L chain CDR1, CDR2, and CDR3 of an antibody variable region functionally equivalent thereto: (a) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 913; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 912; (b) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 916; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 915; (c) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 929; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 912; (d) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 930; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 912; and (e) H-chain and L-chain CDR1, CDR2, and CDR3 contained in antibody variable regions that compete with the antibody heavy chain variable region and antibody light chain variable region described in (a) to (d). [21-2b] The fusion protein of any one of [1] to

[20] , wherein the at least one ligand moiety comprises IL-22, and the ligand-binding moiety comprises any one of the combinations of a heavy chain variable region (VH) and a light chain variable region (VL) selected from the following (a) to (e): (a) VH comprised in SEQ ID NO: 913; and VL comprised in SEQ ID NO: 912; (b) VH comprised in SEQ ID NO: 916; and VL comprised in SEQ ID NO: 915; (c) VH comprised in SEQ ID NO: 929; and VL comprised in SEQ ID NO: 912; (d) a VH comprised in SEQ ID NO: 930; and a VL comprised in SEQ ID NO: 912; and (e) VH and VL that compete with the VH and VL described in (a) to (d). [21-3] The fusion protein of any one of [1] to

[20] , wherein the at least one ligand moiety comprises IL-2, and the fusion protein comprises an antibody heavy chain and a light chain selected from the group consisting of: (a) a light chain comprising the amino acid sequence of SEQ ID NO: 920, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 919; (b) a light chain comprising the amino acid sequence of SEQ ID NO: 923, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 922; and (c) An antibody heavy chain and a light chain that compete with the antibody heavy chain and the antibody light chain described in (a) to (b). [21-3a] Any one of the fusion proteins of [1] to

[20] , wherein the at least one ligand moiety comprises IL-2, and the ligand-binding moiety comprises an antibody variable region comprising any one of the combinations of H chain CDR1, CDR2, and CDR3 and L chain CDR1, CDR2, and CDR3 selected from the following (a) to (e), or an antibody variable region comprising any one of the combinations of H chain CDR1, CDR2, and CDR3 and L chain CDR1, CDR2, and CDR3 of an antibody variable region functionally equivalent thereto: (a) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 919; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 920; (b) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 922; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 923; and (c) H-chain and L-chain CDR1, CDR2, and CDR3 contained in antibody variable regions that compete with the antibody heavy chain variable region and antibody light chain variable region described in (a) to (b). [21-3b] The fusion protein of any one of [1] to

[20] , wherein the at least one ligand moiety comprises IL-2, and the ligand-binding moiety comprises any one of the following combinations of a heavy chain variable region (VH) and a light chain variable region (VL) selected from the following (a) to (c): (a) VH comprised in SEQ ID NO: 919; and VL comprised in SEQ ID NO: 920; (b) VH comprised in SEQ ID NO: 922; and VL comprised in SEQ ID NO: 923; and (c) VH and VL that compete with the VH and VL described in (a)-(b).

[22] A fusion protein of any one of [1] to [21-2], in which each protease cleavage site is independently cleavable by a protease specific to a target tissue.

[23] The fusion protein of

[22] , wherein the target tissue is a cancer tissue.

[24] The fusion protein of

[23] , wherein each protease cleavage site is independently cleavable by a protease selected from the group consisting of matriptase, urokinase-type plasminogen activator (uPA), and a metalloprotease.

[25] The fusion protein of any one of [1] to

[24] , wherein each protease cleavage site independently comprises a protease cleavage sequence selected from the group consisting of SEQ ID NOs: 2 to 135 and 160 to 870.

[26] A fusion protein according to any one of [1] to

[25] , wherein each protease cleavage site is cleavable by the same protease.

[27] A fusion protein selected from any one of [1] to

[26] , wherein each protease cleavage site contains the same protease cleavage sequence.

[28] The fusion protein of

[27] , wherein each protease cleavage site comprises the amino acid sequence of SEQ ID NO: 873.

[29] The fusion protein of [1], wherein the ligand-binding portion further comprises a first flexible linker attached to one end of the at least one first protease cleavage site.

[30] The fusion protein of

[29] , wherein the ligand-binding domain further comprises a second flexible linker attached to the other end of the at least one first protease cleavage site.

[31] The fusion protein of

[29] or

[30] , wherein the first flexible linker is a glycine-serine polymer.

[32] The fusion protein of

[30] or

[31] , wherein the second flexible linker is a glycine-serine polymer.

[33] The fusion protein of [2], wherein the at least one peptide linker comprises a flexible linker.

[34] The fusion protein according to

[33] , wherein the flexible linker is a glycine-serine polymer.

[35] The fusion protein of [3], wherein the at least one peptide linker further comprises a third flexible linker added to one end of the second protease cleavage site.

[36] The fusion protein of

[35] , wherein the at least one peptide linker further comprises a fourth flexible linker added to the other end of the second protease cleavage site.

[37] The fusion protein of

[35] or

[36] , wherein the third flexible linker is a glycine-serine polymer.

[38] The fusion protein of

[36] or

[37] , wherein the fourth flexible linker is a glycine-serine polymer.

[39] The fusion protein of

[15] , wherein the cleavable linker further comprises a fifth flexible linker attached to one end of the third protease cleavage site.

[40] The fusion protein of

[39] , wherein the cleavable linker further comprises a sixth flexible linker attached to the other end of the third protease cleavage site.

[41] The fusion protein of

[39] or

[40] , wherein the fifth flexible linker is a glycine-serine polymer.

[42] The fusion protein of

[40] or

[41] , wherein the sixth flexible linker is a glycine-serine polymer.

[43] The glycine-serine polymer 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: 136); Gly Gly Ser Gly (GGSG, SEQ ID NO: 137); Gly Ser Gly Gly (GSGG, SEQ ID NO: 138); Ser Gly Gly Gly (SGGG, SEQ ID NO: 139); Gly Ser Ser Gly (GSSG, SEQ ID NO: 140); Gly Gly Gly Gly Ser (GGGGS, SEQ ID NO: 141); Gly Gly Gly Ser Gly (GGGSG, SEQ ID NO: 142); Gly Gly Ser Gly Gly (GGSGG, SEQ ID NO: 143); Gly Ser Gly Gly Gly (GSGGG, SEQ ID NO: 144); Gly Ser Gly Gly Ser (GSGGS, SEQ ID NO: 145); Ser Gly Gly Gly Gly (SGGGG, SEQ ID NO: 146); Gly Ser Ser Gly Gly (GSSGG, SEQ ID NO: 147); Gly Ser Gly Ser Gly (GSGSG, SEQ ID NO: 148); Ser Gly Gly Ser Gly (SGGSG, SEQ ID NO: 149); Gly Ser Ser Ser Gly (GSSSG, SEQ ID NO: 150); Gly Gly Gly Gly Gly Ser (GGGGGS, SEQ ID NO: 151); Ser Gly Gly Gly Gly Gly (SGGGGG, SEQ ID NO: 152); Gly Gly Gly Gly Gly Ser (GGGGGGS, SEQ ID NO: 153); Ser Gly Gly Gly Gly Gly Gly (SGGGGGG, SEQ ID NO: 154); (Gly Gly Gly Gly Ser (GGGGS, SEQ ID NO: 141)); and (Ser Gly Gly Gly Gly (SGGGG, SEQ ID NO: 146)) (n is an integer greater than or equal to 1)

[31] ,

[32] ,

[34] ,

[37] ,

[38] ,

[41] , and

[42] .

[44] The fusion protein of any one of [1] to [3], wherein the ligand-binding domain comprises a non-antibody protein or polypeptide.

[45] The fusion protein of

[44] , wherein the non-antibody protein or polypeptide is selected from the group consisting of a scaffold peptide, a peptide aptamer, and an IL-12 receptor.

[46] A pharmaceutical composition comprising any one of the fusion proteins according to [1] to

[45] .

[47] The pharmaceutical composition of

[46] , for use in the treatment of cancer.

[48] ​​Use of a fusion protein of any one of [1] to

[45] for the manufacture of a pharmaceutical composition for the treatment of cancer.

[49] (a) a ligand-binding molecule comprising a ligand-binding domain and at least one first protease cleavage site; (b) at least one ligand molecule, and (c) at least one peptide linker providing connecting at least one ligand molecule to the C-terminal region of the ligand-binding molecule via at least one peptide linker; A method for producing a fusion protein according to any one of [1] to

[45] , comprising: the ligand-binding domain binds to the ligand molecule; the ligand-binding domain is capable of releasing the ligand molecule in the presence of a protease; method.

[50] The method of

[49] , further comprising the step of connecting at least one ligand molecule to the N-terminal region of the ligand-binding molecule via a cleavable linker.

[51] A method for treating cancer, comprising administering to a subject the fusion protein of any one of [1] to

[41] or the pharmaceutical composition of

[46] or

[47] .

[52] A polynucleotide encoding any one of the fusion proteins [1] to

[45] .

[53]

[52] A vector comprising the polynucleotide.

[54] A host cell containing the polynucleotide of

[52] or the vector of

[53] .

[55] A method for producing a fusion protein according to any one of [1] to

[45] , comprising culturing a host cell according to

[54] .

[0101] comprising a ligand-binding moiety and having the general formula (I): [Ligand-binding domain]-[Lx]-[Cx]-[Ly]-[ligand moiety] (I) A fusion protein represented by During the ceremony, Lx represents a first peptide linker optionally containing a first protease cleavage site, or Lx is absent; Cx represents a constant region comprising a second peptide linker and, optionally, one or more amino acid residues that are modified to or from cysteine; Ly represents the third peptide linker; the ligand-binding domain is capable of binding to the ligand moiety and releasing the ligand moiety in the presence of a protease; Fusion proteins. [101-1] A fusion protein according to

[0101] , comprising two sets of ligand-binding domains, a ligand portion, a first peptide linker, a constant region, and a third peptide linker. [101-2]General formula (II): [Ligand-binding domain]-[Lx]-[Cx]-[Ly]-[ligand portion] / / [Non-ligand-binding domain]-[Lz]-[Cz] (II) A fusion protein represented by During the ceremony, Lx, Cx, and Ly are as defined in claim 1; Lz represents a fourth peptide linker optionally containing a first protease cleavage site, or Lz is absent; Cz represents a second constant region, optionally including a fifth peptide linker and, optionally, one or more amino acid residues that are modified to or from cysteine; A fusion protein of

[0101] . [101-3] A fusion protein of any one of

[0101] or [101-2], wherein the ligand portion comprises CXCL10, IL-2, IL-12, IL-22, PD-1, or IL-6R. [101-11] A fusion protein of any one of

[0101] to [101-3], wherein the first (or fourth) peptide linker (Lx (or Lz)) is a linker comprising the sequence of SEQ ID NO: 873 (L1). [101-12] A fusion protein of any one of

[0101] to [101-3], wherein the constant region (or the second constant region) comprises the sequence of SEQ ID NO: 901 (C1). [101-13] A fusion protein of any one of

[0101] to [101-3], wherein the third peptide linker (Ly) is a linker comprising the sequence of SEQ ID NO: 903 (L4), or SEQ ID NO: 873 or 879 (L3), or SEQ ID NO: 927 (L5). [101-14] A fusion protein of any one of

[0101] , [101-1], and [101-3], which is a homodimer (Mab80-L1-C1-L4-IL12) comprising a light chain of SEQ ID NO: 876 and a heavy chain of SEQ ID NO: 885. [101-15] A fusion protein of any one of

[0101] , [101-1], and [101-3], which is a homodimer (087B03-L1-C1-L3-IL22) comprising a light chain of SEQ ID NO: 912 and a heavy chain of SEQ ID NO: 913. [101-21] A fusion protein of any one of

[0101] to [101-3], wherein the constant region (or the second constant region) comprises the sequence of SEQ ID NO: 905 (C2) or 932 (C5). [101-22] A fusion protein of any one of

[0101] to [101-3], comprising a heavy chain and a light chain, wherein the second linker is positioned in the hinge region so as to promote disulfide bond formation between Cys at position 220 (C220) (EU numbering) of the heavy chain and Cys at position 214 (C214) (EU numbering) of the light chain. [101-23] The fusion protein of [101-22], wherein the hinge region comprises the following amino acid sequence from position 216 (EU numbering): EPKSCGGGGSGGGGSDKTHTCPPCP (sequence number: 935). [101-24] A fusion protein of any one of

[0101] , [101-1], and [101-3], which is a homodimer (Mab80-L1-C2-L4-IL12) comprising a light chain of SEQ ID NO: 876 and a heavy chain of SEQ ID NO: 904. [101-25] A fusion protein of any one of

[0101] , [101-1], and [101-3], which is a homodimer (087B03-L1-C2-L3-IL22) comprising a light chain of SEQ ID NO: 912 and a heavy chain of SEQ ID NO: 929. [101-26] A fusion protein of any one of

[0101] , [101-1], and [101-3], which is a homodimer (Cx-L1-C5-L5-IL2.N88D) comprising a light chain of SEQ ID NO: 920 and a heavy chain of SEQ ID NO: 919. [101-27] A fusion protein of any one of

[0101] , [101-1], and [101-3], which is a homodimer (16C3-L1-C5-L5-IL2.N88D) comprising a light chain of SEQ ID NO: 923 and a heavy chain of SEQ ID NO: 922. [101-31] ​​A fusion protein according to any one of

[0101] to [101-3], wherein the ligand-binding portion comprises a heavy chain and a light chain, and amino acid residues in the heavy chain and the light chain have been modified so that no disulfide bond is formed between position 220 (EU numbering) of the heavy chain and position 214 (EU numbering) of the light chain. [101-32] The fusion protein of [101-31], wherein the light chain comprises a C214S (EU numbering) modification and the heavy chain comprises a C220S (EU numbering) modification. [101-33] A fusion protein of any one of

[0101] to [101-3], wherein the constant region (or the second constant region) comprises the sequence of SEQ ID NO: 908 (C3). [101-34] A fusion protein of any one of

[0101] , [101-1], and [101-3], which is a homodimer (Mab80-L1-C3-L4-IL12) comprising a light chain of SEQ ID NO: 906 and a heavy chain of SEQ ID NO: 907. [101-35] A fusion protein of any one of

[0101] , [101-1], and [101-3], which is a homodimer (087B03-L1-C3-L3-IL22) comprising a light chain of SEQ ID NO: 915 and a heavy chain of SEQ ID NO: 916. [101-41] A fusion protein of any one of

[0101] to [101-3], wherein the ligand-binding portion comprises a heavy chain and a light chain, and the heavy chain is modified to enable disulfide bond formation between position 131 (EU numbering) of the heavy chain and position 214 (EU numbering) of the light chain. [101-42] The fusion protein of [101-41], wherein the heavy chain comprises the modifications S131C (EU numbering) and C220S (EU numbering). [101-43] A fusion protein of any one of

[0101] to [101-3], wherein the constant region (or the second constant region) comprises the sequence of SEQ ID NO: 910 (C4). [101-44] A fusion protein of any one of

[0101] , [101-1], and [101-3], which is a homodimer (Mab80-L1-C4-L4-IL12) comprising a light chain of SEQ ID NO: 876 and a heavy chain of SEQ ID NO: 909. [101-45] A fusion protein of any one of

[0101] , [101-1], and [101-3], which is a homodimer (087B03-L1-C4-L3-IL22) comprising a light chain of SEQ ID NO: 912 and a heavy chain of SEQ ID NO: 930.

[0102] The fusion protein of any one of

[0101] to [101-3], wherein the at least one peptide linker does not contain any protease cleavage site. [102-1] A fusion protein of any one of

[0101] to [101-3], wherein Cx and / or Ly do not contain any protease cleavage site.

[0103] The fusion protein of any one of

[0101] to [101-3], wherein the at least one peptide linker comprises a second protease cleavage site. [103-1] A fusion protein of any one of

[0101] to [101-3], wherein Ly contains a second protease cleavage site.

[0104] The fusion protein of any one of

[0101] to [103-1], wherein the ligand-binding domain comprises an antibody variable region.

[0105] A fusion protein according to

[0104] , wherein the ligand-binding domain comprises a VH region and a VL region that associate with each other.

[0106] A fusion protein of

[0104] or

[0105] , wherein Cx comprises a CH1 region and a CL region.

[0107] A fusion protein of

[0106] , wherein at least one protease cleavage site is located near the boundary between the VH or VL domain and the CH1 or CL domain. [107-1] A fusion protein of

[0106] , wherein Lx comprises at least one protease cleavage site located near the boundary between the VH or VL domain and the CH1 or CL domain.

[0108] A fusion protein of any one of

[0104] to [107-1], which contains an Fc region.

[0109] A fusion protein of

[0108] containing a full-length antibody.

[0110] A fusion protein of

[0109] , wherein the full-length antibody is an IgG antibody.

[0111] A fusion protein of

[0109] or

[0110] , wherein the fusion protein comprises two ligand moieties and two peptide linkers, each of the ligand moieties being connected to the C-terminal region of the ligand-binding moiety via a respective peptide linker. [111-1] A fusion protein of

[0109] or

[0110] , wherein the fusion protein comprises two ligand moieties and two peptide linkers, each of which is connected to the C-terminal region of Cx via Ly.

[0112] A fusion protein according to any one of

[0108] to [111-2], wherein at least one ligand moiety is connected to an amino acid residue exposed on the surface of the CH3 region of the Fc region via at least one peptide linker. [112-1] A fusion protein of any one of

[0108] to [111-1], wherein at least one ligand moiety is connected via Ly to an amino acid residue exposed on the surface of the CH3 region of the Fc region.

[0113] A fusion protein according to any one of

[0101] to [112-1], wherein at least one ligand moiety is connected to the C-terminal amino acid residue of the ligand-binding moiety via at least one peptide linker. [113-1] A fusion protein according to any one of

[0101] to [112-1], wherein at least one ligand moiety is connected to the C-terminal amino acid residue of Cx via Ly.

[0114] The fusion protein of any one of

[0101] to [113-1], further comprising a cleavable linker comprising a third protease cleavage site, wherein at least one ligand moiety is connected to the N-terminal region of the ligand binding moiety (domain) via the cleavable linker.

[0115] The fusion protein of any one of

[0108] to [113-1], further comprising a cleavable linker comprising a third protease cleavage site, wherein at least one ligand moiety is connected via the cleavable linker to one of amino acid residues 1 to 230 from the N-terminus of the ligand-binding portion (domain).

[0116] A fusion protein according to

[0114] or

[0115] , wherein the at least one ligand moiety is connected to the N-terminal amino acid residue of the ligand-binding moiety (domain) via a cleavable linker.

[0117] The fusion protein of any one of

[0101] to

[0116] , wherein at least one ligand moiety has biological activity, and binding of the ligand-binding domain to the ligand moiety inhibits the biological activity of the ligand moiety.

[0118] A fusion protein according to

[0117] , wherein the at least one ligand moiety comprises a protein or polypeptide having biological activity.

[0119] The fusion protein of

[0118] , wherein the at least one ligand moiety comprises a cytokine or chemokine.

[0120] A fusion protein of

[0118] , wherein the at least one ligand portion comprises a ligand protein or polypeptide selected from the group consisting of CXCL10, IL-2, IL-12, IL-22, PD-1, or IL-6R.

[0121]

[0101] to

[0120] , wherein at least one ligand moiety comprises IL-12, and the fusion protein comprises an antibody heavy chain and a light chain selected from the group consisting of: (a) a light chain comprising the amino acid sequence of SEQ ID NO: 874, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 875; (b) a light chain comprising the amino acid sequence of SEQ ID NO: 876, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 880; (c) a light chain comprising the amino acid sequence of SEQ ID NO: 876, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 881; (d) a light chain comprising the amino acid sequence of SEQ ID NO: 874, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 884; (e) a light chain comprising the amino acid sequence of SEQ ID NO: 876, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 885; (f) a light chain comprising the amino acid sequence of SEQ ID NO: 876, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 886; (g) a light chain comprising the amino acid sequence of SEQ ID NO: 887, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 888; (h) a light chain comprising the amino acid sequence of SEQ ID NO: 890, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 891; (i) a light chain comprising the amino acid sequence of SEQ ID NO: 876, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 904; (j) a light chain comprising the amino acid sequence of SEQ ID NO: 906, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 907; (k) a light chain comprising the amino acid sequence of SEQ ID NO: 876, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 909; and (l) An antibody heavy chain and a light chain that compete with the antibody heavy chain and the antibody light chain described in (a) to (k). [121-a] Any one of the fusion proteins of

[0101] to

[0120] , wherein the at least one ligand moiety comprises IL-12, and the ligand-binding moiety comprises an antibody variable region comprising any one of the combinations of H chain CDR1, CDR2, and CDR3 and L chain CDR1, CDR2, and CDR3 selected from the following (a) to (l), or an antibody variable region comprising any one of the combinations of H chain CDR1, CDR2, and CDR3 and L chain CDR1, CDR2, and CDR3 of antibody variable regions functionally equivalent thereto: (a) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 875; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 874; (b) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 880; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 876; (c) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 881; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 876; (d) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 884; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 874; (e) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 885; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 876; (f) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 886; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 876; (g) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 888; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 887; (h) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 891; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 890; (i) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 904; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 876; (j) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 907; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 906; (k) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 909; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 876; and (l) H-chain and L-chain CDR1, CDR2, and CDR3 contained in antibody variable regions that compete with the antibody heavy chain variable region and antibody light chain variable region described in (a) to (k). [121-b] Any one of the fusion proteins of

[0101] to

[0120] , wherein the at least one ligand moiety comprises IL-12, and the ligand-binding moiety comprises any one of the combinations of heavy chain variable region (VH) and light chain variable region (VL) selected from the following (a) to (l): (a) VH comprised in SEQ ID NO: 875; and VL comprised in SEQ ID NO: 874; (b) VH comprised in SEQ ID NO: 880; and VL comprised in SEQ ID NO: 876; (c) VH comprised in SEQ ID NO: 881; and VL comprised in SEQ ID NO: 876; (d) VH comprised in SEQ ID NO: 884; and VL comprised in SEQ ID NO: 874; (e) VH comprised in SEQ ID NO: 885; and VL comprised in SEQ ID NO: 876; (f) VH comprised in SEQ ID NO: 886; and VL comprised in SEQ ID NO: 876; (g) VH comprised in SEQ ID NO: 888; and VL comprised in SEQ ID NO: 887; (h) VH comprised in SEQ ID NO: 891; and VL comprised in SEQ ID NO: 890; (i) VH comprised in SEQ ID NO: 904; and VL comprised in SEQ ID NO: 876; (j) VH comprised in SEQ ID NO: 907; and VL comprised in SEQ ID NO: 906; (k) VH comprised in SEQ ID NO: 909; and VL comprised in SEQ ID NO: 876; and (l) VH and VL that compete with the VH and VL described in (a) to (k). [121-c] Any one of the fusion proteins of

[0101] to

[0120] , wherein the at least one ligand moiety comprises IL-12, and the fusion protein comprises any one of the combinations selected from the following (a) to (d): (a) a first light chain comprising the sequence of SEQ ID NO: 876, a first heavy chain comprising the sequence of SEQ ID NO: 881, a second light chain comprising the sequence of SEQ ID NO: 882, and a second heavy chain comprising the sequence of SEQ ID NO: 883; (b) a first light chain comprising the sequence of SEQ ID NO: 876, a first heavy chain comprising the sequence of SEQ ID NO: 886, a second light chain comprising the sequence of SEQ ID NO: 882, and a second heavy chain comprising the sequence of SEQ ID NO: 883; (c) a first light chain comprising the sequence of SEQ ID NO: 887, a first heavy chain comprising the sequence of SEQ ID NO: 888, a second light chain comprising the sequence of SEQ ID NO: 882, and a second heavy chain comprising the sequence of SEQ ID NO: 883; and (d) a first light chain comprising the sequence of SEQ ID NO: 890, a first heavy chain comprising the sequence of SEQ ID NO: 891, a second light chain comprising the sequence of SEQ ID NO: 882, and a second heavy chain comprising the sequence of SEQ ID NO: 883. [121-2] Any one of the fusion proteins of

[0101] to

[0120] , wherein at least one ligand moiety comprises IL-22, and the fusion protein comprises an antibody heavy chain and a light chain selected from the group consisting of: (a) a light chain comprising the amino acid sequence of SEQ ID NO: 912, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 913; (b) a light chain comprising the amino acid sequence of SEQ ID NO: 915, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 916; (c) a light chain comprising the amino acid sequence of SEQ ID NO: 912, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 929; (d) a light chain comprising the amino acid sequence of SEQ ID NO: 912, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 930; and (e) An antibody heavy chain and a light chain that compete with the antibody heavy chain and the antibody light chain described in (a) to (d). [121-2a] Any one of the fusion proteins of

[0101] to

[0120] , wherein the at least one ligand moiety comprises IL-22, and the ligand-binding moiety comprises an antibody variable region comprising any one of the combinations of H chain CDR1, CDR2, and CDR3 and L chain CDR1, CDR2, and CDR3 selected from the following (a) to (e), or an antibody variable region comprising any one of the combinations of H chain CDR1, CDR2, and CDR3 and L chain CDR1, CDR2, and CDR3 of antibody variable regions functionally equivalent thereto: (a) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 913; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 912; (b) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 916; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 915; (c) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 929; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 912; (d) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 930; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 912; and (e) H-chain and L-chain CDR1, CDR2, and CDR3 contained in antibody variable regions that compete with the antibody heavy chain variable region and antibody light chain variable region described in (a) to (d). [121-2b] Any one of the fusion proteins of

[0101] to

[0120] , wherein the at least one ligand moiety comprises IL-22, and the ligand-binding moiety comprises any one of the combinations of heavy chain variable region (VH) and light chain variable region (VL) selected from the following (a) to (e): (a) VH comprised in SEQ ID NO: 913; and VL comprised in SEQ ID NO: 912; (b) VH comprised in SEQ ID NO: 916; and VL comprised in SEQ ID NO: 915; (c) VH comprised in SEQ ID NO: 929; and VL comprised in SEQ ID NO: 912; (d) a VH comprised in SEQ ID NO: 930; and a VL comprised in SEQ ID NO: 912; and (e) VH and VL that compete with the VH and VL described in (a) to (d). [121-3] Any one of the fusion proteins of

[0101] to

[0120] , wherein the at least one ligand moiety comprises IL-2, and the fusion protein comprises an antibody heavy chain and a light chain selected from the group consisting of: (a) a light chain comprising the amino acid sequence of SEQ ID NO: 920, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 919; (b) a light chain comprising the amino acid sequence of SEQ ID NO: 923, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 922; and (c) An antibody heavy chain and a light chain that compete with the antibody heavy chain and the antibody light chain described in (a) to (b). [121-3a] Any one of the fusion proteins of

[0101] to

[0120] , wherein the at least one ligand moiety comprises IL-2, and the ligand-binding moiety comprises an antibody variable region comprising any one of the combinations of H chain CDR1, CDR2, and CDR3 and L chain CDR1, CDR2, and CDR3 selected from the following (a) to (e), or an antibody variable region comprising any one of the combinations of H chain CDR1, CDR2, and CDR3 and L chain CDR1, CDR2, and CDR3 of an antibody variable region functionally equivalent thereto: (a) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 919; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 920; (b) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 922; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 923; and (c) H-chain and L-chain CDR1, CDR2, and CDR3 contained in antibody variable regions that compete with the antibody heavy chain variable region and antibody light chain variable region described in (a) to (b). [121-3b] Any one of the fusion proteins of

[0101] to

[0120] , wherein the at least one ligand moiety comprises IL-2, and the ligand-binding moiety comprises any one of the combinations of heavy chain variable region (VH) and light chain variable region (VL) selected from the following (a) to (c): (a) VH comprised in SEQ ID NO: 919; and VL comprised in SEQ ID NO: 920; (b) VH comprised in SEQ ID NO: 922; and VL comprised in SEQ ID NO: 923; and (c) VH and VL that compete with the VH and VL described in (a)-(b).

[0122] A fusion protein of any one of

[0101] to [121-3b], wherein each protease cleavage site is independently cleavable by a protease specific to a target tissue.

[0123] The fusion protein of

[0122] , wherein the target tissue is cancer tissue.

[0124] A fusion protein of

[0123] , wherein each protease cleavage site is independently cleavable by a protease selected from the group consisting of matriptase, urokinase-type plasminogen activator (uPA), and a metalloprotease.

[0125] A fusion protein according to any one of

[0101] to

[0124] , wherein each protease cleavage site independently comprises a protease cleavage sequence selected from the group consisting of SEQ ID NOs: 2 to 135 and 160 to 870.

[0126] A fusion protein according to any one of

[0101] to

[0125] , wherein each protease cleavage site is cleavable by the same protease.

[0127] A fusion protein selected from any one of

[0101] to

[0126] , wherein each protease cleavage site contains the same protease cleavage sequence.

[0128] A fusion protein of

[0127] , wherein each protease cleavage site comprises the amino acid sequence of SEQ ID NO: 873.

[0129] The fusion protein of any one of

[0101] to [101-3], wherein the ligand-binding portion further comprises a first flexible linker added to one end of the first protease cleavage site.

[0130] A fusion protein of

[0129] , wherein the ligand-binding domain further comprises a second flexible linker attached to the other end of the first protease cleavage site.

[0131] A fusion protein of

[0129] or

[0130] , wherein the first flexible linker consists of a glycine-serine polymer.

[0132] A fusion protein of

[0130] or

[0131] , wherein the second flexible linker consists of a glycine-serine polymer.

[0133] A fusion protein according to

[0102] , wherein at least one peptide linker comprises a flexible linker.

[0134] A fusion protein according to

[0133] , wherein the flexible linker consists of a glycine-serine polymer.

[0135] The fusion protein of

[0103] , wherein the at least one peptide linker further comprises a third flexible linker attached to one end of the second protease cleavage site.

[0136] A fusion protein of

[0135] , wherein the at least one peptide linker further comprises a fourth flexible linker added to the other end of the second protease cleavage site.

[0137] A fusion protein of

[0135] or

[0136] , wherein the third flexible linker consists of a glycine-serine polymer.

[0138] A fusion protein of

[0136] or

[0137] , wherein the fourth flexible linker consists of a glycine-serine polymer.

[0139] A fusion protein of

[0115] , wherein the cleavable linker further comprises a fifth flexible linker attached to one end of the third protease cleavage site.

[0140] A fusion protein of

[0139] , wherein the cleavable linker further comprises a sixth flexible linker added to the other end of the third protease cleavage site.

[0141] A fusion protein of

[0139] or

[0140] , wherein the fifth flexible linker consists of a glycine-serine polymer.

[0142] A fusion protein of

[0140] or

[0141] , wherein the sixth flexible linker consists of a glycine-serine polymer.

[0143] the glycine-serine polymer 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: 136); Gly Gly Ser Gly (GGSG, SEQ ID NO: 137); Gly Ser Gly Gly (GSGG, SEQ ID NO: 138); Ser Gly Gly Gly (SGGG, SEQ ID NO: 139); Gly Ser Ser Gly (GSSG, SEQ ID NO: 140); Gly Gly Gly Gly Ser (GGGGS, SEQ ID NO: 141); Gly Gly Gly Ser Gly (GGGSG, SEQ ID NO: 142); Gly Gly Ser Gly Gly (GGSGG, SEQ ID NO: 143); Gly Ser Gly Gly Gly (GSGGG, SEQ ID NO: 144); Gly Ser Gly Gly Ser (GSGGS, SEQ ID NO: 145); Ser Gly Gly Gly Gly (SGGGG, SEQ ID NO: 146); Gly Ser Ser Gly Gly (GSSGG, SEQ ID NO: 147); Gly Ser Gly Ser Gly (GSGSG, SEQ ID NO: 148); Ser Gly Gly Ser Gly (SGGSG, SEQ ID NO: 149); Gly Ser Ser Ser Gly (GSSSG, SEQ ID NO: 150); Gly Gly Gly Gly Gly Ser (GGGGGS, SEQ ID NO: 151); Ser Gly Gly Gly Gly Gly (SGGGGG, SEQ ID NO: 152); Gly Gly Gly Gly Gly Ser (GGGGGGS, SEQ ID NO: 153); Ser Gly Gly Gly Gly Gly Gly (SGGGGGG, SEQ ID NO: 154); (Gly Gly Gly Gly Ser (GGGGS, SEQ ID NO: 141)); and (Ser Gly Gly Gly Gly (SGGGG, SEQ ID NO: 146)) (n is an integer greater than or equal to 1) A fusion protein selected from the group consisting of any one of

[0131] ,

[0132] ,

[0134] ,

[0137] ,

[0138] ,

[0141] , and

[0142] .

[0144] A fusion protein according to any one of

[0101] to

[0103] , wherein the ligand-binding domain comprises a non-antibody protein or polypeptide.

[0145] The fusion protein of

[0144] , wherein the non-antibody protein or polypeptide is selected from the group consisting of a scaffold peptide, a peptide aptamer, and an IL-12 receptor.

[0146] A pharmaceutical composition comprising any one of the fusion proteins of

[0101] to

[0145] .

[0147] A pharmaceutical composition of

[0146] for use in the treatment of cancer.

[0148] Use of any one of the fusion proteins

[0101] to

[0145] for the manufacture of a pharmaceutical composition for the treatment of cancer.

[0149] (a) a ligand-binding molecule comprising a ligand-binding domain and at least one first protease cleavage site; (b) at least one ligand moiety, and (c) at least one peptide linker providing connecting at least one ligand moiety to the C-terminal region of the ligand-binding molecule via at least one peptide linker; A method for producing a fusion protein according to any one of

[0101] to

[0145] , comprising: the ligand binding domain binds to the ligand moiety; the ligand-binding domain is capable of releasing the ligand moiety in the presence of a protease; method.

[0150] The method of

[0149] , further comprising the step of connecting at least one ligand moiety to the N-terminal region of the ligand-binding molecule via a cleavable linker.

[0151] A method for treating cancer, comprising administering to a subject a fusion protein of any one of

[0101] to

[0141] or a pharmaceutical composition of

[0146] or

[0147] .

[0152] A polynucleotide encoding any one of the fusion proteins of

[0101] to

[0145] .

[0153] A vector comprising the polynucleotide of

[0152] .

[0154] A host cell containing the polynucleotide of

[0152] or the vector of

[0153] .

[0155] A method for producing a fusion protein of any one of

[0101] to

[0145] , comprising a step of culturing a host cell of

[0154] . [Brief explanation of the drawings]

[0012] [Figure 1A] 1 shows a fusion protein of an IgG antibody with IL-12, in which the antibody and the IL-12 molecule are fused via a cleavable linker, which is indicated by a black triangle and is cleaved by a protease, liberating free, active IL-12 molecules after cleavage. [Figure 1B]1 shows a fusion protein of an IgG antibody with IL-12, in which the antibody and the IL-12 molecule are fused via a cleavable linker, which is indicated by a black triangle and is cleaved by a protease, liberating free, active IL-12 molecules after cleavage. [Figure 1C] 1 shows a fusion protein of an IgG antibody with IL-12, in which the antibody and the IL-12 molecule are fused via a cleavable linker, which is indicated by a black triangle and is cleaved by a protease, liberating free, active IL-12 molecules after cleavage. [Figure 2A] 1 shows a fusion protein of an IgG antibody and IL-12, in which the antibody Fc domain and the IL-12 molecule are fused via a non-cleavable linker. The cleavable linker, indicated by a black triangle, is cleaved by a protease, and active IL-12 molecules are released as antibody fusion products after cleavage. [Figure 2B] 1 shows a fusion protein of an IgG antibody and IL-12, in which the antibody Fc domain and the IL-12 molecule are fused via a non-cleavable linker. The cleavable linker, indicated by a black triangle, is cleaved by a protease, and active IL-12 molecules are released as antibody fusion products after cleavage. [Figure 2C] 1 shows a fusion protein of an IgG antibody and IL-12, in which the antibody Fc domain and the IL-12 molecule are fused via a non-cleavable linker. The cleavable linker, indicated by a black triangle, is cleaved by a protease, and active IL-12 molecules are released as antibody fusion products after cleavage. [Figure 2D] 1 shows a fusion protein of an IgG antibody and IL-12, in which the antibody Fc domain and the IL-12 molecule are fused via a non-cleavable linker. The cleavable linker, indicated by a black triangle, is cleaved by a protease, and active IL-12 molecules are released as antibody fusion products after cleavage. [Figure 2E]1 shows a fusion protein of an IgG antibody and IL-12, in which the antibody Fc domain and the IL-12 molecule are fused via a non-cleavable linker. The cleavable linker, indicated by a black triangle, is cleaved by a protease, and active IL-12 molecules are released as antibody fusion products after cleavage. [Figure 3A] 1 shows an SDS-PAGE of the cleavage products obtained after MTSP1 digestion of the free IL-12 molecule. [Figure 3B] 1 shows an SDS-PAGE of the cleavage products obtained after MTSP1 digestion of the free IL-12 molecule. [Figure 3C] 1 shows an SDS-PAGE of the cleavage products obtained after MTSP1 digestion of the free IL-12 molecule. [Figure 4A] 1 shows an SDS-PAGE of the cleavage products obtained after MTSP1 digestion of the IL-12 fusion molecule. [Figure 4B] 1 shows an SDS-PAGE of the cleavage products obtained after MTSP1 digestion of the IL-12 fusion molecule. [Figure 4C] 1 shows an SDS-PAGE of the cleavage products obtained after MTSP1 digestion of the IL-12 fusion molecule. [Figure 4D] 1 shows an SDS-PAGE of the cleavage products obtained after MTSP1 digestion of the IL-12 fusion molecule. [Figure 4E] 1 shows an SDS-PAGE of the cleavage products obtained after MTSP1 digestion of the IL-12 fusion molecule. [Figure 5] IL-12 bioactivity of intact and MTSP1-cleaved free IL-12 antibodies is shown. [Figure 6] IL-12 bioactivity of intact and MTSP1-cleaved IL-12 fusion antibodies is shown. [Figure 7A] The results of SDS-PAGE under reducing and non-reducing conditions for Mab80-L1-C1-L4-IL12 (F4 bivalent IL-12 fused Mab80) are shown. [Figure 7B]The results of SDS-PAGE under reducing and non-reducing conditions for Mab80-L1-C2-L4-IL12 (lane 4), Mab80-L1-C3-L4-IL12 (lane 5), and Mab80-L1-C4-L4-IL12 (lane 6) are shown. [Figure 8] IL-12 bioactivity of intact and MTSP1-cleaved Mab80-L1-C1-L4-IL12, Mab80-L1-C2-L4-IL12, and Mab80-L1-C3-L4-IL12 is shown. [Figure 9] Figure 1 shows IL-22 fusion antibodies in which IL-22 is fused to the C-terminus of Fc via a cleavable linker (A) or a non-cleavable linker (B). The cleavable linker is indicated by a black triangle and, when cleaved by a protease, releases active IL-22 molecules. [Figure 10A] 1 shows the SDS-PAGE results of the cleavage products obtained after uPA digestion of the free IL-22 molecule. [Figure 10B] 1 shows the results of Western blotting demonstrating IL-22 release from IL-22 free molecules after uPA digestion. [Figure 11] IL-22 bioactivity of uPA-cleaved and intact free IL-22 molecules is shown. [Figure 12] 1 shows SDS-PAGE analysis of IL-22 free antibodies with improved homogeneity. [Figure 13] 1 shows SDS-PAGE results for cleavage products obtained after uPA digestion of the IL-22 free molecules 087B03-L1-C2-L3-IL22 and 087B03-L1-C4-L3-IL22. [Figure 14] 1 shows the results of Western blotting demonstrating IL-22 release from the IL-22 free molecules 087B03-L1-C2-L3-IL22 and 087B03-L1-C4-L3-IL22 after uPA digestion. [Figure 15]IL-22 bioactivity of uPA-cleaved and intact IL-22 free molecules 087B03-L1-C2-L3-IL22 and 087B03-L1-C4-L3-IL22 is shown. [Figure 16] 1 shows a schematic diagram of an exemplary IL-2 N88D fusion antibody with and without a protease cleavage site to illustrate protease cleavage. [Figure 17] 1 shows VH release from antibodies Cx-L1-C5-L5-IL2.N88D and 16C3-L1-C5-L5-IL2.N88D as a result of protease digestion. [Figure 18] 1 shows the results of an IL-2 bioassay to evaluate the biological activity of IL-2 released from an IL-2 fusion antibody. DETAILED DESCRIPTION OF THE INVENTION

[0013] Details of the aspect As used herein, the term "polypeptide" or "protein" generally refers to a peptide having a length of about four amino acids or more. Furthermore, the polypeptide or protein referred to herein is typically, but not limited to, a polypeptide consisting of an artificially designed sequence. For example, the polypeptide or protein may be of biological origin. Alternatively, the polypeptide or protein referred to herein may be any of a natural polypeptide, a synthetic polypeptide, a recombinant polypeptide, etc. Furthermore, fragments of such polypeptides or proteins are also included in the term "polypeptide" or "protein" used herein.

[0014] As used herein, each amino acid is designated by a one-letter code or a three-letter code, or both, as represented, for example, by 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, or Val / V.

[0015] The present specification refers to amino acid modifications. Amino acid modifications refer to substitutions, deletions, additions, and insertions, or any combination thereof. In the present disclosure, amino acid modifications may also be referred to as amino acid mutations or amino acid modifications. For amino acid modifications in the amino acid sequence of an antigen-binding molecule, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR may be appropriately employed. Furthermore, several known methods for amino acid modification, such as substitution with unnatural amino acids, may also be employed (Annu Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; and Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, it is preferable to use a cell-free translation system (Clover Direct (Protein Express)) containing a tRNA in which an unnatural amino acid is bound to an amber suppressor tRNA complementary to the UAG codon (amber codon), which is one of the termination codons.

[0016] As used herein, the term "and / or" used to refer to amino acid modification sites is intended to include all combinations of amino acid modification sites, where appropriate, combined with "and" and "or." Specifically, for example, the phrase "amino acids at positions 37, 45, and / or 47 are substituted" includes the following amino acid modification variations: (a) 37th place, (b) 45th place, (c) 47th place, (d) 37th and 45th place, (e) 37th and 47th place, (f) 45th and 47th place, and (g) 37th, 45th, and 47th place.

[0017] Herein, where appropriate, amino acid modifications can be represented by a number representing a specific position, preceded and followed by the one-letter or three-letter code of the amino acid before and after modification, respectively. For example, the modification F37V or Phe37Val used to replace an amino acid contained in an antibody variable region represents the replacement of Phe at position 37, as defined by Kabat numbering, with Val. Specifically, the number represents the amino acid position as defined by Kabat numbering; the one-letter or three-letter code of the amino acid before the number represents the amino acid before replacement; and the one-letter or three-letter code of the amino acid following the number represents the amino acid after replacement. Similarly, the modification P238A or Pro238Ala used to replace an amino acid in the Fc region contained in an antibody constant region represents the replacement of Pro at position 238, as defined by EU numbering, with Ala. Specifically, the number represents the position of the amino acid as defined by EU numbering; the one-letter or three-letter code of the amino acid preceding the number represents the amino acid before substitution; and the one-letter or three-letter code of the amino acid following the number represents the amino acid after substitution.

[0018] In one aspect, the present invention provides a method for producing a composition comprising: (a) a ligand-binding portion comprising a ligand-binding domain and at least one first protease cleavage site; (b) at least one ligand moiety; and (c) at least one peptide linker connecting at least one ligand moiety to the C-terminal region of the ligand-binding moiety; In a fusion protein comprising The ligand binding domain is capable of binding to the at least one ligand moiety and releasing the at least one ligand moiety in the presence of a protease.

[0019] As used herein, the term "ligand-binding moiety" or "ligand-binding molecule" refers to a moiety or molecule capable of binding to a ligand (e.g., a ligand moiety in a fusion protein of the present invention), particularly a moiety or molecule capable of binding to a ligand when the moiety or molecule is in an uncleaved state. In this context, "binding" generally refers to binding by interactions primarily based on non-covalent bonds, such as electrostatic forces, van der Waals forces, or hydrogen bonds. Preferred examples of binding modes of ligand-binding moieties or molecules include, but are not limited to, antigen-antibody reactions through which antigen-binding domains, antigen-binding molecules, antibodies, antibody fragments, and the like bind to antigens. In certain embodiments, ligand-binding moieties or molecules include, but are not limited to, antibody fragments, antibodies, and molecules formed from antibody fragments (e.g., diabodies, chimeric antigen receptors (CARs)), including multispecific binding molecules (e.g., bispecific diabodies and bispecific antibodies).

[0020] The phrase "capable of binding to a ligand" means that the ligand-binding moiety / molecule or the ligand-binding domain in the molecule can bind to the ligand, even if the ligand-binding moiety / molecule and the ligand are not bound to each other in a single molecule but are separate molecules. That is, the ligand-binding moiety / molecule and the ligand may or may not be connected through a covalent bond. For example, the phrase "capable of binding to a ligand" does not necessarily mean that the ligand and the ligand-binding moiety / molecule are connected through a covalent bond via a linker. Also, the phrase "ligand binding is attenuated" means that the ability of the above-mentioned binding (i.e., the binding ability of the ligand-binding domain) is attenuated. For example, when the ligand and the ligand-binding molecule are connected through a covalent bond via a linker, cleavage of the linker does not mean attenuation of ligand binding. In the present invention, the ligand-binding moiety / molecule is connected to the ligand moiety / molecule via a peptide linker in a manner that binds the ligand-binding domain to the ligand moiety / molecule.

[0021] As used herein, the term "ligand-binding domain" refers to a portion of a ligand-binding moiety or molecule that binds to only a portion (epitope) of a ligand when the ligand-binding moiety / molecule is bound to the ligand. In the present invention, a ligand-binding domain is limited only by the fact that the domain binds to a ligand when the ligand-binding moiety / molecule is in an uncleaved state, and can have any structure, so long as the domain is capable of binding to a ligand of interest when the ligand-binding moiety / molecule is in an uncleaved state.Examples of ligand-binding domains include antibody heavy chain variable regions (VH), antibody light chain variable regions (VL), antibody Fv regions, single domain antibodies (sdAb), scaffold peptides, and peptide aptamers (Reverdatto S. et al., Curr Top Med Chem. 2015; 15(12): 1082-1101), IL-12 receptor, avimer (WO2004 / 044011 and WO2005 / 040229), a module called an A domain of approximately 35 amino acids contained in in vivo cell membrane proteins, adnectin (WO2002 / 032925), which contains the Fn3 domain that acts as a protein-binding domain derived from the glycoprotein fibronectin expressed on the cell membrane, affibody (WO1995 / 001937), which contains an IgG-binding domain scaffold consisting of a three-helix bundle of 58 amino acids from protein A, and ankyrin repeat (AR), each of which has a structure of 33 amino acid residues and is folded into a subunit of a turn, two antiparallel helices, and a loop. Examples of such a region include, but are not limited to, DARPins (designed ankyrin repeat proteins) (WO 2002 / 020565), which are regions exposed on the molecular surface of a protein; anticalins (WO 2003 / 029462), which have four loop regions connecting eight antiparallel strands curved toward the central axis at one end of a barrel structure, which are highly conserved in lipocalin molecules such as neutrophil gelatinase-binding lipocalin (NGAL); and concave regions in the internal parallel sheet structure of a horseshoe-shaped fold composed of repeated leucine-rich repeat (LRR) modules of variable lymphocyte receptors (VLRs) that do not have the structure of immunoglobulins, such as those found in the adaptive immune systems of jawless vertebrates such as lampreys or hagfish (WO 2008 / 016854).

[0022] As used herein, the term "antibody" is used 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 the antibody exhibits the desired antigen-binding activity.

[0023] Methods for preparing antibodies with the desired binding activity are known to those skilled in the art. A method for preparing an antibody that binds to IL-6R (anti-IL-6R antibody) is shown below as an example. Antibodies that bind to antigens other than IL-6R can also be prepared appropriately according to the examples shown below.

[0024] Anti-IL-6R antibodies can be obtained as polyclonal or monoclonal antibodies using approaches known in the art. Preferably, anti-IL-6R antibodies are prepared as mammalian-derived monoclonal antibodies. Mammalian-derived monoclonal antibodies include, for example, those produced by hybridomas and those produced by host cells transformed with expression vectors containing antibody genes by genetic engineering approaches. The antibodies described herein include "humanized antibodies" and "chimeric antibodies."

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

[0026] Specifically, monoclonal antibodies can be prepared, for example, as follows: First, the IL-6R gene is expressed to obtain IL-6R protein, which can be used as a sensitizing antigen for antibody production. Specifically, the gene sequence encoding IL-6R is inserted into an expression vector known in the art, and then an appropriate host cell is transformed with it. The desired human IL-6R protein is purified from the host cells or their culture supernatant by methods known in the art. To obtain soluble IL-6R from the culture supernatant, for example, soluble IL-6R is expressed as described by Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968). Alternatively, purified native IL-6R protein can also be used as a sensitizing antigen.

[0027] Purified IL-6R protein can be used as a sensitizing antigen for use in mammalian immunization. A partial peptide of IL-6R can also be used as a sensitizing antigen. This partial peptide can be obtained by chemical synthesis from the amino acid sequence of human IL-6R. Alternatively, the partial peptide can be obtained by incorporating a portion of the IL-6R gene into an expression vector and subsequently expressing it. Furthermore, a partial peptide can also be obtained by digesting the IL-6R protein with a protease. The region and size of the IL-6R peptide for use as such a partial peptide are not particularly limited, depending on the specific embodiment. The number of amino acids constituting the peptide as a sensitizing antigen is preferably at least 5 or more, for example, 6 or more or 7 or more. More specifically, a peptide of 8 to 50 residues, preferably 10 to 30 residues, can be used as a sensitizing antigen.

[0028] Fusion proteins of desired partial polypeptides or peptides of the IL-6R protein fused with different polypeptides can also be used as sensitizing antigens. For example, antibody Fc fragments or peptide tags can be preferably used to prepare fusion proteins for use as sensitizing antigens. A vector for expressing a fusion protein can be prepared by fusing genes encoding two or more desired polypeptide fragments in frame and inserting the fusion gene into an expression vector as described above. Methods for preparing fusion proteins are described in Molecular Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed., pp. 9.47-9.58 (1989), Cold Spring Harbor Lab. Press). Methods for obtaining IL-6R for use as a sensitizing antigen and immunization methods using this sensitizing antigen are also specifically described in WO2003 / 000883, WO2004 / 022754, WO2006 / 006693, etc.

[0029] The mammal to be immunized with the sensitizing antigen is not limited to a specific animal. The mammal to be immunized is preferably selected taking into consideration compatibility with the parent cells to be used in cell fusion. Generally, rodents (e.g., mice, rats, and hamsters), rabbits, monkeys, etc. are preferably used.

[0030] These animals are immunized with a sensitizing antigen according to methods known in the art. For example, a common immunization method involves administering the sensitizing antigen to a mammal by intraperitoneal or subcutaneous injection. Specifically, the sensitizing antigen is diluted to an appropriate dilution rate with PBS (phosphate-buffered saline), physiological saline, or the like, and, if desired, mixed with a conventional adjuvant, such as Freund's complete adjuvant, and emulsified. The resulting sensitizing antigen is then administered to the mammal several times at intervals of 4 to 21 days. Furthermore, an appropriate carrier can be used in immunization with the sensitizing antigen. In particular, when a partial peptide with a small molecular weight is used as the sensitizing antigen, immunization with the sensitizing antigen peptide bound to a carrier protein such as albumin or keyhole limpet hemocyanin may be desirable in some cases.

[0031] Alternatively, hybridomas producing the desired antibodies can also be prepared by DNA immunization, as described below. DNA immunization is an immunization method that involves immune stimulation of an immunized animal by in vivo expression of a sensitizing antigen in the immunized animal, which has been given vector DNA constructed in a form that allows expression of a gene encoding an antigen protein in the immunized animal. DNA immunization is expected to be superior to common immunization methods that use the administration of protein antigens to the immunized animal, as follows: DNA immunization can provide immune stimulation while maintaining the structure of membrane proteins (e.g., IL-6R); and - DNA immunization eliminates the need to purify the immunizing antigen.

[0032] To obtain the monoclonal antibody of the present invention by DNA immunization, first, DNA for expressing IL-6R protein is administered to the animal to be immunized. DNA encoding IL-6R can be synthesized by methods known in the art, such as PCR. The resulting DNA is inserted into an appropriate expression vector and then administered to the animal to be immunized. For example, commercially available expression vectors such as pcDNA3.1 are preferably used as the expression vector. The vector can be administered to an organism by commonly used methods. For example, DNA immunization of an animal can be performed by introducing gold particles onto which the expression vector is adsorbed into the animal's cells using a gene gun. Furthermore, antibodies that recognize IL-6R can also be prepared using the method described in WO 2003 / 104453.

[0033] The serum of the immunized mammal is then examined for an increase in the titer of antibodies that bind to IL-6R. Immune cells are then collected from the mammal and subjected to cell fusion. In particular, splenocytes can be used as preferred immune cells.

[0034] Mammalian myeloma cells are used in cell fusion with immune cells. Myeloma cells preferably have an appropriate selection marker for screening. A selection marker refers to a trait that allows (or prevents) survival under specific culture conditions. For example, hypoxanthine-guanine phosphoribosyltransferase deficiency (hereinafter referred to as HGPRT deficiency) or thymidine kinase deficiency (hereinafter referred to as TK deficiency) are known in the art as selection markers. Cells with HGPRT deficiency or TK deficiency are sensitive to hypoxanthine-aminopterin-thymidine (hereinafter referred to as HAT-sensitive). HAT-sensitive cells die in HAT-selective medium because they cannot synthesize DNA. In contrast, when these cells are fused with normal cells, the fused cells can continue DNA synthesis by using the salvage pathway of normal cells, allowing them to grow even in HAT-selective medium.

[0035] Cells with HGPRT or TK deficiency can be selected in media containing 6-thioguanine or 8-azaguanine (hereinafter abbreviated as 8AG) for HGPRT deficiency, or 5'-bromodeoxyuridine for TK deficiency. Normal cells die by incorporating these pyrimidine analogs into their DNA. In contrast, cells deficient in these enzymes can survive in selective media because they cannot incorporate the pyrimidine analogs. In addition, a selectable marker called G418 resistance confers resistance to the 2-deoxystreptamine antibiotic (a gentamicin analog) via the neomycin resistance gene. Various myeloma cells suitable for cell fusion are known in the art.

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

[0037] Basically, cell fusion between immune cells and myeloma cells is carried out according to methods known in the art, for example, the method of Kohler and Milstein et al. (Methods Enzymol. (1981) 73, 3-46). More specifically, cell fusion can be carried out in a normal nutrient medium in the presence of a cell fusion promoter. For example, polyethylene glycol (PEG) or Sendai virus (HVJ) can be used as a fusion promoter. In addition, if desired, an auxiliary agent such as dimethyl sulfoxide can be added thereto to enhance fusion efficiency.

[0038] The ratio of immune cells to myeloma cells used can be set arbitrarily. For example, the amount of immune cells is preferably set to 1 to 10 times the amount of myeloma cells. For example, RPMI1640 medium or MEM medium, which is suitable for growing myeloma cell lines, as well as regular media for use in this type of cell culture, can be used as the medium for cell fusion. Preferably, a solution supplemented with serum (e.g., fetal calf serum (FCS)) can be further added to the medium.

[0039] For cell fusion, immune cells and myeloma cells are thoroughly mixed in a predetermined amount in a medium. A PEG solution (e.g., PEG with an average molecular weight of approximately 1,000 to 6,000) preheated to approximately 37°C is added, usually at a concentration of 30 to 60% (w / v). The mixed solution is gently mixed to form the desired fused cells (hybridomas). The appropriate medium listed above is then sequentially added to the cell culture, and the supernatant is removed by centrifugation. This procedure can be repeated to remove cell fusion agents and other substances that are undesirable for hybridoma growth.

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

[0041] The hybridomas thus obtained can be selected by using a selection medium appropriate for the selection marker of the myeloma cells used in cell fusion. For example, cells with HGPRT deficiency or TK deficiency can be selected by culturing in HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). Specifically, when HAT-sensitive myeloma cells are used in cell fusion, only cells that successfully fuse with normal cells can selectively grow in HAT medium. Culture in HAT medium is continued for a sufficient time to allow cells other than the desired hybridoma (unfused cells) to die. Specifically, to select the desired hybridoma, culture can generally be carried out for several days to several weeks. Hybridomas producing the desired antibody are then screened and single-cell cloned by conventional limiting dilution.

[0042] Screening for the desired antibody and single-cell cloning can be preferably performed by screening methods based on antigen-antibody reactions known in the art. For example, a monoclonal antibody that binds to IL-6R can bind to IL-6R expressed on the cell surface. Such monoclonal antibodies can be screened, for example, by FACS (fluorescence-activated cell sorting). FACS is a system in which cells contacted with a fluorescent antibody are analyzed using laser light and the fluorescence emitted by individual cells is measured, thereby measuring the binding of the antibody to the cell surface.

[0043] To screen for hybridomas producing the desired monoclonal antibody by FACS, IL-6R-expressing cells are first prepared. The preferred cells for screening are mammalian cells overexpressing IL-6R. Using untransformed host mammalian cells as a control, the binding activity of antibodies to IL-6R on the cell surface can be selectively detected. Specifically, hybridomas producing antibodies that do not bind to control host cells but bind to cells overexpressing IL-6R are selected to obtain hybridomas producing monoclonal antibodies against IL-6R.

[0044] Alternatively, based on the principle of ELISA, antibodies can be evaluated for their binding activity to immobilized IL-6R-expressing cells. IL-6R-expressing cells are immobilized, for example, on each well of an ELISA plate. The culture supernatant of hybridomas is contacted with the immobilized cells in the wells to detect antibodies that bind to the immobilized cells. If the monoclonal antibody is derived from a mouse, the antibody bound to the cells can be detected using an anti-mouse immunoglobulin antibody. Hybridomas that produce the desired antibody that can bind to the antigen and are selected by these screening methods can be cloned by limiting dilution or other methods.

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

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

[0047] Antibodies encoded by antibody genes cloned from antibody-producing cells such as hybridomas can also be preferably used. The cloned antibody gene is inserted into an appropriate vector, which is then transfected into a host so that the antibody encoded by the gene is expressed. Methods for isolating antibody genes, inserting 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, such as those described below, are also known in the art.

[0048] For example, cDNA encoding the variable region (V region) of an anti-IL-6R antibody is obtained from hybridoma cells producing the anti-IL-6R antibody. To this end, total RNA is usually first extracted from the hybridoma. For example, mRNA can be extracted from the cells using any of the following methods: - guanidine ultracentrifugation (Biochemistry (1979) 18 (24), 5294-5299), and - AGPC method (Anal. Biochem. (1987) 162 (1), 156-159).

[0049] The extracted mRNA can be purified using an mRNA Purification Kit (GE Healthcare Bio-Sciences Corp.) or similar. Alternatively, kits for extracting total mRNA directly from cells, such as the QuickPrep mRNA Purification Kit (GE Healthcare Bio-Sciences Corp.), are also commercially available. Using such kits, mRNA can be obtained from hybridomas. cDNA encoding antibody V regions can be synthesized from the obtained mRNA using reverse transcriptase. cDNA can be synthesized, for example, using the AMV Reverse Transcriptase First-Strand cDNA Synthesis Kit (Seikagaku Corp.). Alternatively, the 5'-RACE method using PCR with a SMART RACE cDNA Amplification Kit (Clontech Laboratories, Inc.) and PCR (Proc. Natl. Acad. Sci. USA (1988) 85(23), 8998-9002; and Nucleic Acids Res. (1989) 17(8), 2919-2932) can be used appropriately for cDNA synthesis and amplification. During such cDNA synthesis, appropriate restriction enzyme sites can be further introduced at both ends of the cDNA, as described below.

[0050] The desired cDNA fragment is purified from the resulting PCR product and then ligated with vector DNA. The recombinant vector thus prepared is transfected into E. coli or other bacteria. After colony selection, the desired recombinant vector can be prepared from the E. coli that formed the colonies. Next, whether the recombinant vector contains the nucleotide sequence of the desired cDNA is confirmed by methods known in the art, such as the dideoxynucleotide chain termination method.

[0051] To obtain genes encoding the variable regions, the 5'-RACE method using primers for amplifying variable region genes is conveniently used. First, a 5'-RACE cDNA library is obtained by cDNA synthesis using RNA extracted from hybridoma cells as a template. A commercially available kit, such as the SMART RACE cDNA Amplification Kit, is used appropriately to synthesize the 5'-RACE cDNA library.

[0052] Antibody genes are amplified by PCR using the resulting 5'-RACE cDNA library as a template. Primers for amplifying mouse antibody genes can be designed based on antibody gene sequences known in the art. These primers have different nucleotide sequences depending on the immunoglobulin subclass. Therefore, the subclass is preferably determined in advance using a commercially available kit such as the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche Diagnostics KK).

[0053] Specifically, for example, to obtain a gene encoding mouse IgG, primers capable of amplifying genes encoding γ1, γ2a, γ2b, and γ3 heavy chains and κ and λ light chains can be used. To amplify IgG variable region genes, 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 a 5' RACE cDNA library preparation kit is used as the 5' primer.

[0054] Using the PCR products obtained by this amplification, immunoglobulins consisting of a combination of heavy and light chains can be reconstructed. To obtain the desired antibody, the reconstructed immunoglobulins can be screened for their binding activity to IL-6R. More preferably, for example, to obtain an antibody against IL-6R, the binding of the antibody to IL-6R is specific. Antibodies that bind to IL-6R can be screened, for example, by the following steps: (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 IL-6R-expressing cells; and (3) A step of selecting an antibody that binds to IL-6R-expressing cells.

[0055] Methods for detecting antibody binding to IL-6R-expressing cells are known in the art. Specifically, antibody binding to IL-6R-expressing cells can be detected by approaches such as FACS, as described above. Fixed preparations of IL-6R-expressing cells can be used as appropriate to assess the binding activity of the antibody.

[0056] Panning methods using phage vectors are also preferred methods for screening antibodies based on binding activity. Screening methods using phage vectors are advantageous when antibody genes are obtained as libraries of heavy and light chain subclasses from a cell population expressing polyclonal antibodies. Genes encoding the heavy and light chain variable regions can be linked via an appropriate linker sequence to form a gene encoding a single-chain Fv (scFv). A gene encoding an scFv can be inserted into a phage vector to obtain a phage that expresses the scFv on its surface. After contacting the phage with the desired antigen, the phage bound to the antigen can be recovered, and DNA encoding the scFv with the desired binding activity can be recovered. This procedure can be repeated, if necessary, to enrich for scFv with the desired binding activity.

[0057] After obtaining cDNA encoding the V region of the anti-IL-6R antibody of interest, the cDNA is digested with a restriction enzyme that recognizes the restriction enzyme sites inserted at both ends of the cDNA. The restriction enzyme preferably recognizes and digests nucleotide sequences that occur at low frequency in the nucleotide sequence constituting the antibody gene. Inserting a restriction enzyme site that provides a sticky end is preferred for inserting a single copy of the digested fragment into the vector in the correct orientation. The cDNA encoding the V region of the anti-IL-6R antibody thus digested can be inserted into an appropriate expression vector to obtain an antibody expression vector. In this case, to obtain a chimeric antibody, a gene encoding the antibody constant region (C region) and a gene encoding the V region are fused in frame. In this context, "chimeric antibody" refers to an antibody having constant and variable regions of different origins. Therefore, in addition to heterologous (e.g., mouse-human) chimeric antibodies, human-human allogeneic chimeric antibodies are also included in the chimeric antibody of the present invention. To construct a chimeric antibody expression vector, a V region gene can be inserted into an expression vector that already contains a constant region gene. Specifically, for example, a recognition sequence for a restriction enzyme that digests a V region gene can be appropriately placed at the 5' end of an expression vector carrying DNA encoding the desired antibody constant region (C region). This expression vector carrying the C region gene and V region gene is digested with the same combination of restriction enzymes and fused in-frame to construct a chimeric antibody expression vector.

[0058] To produce an anti-IL-6R monoclonal antibody, the antibody gene is inserted into an expression vector so that it is expressed under the control of an expression control region. Expression control regions for antibody expression include, for example, enhancers and promoters. Furthermore, an appropriate signal sequence can be added to the amino terminus so that the expressed antibody is secreted extracellularly. For example, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 1) can be used as a signal sequence. Any other suitable signal sequence may also be added thereto. The expressed polypeptide is cleaved at the carboxyl-terminal portion of this sequence. The cleaved polypeptide can be secreted extracellularly as a mature polypeptide. Subsequently, appropriate host cells can be transformed with this expression vector to obtain recombinant cells expressing DNA encoding the anti-IL-6R antibody.

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

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

[0061] The term "variable region" or "variable domain" refers to the region or domain of an antibody heavy or light chain that is involved in binding the antibody to its antigen. Typically, the variable domains of an antibody heavy and light chain (VH and VL, respectively) are structurally similar, each containing four conserved framework regions (FR) and three complementarity-determining regions (CDR) (see, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). One VH or VL domain may be sufficient to confer antigen-binding specificity.

[0062] As used herein, the term "complementarity determining region" or "CDR" refers to each region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops ("hypervariable loops") and / or antigen contact residues ("antigen contact sites"). Typically, antibodies contain six CDRs: three in VH (H1, H2, and H3) and three in VL (L1, L2, and L3). As used herein, exemplary CDRs include: (a) hypervariable loops formed by 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 formed by amino acid residues 24–34 (L1), 50–56 (L2), 89–97 (L3), 31–35b (H1), 50–65 (H2), and 95–102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigen contacts formed by amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) A combination of (a), (b), and / or (c) containing 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). Herein, unless otherwise specified, CDR residues and other residues in the variable domain (eg, FR residues) are numbered according to Kabat et al. (supra).

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

[0064] As used herein, the term "constant region" or "constant domain" refers to a region or domain in an antibody other than the variable region. For example, an IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 Da, composed of two identical light chains and two identical heavy chains connected by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or a heavy chain variable domain, followed by a heavy chain constant region (CH) containing a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain. The light chain of a natural antibody can be assigned to one of two types, called kappa and lambda, based on the amino acid sequence of its constant domain.

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

[0066] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one aspect, the heavy chain Fc region of human IgG1 extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, amino acid residues in the Fc region or constant region are numbered according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0067] In the present invention, a ligand-binding moiety or molecule contains at least one protease cleavage site. The protease cleavage site may be located anywhere within the ligand-binding moiety / molecule, as long as the ligand-binding domain of the ligand-binding moiety / molecule can release the ligand moiety / molecule in the presence of a protease. For example, the protease cleavage site may be located within the ligand-binding domain of the ligand-binding moiety / molecule. As used herein, the phrase "liberating / releasing a ligand moiety / molecule" or "the ligand moiety / molecule is released" means that the ligand moiety / molecule is able to exert and / or increase its biological activity through interaction with its binding partner compared to the biological activity of the ligand moiety / molecule bound to the uncleaved ligand-binding moiety / molecule, but does not refer to any specific level of release or any specific mode of action by which the ligand moiety / molecule is released. In some embodiments, in the presence of a protease, the ligand moiety / molecule can be released from the ligand-binding domain of the ligand-binding moiety / molecule due to cleavage at a protease cleavage site located within or near the ligand-binding domain of the ligand-binding moiety / molecule. In this case, even after cleavage, the ligand moiety / molecule may still be linked to the C-terminal region (e.g., Fc region / domain) of the ligand binding moiety / molecule. In some embodiments, in the presence of a protease, the ligand moiety / molecule may be released from the (whole) ligand binding moiety / molecule due to cleavage at a protease cleavage site located between the ligand moiety / molecule and the C-terminal region (e.g., Fc region / domain) of the ligand binding moiety / molecule. In this case, after cleavage, the ligand moiety / molecule is no longer linked to the C-terminal region (e.g., Fc region / domain) of the ligand binding moiety / molecule.

[0068] In one embodiment, the ligand-binding moiety or molecule binds to the ligand or ligand moiety less strongly in the cleaved state compared to the uncleaved state (i.e., ligand binding is attenuated). In embodiments in which the ligand-binding moiety / molecule binds to the ligand or ligand moiety via an antigen-antibody reaction, attenuation of ligand binding can be assessed based on the ligand binding activity of the ligand-binding moiety / molecule.

[0069] The ligand-binding activity of the ligand-binding moiety / molecule can be confirmed by well-known methods such as FACS, ELISA format, ALPHA (amplified luminescence proximity homogeneous assay) screen, BIACORE method using surface plasmon resonance (SPR) phenomenon, or BLI (biolayer interferometry) (Octet) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010). The ALPHA screen is performed using two beads, donor and acceptor, according to ALPHA technology, based on the following principle: A luminescent signal is detected only when the two beads are positioned in close proximity through the interaction between molecules bound to the donor bead and molecules bound to the acceptor bead. A photosensitizer in the donor bead, excited by a laser, converts ambient oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and reaches the nearby acceptor bead, thereby triggering a chemiluminescent reaction in the bead, which ultimately emits light. In the absence of an interaction between the molecules bound to the donor bead and the molecules bound to the acceptor bead, the singlet oxygen produced by the donor bead does not reach the acceptor bead, and therefore no chemiluminescent reaction occurs.

[0070] For example, a biotin-labeled ligand-binding molecule is bound to donor beads, and a glutathione S-transferase (GST)-tagged ligand is bound to acceptor beads. In the absence of an untagged competing ligand-binding molecule, the ligand-binding molecule interacts with the ligand, generating a signal at 520-620 nm. The untagged ligand-binding molecule competes with the tagged ligand-binding molecule for interaction with the ligand. The resulting decrease in fluorescence can be quantified to determine relative binding affinity. Biotinylation of ligand-binding molecules, such as antibodies, using sulfo-NHS-biotin or similar is known in the art. GST-tagging of a ligand can be achieved, for example, by fusing a polynucleotide encoding the ligand with a polynucleotide encoding GST in frame; expressing the resulting GST-fusion ligand from cells carrying a vector enabling expression of the fusion gene; and purifying the GST-fusion ligand using a glutathione column. The resulting signals are preferably analyzed by fitting to a one-site competition model based on nonlinear regression analysis, for example, using the software GRAPHPAD PRISM (GraphPad Software, Inc., San Diego).

[0071] One of the substances whose interaction is to be monitored (the ligand) is immobilized on a thin gold film on a sensor chip. Light is shone on the back of the sensor chip to cause total internal reflection at the interface between the gold film and the glass. As a result, a region of reduced reflection intensity (the SPR signal) is formed in a portion of the reflected light. The other substance whose interaction is to be monitored (the analyte) is then flowed over the surface of the sensor chip and allowed to bind to the ligand. The mass of the immobilized ligand molecule increases, causing a change in the refractive index of the solvent on the sensor chip surface. This change in refractive index shifts the position of the SPR signal (conversely, dissociation of the bound molecule returns the signal to its original position). The Biacore system plots the amount of shift, i.e., the change in mass on the sensor chip surface, on the vertical axis, and displays the change in mass over time as assay data (a sensorgram). Kinetics: The association rate constant (ka) and dissociation rate constant (kd) are determined from the sensorgram curve, and the dissociation constant (KD) is determined from the ratio between these constants. Inhibition assays or equilibrium analysis 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 analysis is described in Methods Enzymol. 2000; 323: 325-40.

[0072] The phrase "the ligand-binding function of a ligand-binding molecule is attenuated" means that the amount of the test ligand-binding molecule bound to the ligand is, for example, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, or 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 of the amount of the control ligand-binding molecule bound to the ligand, based on the above-mentioned measurement method. Any desired index may be used as an index of binding activity. For example, the dissociation constant (KD) may be used. When the dissociation constant (KD) is used as an index for evaluating binding activity, a larger dissociation constant (KD) of the test ligand-binding molecule for the ligand than that of the control ligand-binding molecule means that the test ligand-binding molecule has a weaker binding activity for the ligand than that of the control ligand-binding molecule. The phrase "ligand binding function is attenuated" means that the dissociation constant (KD) of the test ligand-binding molecule for the ligand is, for example, at least 2-fold, preferably at least 5-fold or at least 10-fold, and particularly preferably at least 100-fold, the dissociation constant (KD) of the control ligand-binding molecule for the ligand. Examples of control ligand-binding molecules include uncleaved forms of the ligand-binding molecule.

[0073] In the fusion proteins of the present invention, the ligand moiety or molecule is connected to the C-terminal region of the ligand-binding moiety or molecule via a peptide linker. As used herein, the term "C-terminal region" refers to a region of a polypeptide extending from an internal amino acid residue in the polypeptide to the C-terminal amino acid residue of the polypeptide. In certain embodiments, for example, when the ligand-binding moiety / molecule is in the form of an antibody or an antibody fragment containing an Fc region, the C-terminal region of the ligand-binding moiety / molecule typically refers to the region of amino acid residues 1 to 250 from the C-terminus of the ligand-binding moiety / molecule. In a preferred embodiment, the ligand-binding moiety / molecule is linked to an amino acid residue exposed on the surface of the CH3 region of the antibody Fc region via a peptide linker. In another preferred embodiment, the ligand moiety / molecule is connected to the C-terminal amino acid residue of the ligand-binding moiety / molecule via a peptide linker. The peptide linker may be attached to the ligand moiety / molecule and the C-terminal region of the ligand-binding moiety / molecule by any covalent bond, such as a peptide bond. The length of the peptide linker is not particularly limited as long as it connects the ligand moiety / molecule to the ligand-binding domain in the ligand-binding moiety / molecule. The peptide linker may or may not contain a protease cleavage site.

[0074] In one embodiment, the ligand moiety / molecule of the present invention is IL-12, which is connected to the C-terminal amino acid residue of the ligand binding moiety / molecule via a peptide linker attached to the p35 subunit of IL-12 or the p40 subunit of IL-12. In one embodiment, the ligand moiety / molecule of the present invention is IL-12, which is connected to the C-terminal amino acid residue of the ligand binding moiety / molecule via a peptide linker added to the N-terminus of the p35 subunit of IL-12 or the p40 subunit of IL-12. In one embodiment, the ligand-binding domain of the present invention is connected to the hinge region contained in the ligand-binding moiety via a peptide linker. In a preferred embodiment, the ligand-binding moiety of the present invention may further comprise a CH1 region connected to the hinge region via a peptide linker. The peptide linker can be inserted between the CH1 and the hinge on both sides of the linker. In some embodiments, the fusion protein (or ligand-binding portion) of the invention comprises a constant region comprising a peptide linker. In some embodiments, the constant region comprises a hinge region comprising a peptide linker. The peptide linker may be included at any position before / within the hinge region. The peptide linker may be included between the CH1 and the hinge region, i.e., before the amino acid sequence EPKSC (SEQ ID NO: 936) in the hinge region (Note: the first residue (E) is at position 216 (EU numbering)). The peptide linker may be included after the amino acid sequence EPKSC (SEQ ID NO: 936) in the hinge region. Examples of peptide linker positions include, but are not limited to, the following: [Peptide linker] EPKSCDKTHTCPPCP (see SEQ ID NO: 901; examples include the "C1" type); EPKSC[peptide linker]DKTHTCPPCP (see SEQ ID NO:905; examples include the "C2" type); and [Peptide linker] EPKSSDKTHTCPPCP (see SEQ ID NOs: 908 and 910; examples include "C3" and "C4" types); and EPKSCDKTHT[peptide linker]CPPCP (see SEQ ID NO:932; examples include the "C5" type). In some embodiments, the peptide linker (shown above as [peptide linker]) is a GS linker as described herein, for example, (GS)2, (GGGGS: SEQ ID NO: 141)2. The above-mentioned suitable peptide linkers can be easily selected, and can be preferably selected from a variety of lengths, such as 1 amino acid (such as Gly) to 300 amino acids, 2 to 200 amino acids, or 3 to 100 amino acids, including 4 to 100 amino acids, 5 to 100 amino acids, 5 to 50 amino acids, 5 to 30 amino acids, 5 to 25 amino acids, or 5 to 20 amino acids. Examples of peptide linkers include, but are not limited to, glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n, (GGGGS: SEQ ID NO:141)n, and (GGGS: SEQ ID NO:136)n, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Examples of constituent peptide linkers include: Gly Gly Gly Gly Ser (GGGGS, SEQ ID NO: 141) (Gly Gly Gly Gly Ser (GGGGS, SEQ ID NO: 141)) Gly Gly Gly Gly Ala (GGGGA, SEQ ID NO: 893) Gly Gly Gly Gly Glu (GGGGE, SEQ ID NO: 894) Gly Gly Gly Ser (GGGS, SEQ ID NO: 136) (Gly Gly Gly Ser (GGGS, SEQ ID NO: 136)) Gly Gly Gly Ala (GGGA, SEQ ID NO: 895) Gly Gly Gly Glu (GGGE, SEQ ID NO: 896) Gln Gln Gln Gly (QQQG, SEQ ID NO: 897) Gln Gln Gln Gln Gly (QQQQG, SEQ ID NO: 898) Ser Ser Ser Gly (SSSG, SEQ ID NO: 899) Ser Ser Ser Ser Gly (SSSSG, SEQ ID NO: 900) (n is an integer greater than or equal to 1) may include, but are not limited to: However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0075] The presence of a linker (such as a GS linker) in the hinge region between Fab and Fc can result in heterogeneity in disulfide bond formation between the HC (heavy chain constant region) and LC (light chain constant region). In some embodiments, the fusion protein of the present invention is a homodimer of a light chain and a heavy chain. The heavy chain comprises a linker ("L1", e.g., SEQ ID NO: 873), such as a cleavable linker, introduced into the elbow hinge region between the heavy chain variable region and constant region 1 ("C1", e.g., SEQ ID NO: 901). A single-chain ligand (such as IL-12 or IL-22) may be attached to the C-terminus of the Fc domain via a linker ("L4", e.g., SEQ ID NO: 903), such as a GS linker; alternatively, the linker may be a cleavable linker ("L3", e.g., SEQ ID NO: 879).

[0076] This type of fusion protein may be referred to as a "C1" variant. In some embodiments, the variant is Mab80-L1-C1-L4-IL12 (F4 bivalent IL-12 fusion Mab80), a homodimer comprising a light chain of SEQ ID NO:876 and a heavy chain of SEQ ID NO:885. In some embodiments, the variant is 087B03-L1-C1-L3-IL22, a homodimer comprising a light chain of SEQ ID NO:912 and a heavy chain of SEQ ID NO:913. In some embodiments, the variant is 087B03-C1-L4-IL22, a homodimer comprising a light chain of SEQ ID NO:912 and a heavy chain of SEQ ID NO:917. To promote homogeneity, improved forms (further variants) can be generated as follows.

[0077] In some embodiments, a "C2" variant is used. The heavy chain of this variant may include a linker ("L1", e.g., SEQ ID NO: 873), such as a cleavable linker, introduced into the elbow hinge region between the heavy chain variable region and constant region 2 ("C2", e.g., SEQ ID NO: 905). A non-limiting example of a position shift of the linker (e.g., a GS linker (GGGGSGGGGS (SEQ ID NO: 141)2) present in the hinge region) in constant region 2 is shown below: From [GGGGSGGGGSEPKSCDKTHTCPPCP] (SEQ ID NO: 937) to [EPKSCGGGGSGGGGSDKTHTCPPCP] (SEQ ID NO: 935) (the first residue (E) is at position 216 (EU numbering)). The shifted position of the linker can be appropriately selected or designed by those skilled in the art depending on the purpose, i.e., to promote homogeneity. The shifted position of the linker can promote or facilitate disulfide (cysteine-cysteine ​​(Cys-Cys)) bond formation between Cys at position 220 (C220) (EU numbering) of the heavy chain and Cys at position 214 (C214) (EU numbering) of the light chain. A single-chain ligand (e.g., IL-12 or IL-22) may be added to the C-terminus of the Fc domain via a linker such as a GS linker ("L4," e.g., SEQ ID NO: 903); alternatively, the linker may be a cleavable linker ("L3," e.g., SEQ ID NO: 879). In some embodiments, the variant is Mab80-L1-C2-L4-IL12, a homodimer comprising a light chain of SEQ ID NO:876 and a heavy chain of SEQ ID NO:904. In some embodiments, the variant is 087B03-L1-C2-L3-IL22, which is a homodimer of a light chain (SEQ ID NO: 912) and a heavy chain (SEQ ID NO: 929).

[0078] In some embodiments, a "C3" variant is used. In this variant, the light chain may contain a C214S (EU numbering) modification, and the heavy chain may contain a C220S (EU numbering) modification, which does not result in any disulfide bond formation between the heavy and light chains, i.e., between position 220 (EU numbering) of the heavy chain and position 214 (EU numbering) of the light chain. The heavy chain of this variant may contain a linker ("L1", e.g., SEQ ID NO: 873), such as a cleavable linker, introduced into the elbow hinge region between the heavy chain variable region and constant region 3 ("C3", e.g., SEQ ID NO: 908). A single-chain ligand (such as IL-12 or IL-22) may be attached to the C-terminus of the Fc domain via a linker ("L4", e.g., SEQ ID NO: 903), such as a GS linker; alternatively, the linker may be a cleavable linker ("L3", e.g., SEQ ID NO: 879). In some embodiments, the variant is Mab80-L1-C3-L4-IL12, a homodimer comprising a light chain of SEQ ID NO: 906 and a heavy chain of SEQ ID NO: 907. In some embodiments, the variant is 087B03-L1-C3-L3-IL22, a homodimer comprising a light chain of SEQ ID NO: 915 and a heavy chain of SEQ ID NO: 916.

[0079] In some embodiments, a "C4" variant is used. In this variant, the light chain may not contain the above-mentioned modifications, while the heavy chain may contain S131C (EU numbering) and C220S (EU numbering) modifications, which result in disulfide bond formation between the heavy and light chains, i.e., between Cys at position 131 (C131) (EU numbering) of the heavy chain and Cys at position 214 (C214) (EU numbering) of the light chain. The heavy chain of this variant may also contain a linker ("L1", e.g., SEQ ID NO: 873), such as a cleavable linker, introduced into the elbow hinge region between the heavy chain variable region and constant region 4 ("C4", e.g., SEQ ID NO: 910). Single-chain ligands (such as IL-12 and IL-22) may be attached to the C-terminus of the Fc domain via a linker such as a GS linker (L4, e.g., SEQ ID NO: 903); alternatively, the linker may be a cleavable linker ("L3," e.g., SEQ ID NO: 879). In some embodiments, the variant is Mab80-L1-C4-L4-IL12, a homodimer comprising a light chain of SEQ ID NO:876 and a heavy chain of SEQ ID NO:909. In some embodiments, the variant is 087B03-L1-C4-L3-IL22, which is a homodimer of a light chain (SEQ ID NO: 912) and a heavy chain (SEQ ID NO: 930).

[0080] In some embodiments, a "C5" variant is used. The heavy chain of this variant may include a linker ("L1", e.g., SEQ ID NO: 873), such as a cleavable linker, introduced into the elbow hinge region between the heavy chain variable region and constant region 5 ("C5", e.g., SEQ ID NO: 932). A non-limiting example of a position shift of the linker (e.g., a GS linker (GGGGSGGGGS (SEQ ID NO: 141)2) present in the hinge region) in constant region 5 is shown below: From [GGGGSGGGGSEPKSCDKTHTCPPCP] (SEQ ID NO: 937) to [EPKSCDKTHTGGGGSGGGGSCPPCP] (SEQ ID NO: 938) (the first residue (E) is at position 216 (EU numbering)). The shifted position of the linker can be appropriately selected or designed by those skilled in the art depending on the purpose, i.e., to promote homogeneity. The shifted position of the linker can promote or facilitate disulfide (cysteine-cysteine ​​(Cys-Cys)) bond formation between Cys at position 220 (C220) (EU numbering) of the heavy chain and Cys at position 214 (C214) (EU numbering) of the light chain. A single-chain ligand (e.g., IL-2) may be added to the C-terminus of the Fc domain via a linker such as a GS linker ("L5", e.g., SEQ ID NO: 927). In some embodiments, the variant is Cx-L1-C5-L5-IL2.N88D, a homodimer of the heavy chain (SEQ ID NO: 919) and the light chain (SEQ ID NO: 920). In some embodiments, the variant is 16C3-L1-C5-L5-IL2.N88D, a homodimer of the heavy chain (SEQ ID NO: 922) and the light chain (SEQ ID NO: 923).

[0081] In some aspects, the fusion proteins of the present invention comprise a ligand binding portion comprising (i) a ligand binding domain, (ii) a first peptide linker, and (iii) a constant region comprising a second peptide linker, a third peptide linker, and a ligand portion. The fusion protein has the following general formula (I): [Ligand-binding domain]-[Lx]-[Cx]-[Ly]-[ligand moiety] (I) is represented by During the ceremony, Lx represents a first peptide linker optionally containing a protease cleavage site, or Lx is absent; Cx represents a constant region comprising a second peptide linker and, optionally, one or more amino acid residues that are modified to or from cysteine; Ly represents a third peptide linker; The ligand binding domain is capable of binding to the ligand moiety and releasing the ligand moiety from the ligand binding domain in the presence of a protease. In some embodiments, the fusion protein is a bivalent ligand-binding fusion protein comprising two sets (e.g., two identical sets) of ligand-binding domains, a ligand portion, a first peptide linker, a constant (or Fc) region (including a second peptide linker), and a third peptide linker. When the fusion protein comprises two Fc regions, the regions dimerize with each other to form the ligand-binding portion. In this case, in some embodiments, the fusion protein may be an IgG-type protein, for example, an IgG-type antibody comprising two dimerizing Fc regions. In some embodiments, the fusion protein comprises an Fc region, eg, the protein comprises at least one (ie, one, two, or more than two) Fc region. Each of the first peptide linker, the second peptide linker, and the third peptide linker may be any linker disclosed herein. In some embodiments, each linker may be a cleavable linker or a non-cleavable linker that can or cannot be cleaved by any protease.

[0082] Alternatively, in some aspects, the fusion proteins of the present invention comprise a ligand-binding portion comprising (i) a ligand-binding domain, (ii) a first peptide linker, and (iii) a first constant region comprising a second peptide linker, a third peptide linker, and a ligand portion; and a second constant region comprising a non-ligand-binding domain, a fourth peptide, and a fifth peptide linker. The fusion protein has the following general formula (II): [Ligand-binding domain]-[Lx]-[Cx]-[Ly]-[ligand portion] / / [Non-ligand-binding domain]-[Lz]-[Cz] (II) is represented by During the ceremony, Lx, Cx, and Ly are as defined in formula (I) above; Lz represents a fourth peptide linker optionally containing a protease cleavage site, or Lz is absent; Cz represents a second constant region optionally comprising a fifth peptide linker and optionally one or more amino acid residues that are modified from or to cysteine. In some embodiments, the fusion protein is a monovalent ligand-binding fusion protein comprising a set of a ligand-binding domain, a ligand moiety, a first peptide linker, a first constant region (including a second peptide linker), and a third peptide linker; and a set of a non-ligand-binding domain, a fourth peptide linker, and a second constant region (including a fifth peptide linker). Each of the first peptide linker, the second peptide linker, the third peptide linker, the fourth peptide linker, and the fifth peptide linker may be any linker disclosed herein. In some embodiments, each linker may be a cleavable linker or a non-cleavable linker that can or cannot be cleaved by any protease.

[0083] Possible variations of the fusion proteins of the present invention may include fusion proteins having a structure in which a ligand moiety attached to a peptide linker is inserted into the C-terminal region of the ligand-binding molecule to divide the ligand-binding molecule into two parts. It is understood that such fusion proteins are also included in the scope of the present invention, as long as the ligand moiety is linked via the peptide linker to the C-terminal amino acid residue of the part containing the ligand-binding domain (which serves as the ligand-binding moiety in the present invention).

[0084] In a further embodiment of the present invention, the ligand moiety or molecule is further connected to the N-terminal region of the ligand-binding moiety or molecule via a cleavable linker. As used herein, the term "N-terminal region" refers to a region of a polypeptide extending from the N-terminus of the polypeptide to an internal amino acid residue within the polypeptide. In certain embodiments, for example, when the ligand-binding moiety / molecule is in the form of an antibody or antibody fragment, the N-terminal region of the ligand-binding moiety / molecule typically refers to the region of amino acid residues 1 to 230 from the N-terminus of the ligand-binding moiety / molecule. In a preferred embodiment, the ligand moiety / molecule is connected to the N-terminal amino acid residue of the ligand-binding moiety / molecule via a cleavable linker. The cleavable linker may be attached to the ligand moiety / molecule and the N-terminal region of the ligand-binding moiety / molecule by any covalent bond, such as a peptide bond. The length of the cleavable linker is not particularly limited, as long as it connects the ligand moiety / molecule to the ligand-binding domain in the ligand-binding moiety / molecule. The cleavable linker may contain a protease cleavage site and can be cleaved by a protease.

[0085] Possible variations of the present invention may include fusion proteins having a structure in which a ligand moiety attached to a cleavable linker is inserted into the N-terminal region of a ligand-binding molecule to divide the ligand-binding molecule into two parts. It is understood that such fusion proteins are also included in the scope of the present invention as long as the ligand moiety is linked via the cleavable linker to the N-terminal amino acid residue of the part containing the ligand-binding domain (which serves as the ligand-binding moiety in the present invention).

[0086] In one embodiment of the present invention, the ligand moiety is released from the ligand-binding domain of the ligand-binding moiety by protease cleavage of the fusion protein. In this context, when the ligand moiety is connected to the C-terminal region of the ligand-binding moiety via a peptide linker having a protease cleavage site, the ligand moiety can be completely released from the fusion protein (see, for example, Figures 1A to 1C). This type of fusion protein is referred to herein as a "free type." On the other hand, when the ligand moiety is connected to the C-terminal region of the ligand-binding moiety via a peptide linker without any protease cleavage site, the ligand moiety can be released from the ligand-binding domain while remaining fused to the C-terminal region of the ligand-binding moiety via the peptide linker (see, for example, Figures 2A to 2E). This type of fusion protein is referred to herein as a "fused type."

[0087] Methods for detecting the release of a ligand moiety or molecule from a ligand-binding domain due to cleavage of a protease cleavage site include, for example, detecting the ligand using a ligand-detecting antibody that recognizes the ligand. When the ligand-binding moiety / molecule is an antibody fragment, the ligand-detecting antibody preferably binds to the same epitope as the ligand-binding domain. The ligand detected using the ligand-detecting antibody can be confirmed by well-known methods such as FACS, ELISA format, ALPHA (amplified luminescence proximity homogeneous assay) screen, BIACORE method using surface plasmon resonance (SPR) phenomenon, or BLI (biolayer interferometry) (Octet) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).

[0088] For example, when detecting ligand release using Octet, a ligand-detecting antibody that recognizes the ligand is biotinylated and contacted with a biosensor. Ligand release can then be detected by measuring binding to the ligand in the sample. Specifically, the amount of ligand is measured using a ligand-detecting antibody in a sample containing a ligand-binding molecule and a ligand before or after protease treatment. The amount of ligand detected in the sample before and after protease treatment can be compared to detect ligand release. Alternatively, the amount of ligand is measured using a ligand-detecting antibody in a sample containing a protease, a ligand-binding molecule, and a ligand, and in a sample containing a ligand-binding molecule and a ligand without a protease. The amount of ligand detected in the sample with and without a protease can be compared to detect ligand release. More specifically, ligand release can be detected by the methods described in the Examples of this application. When the ligand-binding molecule is fused with a ligand to form a fusion protein, the amount of ligand is measured using a ligand-detecting antibody in a sample containing the fusion protein before or after protease treatment. The amount of ligand detected in the sample before and after protease treatment can be compared to detect ligand release. Alternatively, the amount of ligand is measured using a ligand-detecting antibody in a sample containing a protease and the fusion protein and in a sample containing the fusion protein without protease. The amount of ligand detected in the sample with and without protease can be compared to detect ligand release. More specifically, ligand release can be detected by the method described in the Examples of this application.

[0089] In embodiments in which the biological activity of a ligand is inhibited upon binding to the ligand-binding domain, release from the ligand-binding molecule can be detected by a method for measuring the biological activity of the ligand in a sample. Specifically, the biological activity of the ligand can be measured in a sample containing the ligand-binding molecule and the ligand before or after protease treatment, and the results before and after protease treatment can be compared to detect ligand release. Alternatively, the biological activity of the ligand can be measured in a sample containing a protease, the ligand-binding molecule, and the ligand, and in a sample containing the ligand-binding molecule and the ligand without protease, and the results can be compared to detect ligand release. When the ligand-binding molecule is fused to the ligand to form a fusion protein, the biological activity of the ligand can be measured in a sample containing the fusion protein before or after protease treatment, and the results before and after protease treatment can be compared to detect ligand release. Alternatively, the biological activity of the ligand can be measured in a sample containing the protease and the fusion protein and a sample containing the fusion protein without the protease, and compared between these samples to detect release of the ligand.

[0090] In the present invention, a protease cleavage site comprises a protease cleavage sequence and is cleaved by a protease. In certain embodiments in which the fusion protein of the present invention has multiple protease cleavage sites, the protease cleavage sites may have the same protease cleavage sequence or different protease cleavage sequences. When the protease cleavage sites have different protease cleavage sequences, the different protease sequences may be cleaved by the same protease or different proteases. In some embodiments of the present invention, the protease cleavage site may also comprise one or more amino acid residues at one or both ends of the protease cleavage sequence, as long as these residues do not inhibit the recognition and cleavage of the protease cleavage sequence by the protease.

[0091] As used herein, the term "protease" refers to an enzyme such as an endopeptidase or exopeptidase that hydrolyzes peptide bonds, and typically refers to an endopeptidase. The protease used in the present invention is limited only by its ability to cleave the protease cleavage sequence, and is not limited to any particular type of protease. In some embodiments, a target tissue-specific protease is used. The target tissue-specific protease may be, for example, (1) a protease that is expressed at a higher level in target tissue than in normal tissue; (2) a protease that has higher activity in target tissue than in normal tissue; (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; It can refer to either of the following. In more specific embodiments, cancer tissue-specific proteases or inflamed tissue-specific proteases are used.

[0092] As used herein, the term "target tissue" refers to a tissue that contains at least one target cell. In some embodiments of the present invention, the target tissue is cancer tissue. In some embodiments of the present invention, the target tissue is inflamed tissue.

[0093] The term "cancer tissue" refers to tissue containing at least one cancer cell. Therefore, for example, considering that cancer tissue contains cancer cells and blood vessels, all cell types that contribute to the formation of a tumor mass containing cancer cells and endothelial cells are included in the scope of the present invention. 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.

[0094] As used herein, examples of "inflamed tissue" include: joint tissue in rheumatoid arthritis or osteoarthritis, Lung (alveolar) tissue in bronchial asthma or COPD, digestive tissue in inflammatory bowel disease, Crohn's disease, or ulcerative colitis; fibrotic tissue in the liver, kidneys, or lungs, tissues undergoing organ transplant rejection, blood vessels or cardiac (myocardial) tissue in arteriosclerosis or heart failure, Visceral adipose tissue in metabolic syndrome, Skin tissue in atopic dermatitis and other dermatitis, and Spinal nerve tissue in herniated discs or chronic lower back pain, Any tissue infiltrated by immune cells.

[0095] Proteases that are specifically expressed or specifically activated, or that are thought to be associated with the disease state of a target tissue (target tissue-specific proteases), are known for some types of target tissue. For example, International Publication Nos. WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846 disclose proteases that are specifically expressed in cancer tissues. Also, 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 disclose proteases that are thought to be involved in inflammation.

[0096] In addition to the 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 inactive form and then converted into active form.Many tissues contain substances that inhibit active proteases, and the activity is controlled by the activation process and the presence of inhibitors (Nat Rev Cancer. 2003 Jul;3 (7):489-501).In target tissues, active proteases can be specifically activated by escaping from inhibition. Active proteases can be measured using antibodies that recognize active proteases (PNAS 2013 Jan 2; 110 (1): 93-98) or by fluorescently labeling protease-recognizable peptides such that the fluorescence is quenched before cleavage but released after cleavage (Nat Rev Drug Discov. 2010 Sep; 9 (9): 690-701. doi: 10.1038 / nrd3053).

[0097] From one perspective, the term "target tissue-specific protease" refers to: (i) a protease that is expressed at a higher level in the target tissue than in normal tissue; (ii) a protease that has higher activity in target tissue than in normal tissue; (iii) a protease that is expressed at a higher level in the target cell than in normal cells, and (iv) a protease that has higher activity in target cells than in normal cells; It can refer to either of the following.

[0098] Specific examples of proteases include cysteine ​​proteases (including cathepsin family B, L, S, etc.), aspartyl proteases (cathepsin D, E, K, O, etc.), serine proteases (including matriptase (including MT-SP1), cathepsin A and G, thrombin, plasmin, urokinase-type plasminogen activator (uPA), tissue plasminogen activator (tPA), elastase, proteinase 3, thrombin, kallikrein, tryptase, and chymase), metalloproteases (including both membrane-bound (MMP14-17 and MMP24-25) and secreted (MMP1-13, MMP18-23, and MMP26-28) metalloproteases (MMP1-28), disintegrin and metalloproteases (A disintegrin and metalloproteinase (ADAM), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), meprin (meprin α and meprin β), CD10 (CALLA), prostate-specific antigen (PSA), legumain, TMPRSS3, TMPRSS4, human 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, cathepsinX / Z / P, cruzipain, otubain 2, kallikrein-related peptidases (KLKs (KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and KLK14)), bone morphogenetic protein 1 (BMP-1), activated protein C, blood coagulation-related proteases (factors VIIa, IXa, Xa, XIa, and XIIa), and HtrA. 1, 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-like protease, AMSH, gamma secretase, antiplasmin cleaving enzyme (APCE), decysin 1, N-acetylated alpha-linked acidic dipeptidase-like 1 (NAALADL1), and furin.

[0099] From another perspective, the target tissue-specific protease can refer to a cancer tissue-specific protease or an inflamed tissue-specific protease.

[0100] Examples of cancer tissue-specific proteases include proteases that are specifically expressed in cancer tissues, as disclosed in International Publication Nos. WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846.

[0101] Regarding the type of cancer tissue-specific protease, a protease with higher expression specificity in the cancer tissue to be treated is more effective in reducing adverse reactions.Preferred cancer tissue-specific proteases have a concentration in cancer tissues that is at least 5 times, more preferably at least 10 times, even more preferably at least 100 times, particularly preferably at least 500 times, and most preferably at least 1000 times higher than their concentration in normal tissues.Preferred cancer tissue-specific proteases also have an activity in cancer tissues that is at least 2 times, more preferably at least 3 times, at least 4 times, at least 5 times, or at least 10 times, more preferably at least 100 times, particularly preferably at least 500 times, and most preferably at least 1000 times higher than their activity in normal tissues.

[0102] The cancer tissue-specific protease may be in a form bound to the cancer cell membrane, or may be in a form secreted outside the cell without binding to the cell membrane. When the cancer tissue-specific protease is not bound to the cancer cell membrane, it is preferable that the cancer tissue-specific protease is present in or near the cancer tissue. As used herein, "near the cancer tissue" means within the range where the cancer tissue-specific protease cleavage sequence is cleaved so as to exert the effect of reducing the ligand binding activity.

[0103] From an alternative perspective, cancer tissue-specific proteases are (i) proteases that are expressed at higher levels in cancer tissues than in normal tissues; (ii) a protease that has higher activity in cancer tissue than in normal tissue; (iii) proteases that are expressed at higher levels in cancer cells than in normal cells; and (iv) a protease that has higher activity in cancer cells than in normal cells; Either: One type of cancer tissue-specific protease may be used alone, or two or more types of cancer tissue-specific proteases may be used in combination. The number of types of cancer tissue-specific proteases can be appropriately determined by those skilled in the art, taking into account the type of cancer to be treated.

[0104] From these perspectives, the cancer tissue-specific protease is preferably a serine protease or a metalloprotease, more preferably matriptase (including MT-SP1), urokinase-type plasminogen activator (uPA), or a metalloprotease, even more preferably MT-SP1, uPA, MMP-2, or MMP-9 among the proteases listed above, and particularly preferably MMP-2 or MMP-9 among the proteases listed above.

[0105] Regarding the type of inflammatory tissue-specific protease, a protease with higher expression specificity in the inflammatory tissue to be treated is more effective in reducing adverse reactions. Preferred inflammatory tissue-specific proteases have a concentration in inflammatory tissue that is at least 5 times, more preferably at least 10 times, even more preferably at least 100 times, particularly preferably at least 500 times, and most preferably at least 1000 times higher than their concentration in normal tissue. Also, preferred inflammatory tissue-specific proteases have an activity in inflammatory tissue that is at least 2 times, more preferably at least 3 times, at least 4 times, at least 5 times, or at least 10 times, more preferably at least 100 times, particularly preferably at least 500 times, and most preferably at least 1000 times higher than their activity in normal tissue.

[0106] The inflammatory tissue-specific protease may be in a form bound to the inflammatory cell membrane, or may be in a form secreted extracellularly without binding to the cell membrane. When the inflammatory tissue-specific protease is not bound to the inflammatory cell membrane, it is preferable that the inflammatory tissue-specific protease is present within or near the inflammatory tissue. As used herein, "near the inflammatory tissue" means within the range where the inflammatory tissue-specific protease cleavage sequence is cleaved so that the effect of reducing ligand binding activity is exerted.

[0107] From an alternative perspective, inflammatory tissue-specific proteases (i) proteases that are expressed at higher levels in inflamed tissues than in normal tissues; (ii) proteases that have higher activity in inflamed tissue than in normal tissue; (iii) proteases that are expressed at higher levels in inflammatory cells than in normal cells; and (iv) a protease that has higher activity in inflammatory cells than in normal cells; Either: One type of inflammatory tissue-specific protease may be used alone, or two or more types of inflammatory tissue-specific proteases may be used 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 to be treated.

[0108] From these viewpoints, the inflammatory tissue-specific protease is preferably a metalloprotease among the proteases listed above, and more preferably ADAMTS5, MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP11, or MMP-13.

[0109] A protease cleavage sequence is a particular amino acid sequence that is specifically recognized by a target tissue-specific protease when a polypeptide is hydrolyzed by the target tissue-specific protease in aqueous solution. The protease cleavage sequence is preferably an amino acid sequence that is hydrolyzed with high specificity by a target tissue-specific protease that is more specifically expressed or more specifically activated in the target tissue or cells to be treated, from the viewpoint of reducing adverse reactions. Specific examples of protease cleavage sequences include target sequences that are specifically hydrolyzed by the above-listed proteases specifically expressed in cancer tissues, inflammatory tissue-specific proteases, and the like, as disclosed in International Publication Nos. WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846. For example, sequences artificially modified by appropriately introducing amino acid mutations into target sequences specifically hydrolyzed by known proteases can also be used. Alternatively, 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 be used. Furthermore, naturally occurring protease cleavage sequences may be used. For example, TGFβ is converted to its latent form by protease cleavage. Similarly, protease cleavage sequences in proteins that change their molecular form upon protease cleavage may also be used.

[0110] Examples of protease cleavage sequences that can be used include those described in WO2015 / 116933, WO2015 / 048329, WO2016 / 118629, WO2016 / 179257, WO2016 / 179285, WO2016 / 179335, WO2016 / 179003, WO2016 / 046778, WO2016 / 014974, U.S. Patent Application Publication No. 2016 / 0289324, U.S. Patent Application Publication No. 2016 / 0311903, PNAS (2000) 97: 7754-7759, Biochemical Journal (2010) 426: 219-228, and Beilstein J Nanotechnol. (2016) 7: 364-373. The protease cleavage sequence is more preferably an amino acid sequence that is specifically hydrolyzed by a suitable target tissue-specific protease as described above. The amino acid sequence that is specifically hydrolyzed by a target tissue-specific protease is preferably any of the following amino acid sequences: LSGRSDNH (SEQ ID NO: 2, cleavable by MT-SP1 or uPA), PLGLAG (SEQ ID NO: 3, cleavable by MMP-2 or MMP-9), and VPLSLTMG (SEQ ID NO: 4, cleavable by MMP-7). Any of the following sequences can also be used as protease cleavage sequences: TSTSGRSANPRG (SEQ ID NO: 5, cleavable by MT-SP1 or uPA), ISSGLLSGRSDNH (SEQ ID NO: 6, cleavable by MT-SP1 or uPA), AVGLLAPPGGLSGRSDNH (SEQ ID NO: 7, cleavable by MT-SP1 or uPA), GAGVPMSMRGGAG (SEQ ID NO: 8, cleavable by MMP-1), GAGIPVSLRSGAG ​​(SEQ ID NO: 9, cleavable by MMP-2), GPLGIAGQ (SEQ ID NO: 10, cleavable by MMP-2), GGPLGMLSQS (SEQ ID NO: 11, cleavable by MMP-2), PLGLWA (SEQ ID NO: 12, cleavable by MMP-2), GAGRPFSMIMGAG (SEQ ID NO: 13, cleavable by MMP-3), GAGVPLSLTMGAG (SEQ ID NO: 14, cleavable by MMP-7), GAGVPLSLYSGAG (SEQ ID NO: 15, cleavable by MMP-9), AANLRN (SEQ ID NO: 16, cleavable by MMP-11), AQAYVK (SEQ ID NO: 17, cleavable by MMP-11), AANYMR (SEQ ID NO: 18, cleavable by MMP-11), AAALTR (SEQ ID NO: 19, cleavable by MMP-11), AQNLMR (SEQ ID NO: 20, cleavable by MMP-11), AANYTK (SEQ ID NO: 21, cleavable by MMP-11), GAGPQGLAGQRGIVAG (SEQ ID NO: 22, cleavable by MMP-13), PRFKIIGG (SEQ ID NO: 23, cleavable by prourokinase), PRFRIIGG (SEQ ID NO: 24, cleavable by prourokinase), GAGSGRSAG (SEQ ID NO: 25, cleavable by uPA), SGRSA (SEQ ID NO: 26, cleavable by uPA), GSGRSA (SEQ ID NO: 27, cleavable by uPA), SGKSA (SEQ ID NO: 28, cleavable by uPA), SGRSS (SEQ ID NO: 29, cleavable by uPA), SGRRA (SEQ ID NO: 30, cleavable by uPA), SGRNA (SEQ ID NO: 31, cleavable by uPA), SGRKA (SEQ ID NO: 32, cleavable by uPA), QRGRSA (SEQ ID NO: 33, cleavable by tPA), GAGSLLKSRMVPNFNAG (SEQ ID NO: 34, cleavable by cathepsin B), TQGAAA (SEQ ID NO: 35, cleavable by cathepsin B), GAAAAAA (SEQ ID NO: 36, cleavable by cathepsin B), GAGAAG (SEQ ID NO: 37, cleavable by cathepsin B), AAAAAG (SEQ ID NO: 38, cleavable by cathepsin B), LCGAAI (SEQ ID NO: 39, cleavable by cathepsin B), FAQALG (SEQ ID NO: 40, cleavable by cathepsin B), LLQANP (SEQ ID NO: 41, cleavable by cathepsin B), LAAANP (SEQ ID NO: 42, cleavable by cathepsin B), LYGAQF (SEQ ID NO: 43, cleavable by cathepsin B), LSQAQG (SEQ ID NO: 44, cleavable by cathepsin B), ASAASG (SEQ ID NO: 45, cleavable by cathepsin B), FLGASL (SEQ ID NO: 46, cleavable by cathepsin B), AYGATG (SEQ ID NO: 47, cleavable by cathepsin B), LAQATG (SEQ ID NO: 48, cleavable by cathepsin B), GAGSGVVIATVIVITAG (SEQ ID NO: 49, cleavable by cathepsin L), APMAEGGG (SEQ ID NO: 50, cleavable by meprin α or meprin β), EAQGDKII (SEQ ID NO: 51, cleavable by meprin α or meprin β), LAFSDAGP (SEQ ID NO: 52, cleavable by meprin α or meprin β), YVADAPK (SEQ ID NO: 53, cleavable by meprin α or meprin β), RRRRR (SEQ ID NO: 54, cleavable by furin), RRRRRR (SEQ ID NO: 55, cleavable by furin), GQSSRHRRAL (SEQ ID NO: 56, cleavable by furin), SSRHRRALD (SEQ ID NO: 57), RKSSIIIRMRDVVL (SEQ ID NO: 58, cleavable by plasminogen), SSSFDKGKYKKGDDA (SEQ ID NO: 59, cleavable by staphylokinase), SSSFDKGKYKRGDDA (SEQ ID NO: 60, cleavable by staphylokinase), IEGR (SEQ ID NO: 61, cleavable by factor IXa), IDGR (SEQ ID NO: 62, cleavable by factor IXa), GGSIDGR (SEQ ID NO: 63, cleavable by factor IXa), GPQGIAGQ (SEQ ID NO: 64, cleavable by collagen), GPQGLLGA (SEQ ID NO: 65, cleavable by collagen), GIAGQ (SEQ ID NO: 66, cleavable by collagen), GPLGIAG (SEQ ID NO: 67, cleavable by collagen), GPEGLRVG (SEQ ID NO: 68, cleavable by collagen), YGAGLGVV (SEQ ID NO: 69, cleavable by collagen), AGLGVVER (SEQ ID NO: 70, cleavable by collagen), AGLGISST (SEQ ID NO: 71, cleavable by collagen), EPQALAMS (SEQ ID NO: 72, cleavable by collagen), QALAMSAI (SEQ ID NO: 73, cleavable by collagen), AAYHLVSQ (SEQ ID NO: 74, cleavable by collagen), MDAFLESS (SEQ ID NO: 75, cleavable by collagen), ESLPVVAV (SEQ ID NO: 76, cleavable by collagen), SAPAVESE (SEQ ID NO: 77, cleavable by collagen), DVAQFVLT (SEQ ID NO: 78, cleavable by collagen), VAQFVLTE (SEQ ID NO: 79, cleavable by collagen), AQFVLTEG (SEQ ID NO: 80, cleavable by collagen), PVQPIGPQ (SEQ ID NO: 81, cleavable by collagen), LVPRGS (SEQ ID NO: 82, cleavable by thrombin), TSTSGRSANPRG (SEQ ID NO: 83), TSTSGRSANPRG (SEQ ID NO: 84), TSGSGRSANARG (SEQ ID NO: 85), TSQSGRSANQRG (SEQ ID NO: 86), TSPSGRSAYPRG (SEQ ID NO: 87), TSGSGRSATPRG (SEQ ID NO: 88), TSQSGRSATPRG (SEQ ID NO: 89), TSASGRSATPRG (SEQ ID NO: 90), TSYSGRSAVPRG (SEQ ID NO: 91), TSYSGRSANFRG (SEQ ID NO: 92), TSSSGRSATPRG (SEQ ID NO: 93), TSTTGRSASPRG (SEQ ID NO: 94), TSTSGRSANPRG (SEQ ID NO: 95).

[0111] The sequences shown in Table 1 may also be used as protease cleavage sequences.

[0112] Table 1: Protease cleavage sequences (cleavable by uPA and MT-SP1) TIFF2026010015000002.tif234134TIFF2026010015000003.tif234170TIFF2026010015000004.t if234170TIFF2026010015000005.tif234170TIFF2026010015000006.tif234170TIFF20260100150 00007.tif234170TIFF2026010015000008.tif234170TIFF2026010015000009.tif234170TIFF202 6010015000010.tif234170TIFF2026010015000011.tif234170TIFF2026010015000012.tif234170

[0113] The following sequences may also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 96) In the sequences, X1 to X8 each represent a single amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; and X5 is A, D, E, F, G, H, I, K, X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0114] The following sequences may also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 97) In the sequences, X1 through X8 each represent a single amino acid, where X1 is an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; and X5 is A, D, E, F, G, H, I, K, L, M , N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0115] The following sequences may also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 98) In the sequence, X1 to X8 each represent a single amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, F, L, M, P, Q, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; and X5 is A, D, E, F, G, H, I, K, L, M, X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0116] The following sequences may also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 99) In the sequences, X1 to X8 each represent a single amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, and Y; X4 is R; and X5 is A, D, E, F, G, H, I, K, L, X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0117] The following sequences may also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 100) In the sequences, X1 to X8 each represent a single amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; and X5 is A, D, E, G, H, I, X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0118] The following sequences may also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 101) In the sequences, X1 through X8 each represent a single amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; and X5 is A, D, E, F , G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from E, F, K, M, N, P, Q, R, S, and W; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0119] The following sequences may also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 102) In the sequence, X1 to X8 each represent a single amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; and X5 is A, D, E, X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, F, G, L, M, P, Q, V, and W; and X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0120] The following sequences may also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 103) In the sequence, X1 to X8 each represent a single amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; and X5 is A, D, E, X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X8 is an amino acid selected from A, D, E, F, G, I, K, N, T, and W.

[0121] The following sequences may also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 104) In the sequence, X1 to X8 each represent a single amino acid, where X1 is an amino acid selected from A, G, I, P, Q, S, and Y; X2 is an amino acid selected from K or T; X3 is G; X4 is R; X5 is S; X6 is A; X7 is an amino acid selected from H, I, and V; and X8 is an amino acid selected from H, V, and Y.

[0122] The following sequences may also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 105) In the sequence, X1 to X8 each represent a single amino acid, where X1 is Y; X2 is an amino acid selected from S and T; X3 is G; X4 is R; X5 is S; X6 is an amino acid selected from A and E; X7 is an amino acid selected from N and V; and X8 is an amino acid selected from H, P, V, and Y.

[0123] The following sequences may also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 106) In the sequences, X1 through X9 each represent a single amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; and X5 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T , V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0124] The following sequences may also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 107) In the sequences, X1 through X9 each represent a single amino acid, where X1 is an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; and X5 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W. , and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0125] The following sequences may also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 108) In the sequences, X1 to X9 each represent a single amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, F, L, M, P, Q, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; and X5 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0126] The following sequences may also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 109) In the sequences, X1 to X9 each represent a single amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, and Y; X4 is R; and X5 is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0127] The following sequences may also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 110) In the sequences, X1 to X9 each represent a single amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; and X5 is A, D, E, G, H, I, K, L, M, N, Q, R, T, V, X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0128] The following sequences may also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 111) In the sequences, X1 through X9 each represent a single amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; and X5 is A, D, E, F, G, H, I, K, L, M, N, P , Q, R, S, T, V, W, and Y; X6 is an amino acid selected from E, F, K, M, N, P, Q, R, S, and W; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0129] The following sequences may also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 112) In the sequences, X1 through X9 each represent a single amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; and X5 is A, D, E, F, G, H, I, K, L, M, N, X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, F, G, L, M, P, Q, V, and W; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0130] The following sequences may also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 113) In the sequences, X1 through X9 each represent a single amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; and X5 is A, D, E, F, G, H, I, K, L, M, N, X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, I, K, N, T, and W; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0131] The following sequences may also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 114) In the sequence, X1 to X9 each represent a single amino acid, where X1 is an amino acid selected from A, G, I, P, Q, S, and Y; X2 is an amino acid selected from K or T; X3 is G; X4 is R; X5 is S; X6 is A; X7 is an amino acid selected from H, I, and V; X8 is an amino acid selected from H, V, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0132] The following sequences may also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 115) In the sequence, X1 to X9 each represent a single amino acid, where X1 is Y; X2 is an amino acid selected from S and T; X3 is G; X4 is R; X5 is S; X6 is an amino acid selected from A and E; X7 is an amino acid selected from N and V; X8 is an amino acid selected from H, P, V, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0133] The following sequences may also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 116) In the sequences, X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0134] The following sequences may also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 117) In the sequences, X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; and X5 is X6 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0135] The following sequences may also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 118) In the sequences, X1 through X11 each represent a single amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, F, L, M, P, Q, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; and X5 is A. , D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0136] The following sequences may also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 119) In the sequences, X1 through X11 each represent a single amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, and Y; X4 is R; and X5 is , A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0137] The following sequences may also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 120) In the sequences, X1 through X11 each represent a single amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X4 is R. X5 is an amino acid selected from A, D, E, G, H, I, K, L, M, N, Q, R, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0138] The following sequences may also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 121) In the sequences, X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; and X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from E, F, K, M, N, P, Q, R, S, and W; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0139] The following sequences may also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 122) In the sequences, X1 through X11 each represent a single amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; and X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, F, G, L, M, P, Q, V, and W; and X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0140] The following sequences may also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 123) In the sequences, X1 through X11 each represent a single amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; and X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X8 is an amino acid selected from A, D, E, F, G, I, K, N, T, and W.

[0141] The following sequences may also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 124) In the sequences, X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, G, I, P, Q, S, and Y; X2 is an amino acid selected from K or T; X3 is G; X4 is R; X5 is S; X6 is A; X7 is an amino acid selected from H, I, and V; and X8 is an amino acid selected from H, V, and Y.

[0142] The following sequences may also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 125) In the sequences, X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is Y; X2 is an amino acid selected from S and T; X3 is G; X4 is R; X5 is S; X6 is an amino acid selected from A and E; X7 is an amino acid selected from N and V; and X8 is an amino acid selected from H, P, V, and Y.

[0143] The following sequences may also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 126) In the sequences, X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; and X5 is A, D, E, F, G, H, X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0144] The following sequences may also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 127) In the sequences, X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; and X5 is A, D, E, F, G, H, I, K, X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0145] The following sequences may also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 128) In the sequences, X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, F, L, M, P, Q, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; and X5 is A, D, E, F, G, H, I, K, L , M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0146] The following sequences may also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 129) In the sequences, X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, and Y; X4 is R; and X5 is A, D, E, F, G, H, I, K X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0147] The following sequences may also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 130) In the sequence, X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; and X5 is A, D, E, G, H , I, K, L, M, N, Q, R, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0148] The following sequences may also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 131) In the sequence, X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; and X5 is A, D, X6 is an amino acid selected from E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0149] The following sequences may also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 132) In the sequences, X1 through X11 each represent a single amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; and X5 is A, D , E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, F, G, L, M, P, Q, V, and W; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0150] The following sequences may also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 133) In the sequences, X1 through X11 each represent a single amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; and X5 is A, D , E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, I, K, N, T, and W; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0151] The following sequences may also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 134) In the sequences, X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, G, I, P, Q, S, and Y; X2 is an amino acid selected from K or T; X3 is G; X4 is R; X5 is S; X6 is A; X7 is an amino acid selected from H, I, and V; X8 is an amino acid selected from H, V, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0152] The following sequences may also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 135) In the sequences, X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is Y; X2 is an amino acid selected from S and T; X3 is G; X4 is R; X5 is S; X6 is an amino acid selected from A and E; X7 is an amino acid selected from N and V; X8 is an amino acid selected from H, P, V, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0153] In addition to using the protease cleavage sequences described above, novel protease cleavage sequences may also be obtained by screening. For example, novel protease cleavage sequences can be discovered by altering the interaction between the cleavage sequence and the active / recognition residues of the enzyme based on the results of crystal structure analysis of known protease cleavage sequences. Novel protease cleavage sequences can also be discovered by modifying amino acids in known protease cleavage sequences and examining the interaction between the modified sequence and the protease. As another example, protease cleavage sequences can be discovered by examining the interaction between proteases and libraries of peptides displayed using in vitro display methods such as phage display and ribosome display, or arrays of peptides immobilized on chips or beads. The interaction between a protease cleavage sequence and a protease can be determined by examining cleavage of the sequence by the protease in vitro or in vivo.

[0154] To evaluate the protease cleavage sequence, protease activity, and cleavage rate of a molecule into which a protease cleavage sequence has been introduced, the cleaved fragments after protease treatment can be separated and quantified by electrophoresis such as SDS-PAGE. Non-limiting examples of methods for evaluating the cleavage rate of a molecule into which a protease cleavage sequence has been introduced include the following. For example, when evaluating the cleavage rate of an antibody variant into which a protease cleavage sequence has been introduced using recombinant human u-plasminogen activator / urokinase (human uPA, huPA) (R&D Systems; 1310-SE-010) or recombinant human matriptase / ST14 catalytic domain (human MT-SP1, hMT-SP1) (R&D Systems; 3946-SE-010), 100 micrograms / mL of the antibody variant is reacted with 40 nM huPA or 3 nM hMT-SP1 in PBS at 37°C for 1 hour, followed by capillary electrophoresis immunoassay. Capillary electrophoresis immunoassay can be performed using Wes (Protein Simple), but the method is not limited thereto. As an alternative to capillary electrophoresis immunoassay, SDS-PAGE or the like can be used for separation, followed by detection by Western blotting. The method is not limited thereto. Before and after cleavage, the light chain can be detected using an anti-human λ chain HRP-labeled antibody (abcam; ab9007), but any antibody capable of detecting cleaved fragments can also be used. The area of ​​each peak obtained after protease treatment can be output using software for Wes (Compass for SW; Protein Simple), and the cleavage rate (%) of the antibody variant can be determined using the following formula: (peak area of ​​cleaved light chain) × 100 / (peak area of ​​cleaved light chain + peak area of ​​uncleaved light chain) The cleavage rate can be determined if protein fragments are detectable before and after protease treatment. The cleavage rate can be determined not only for antibody variants but also for various protein molecules into which a protease cleavage sequence has been introduced.

[0155] The in vivo cleavage rate of a molecule incorporating a protease cleavage sequence can be determined by administering the molecule to an animal and detecting the administered molecule in a blood sample. For example, an antibody variant incorporating a protease cleavage sequence is administered to mice, and plasma is collected from the blood samples. The antibody is purified from the plasma using Dynabeads Protein A (Thermo; 10001D) by methods known to those skilled in the art, and then subjected to capillary electrophoresis immunoassay to evaluate the protease cleavage rate of the antibody variant. Capillary electrophoresis immunoassay can be performed using Wes (Protein Simple), but this method is not limited thereto. As an alternative to capillary electrophoresis immunoassay, SDS-PAGE or the like can be used for separation, followed by detection by Western blotting. This method is not limited thereto. An anti-human λ chain HRP-labeled antibody (abcam; ab9007) can be used to detect the light chain of the antibody variant collected from mice, but any antibody capable of detecting cleavage fragments can be used. Once the area of ​​each peak obtained by capillary electrophoresis immunoassay is output using software for Wes (Compass for SW; Protein Simple), the proportion of remaining light chains can be calculated as [light chain peak area] / [heavy chain peak area], thereby determining the proportion of full-length light chains remaining uncleaved in the mouse body. In vivo cleavage efficiency can be determined if protein fragments collected from the living body are detectable. The cleavage rate can be determined not only for antibody variants but also for various protein molecules into which protease cleavage sequences have been introduced. Calculating the cleavage rate using the above method makes it possible, for example, to compare the in vivo cleavage rates of antibody variants into which different cleavage sequences have been introduced, and to compare the cleavage rates of a single antibody variant between different animal models, such as normal mouse models and tumor-bearing mouse models.

[0156] For example, all of the protease cleavage sequences shown in Table 1 are disclosed in WO2019 / 107384. Polypeptides containing these protease cleavage sequences are useful as protease substrates that are hydrolyzed by the action of a protease. Accordingly, the present invention provides protease substrates comprising a sequence selected from SEQ ID NOs: 96-135 and the sequences listed in Table 1. The protease substrates of the present invention can be utilized, for example, as a library from which substrates with properties suitable for a specific purpose can be selected for incorporation into a ligand-binding moiety or molecule. Specifically, substrates can be evaluated for their susceptibility to proteases localized in lesions in order to selectively cleave the ligand-binding moiety / molecule with a protease localized in lesions. When a ligand-binding moiety / molecule connected to a ligand moiety / molecule is administered in vivo, the molecule may come into contact with various proteases before reaching the lesion. Therefore, the molecule is preferably sensitive to the protease localized in the lesion and as highly resistant as possible to other proteases. To select a desired protease cleavage sequence for a particular purpose, each protease substrate can be comprehensively analyzed in advance for its sensitivity to various proteases to determine its protease resistance. Based on the obtained protease resistance spectrum, it is possible to find a protease cleavage sequence with the required sensitivity and resistance. Alternatively, before delivery to a lesion, a ligand-binding molecule incorporating a protease cleavage sequence may be subjected to various environmental stresses, such as pH changes, temperature, and oxidation / reduction stress, in addition to the enzymatic action of a protease. Resistance to these external factors can also be compared among protease substrates, and this comparative information can be used to select a protease cleavage sequence with desirable properties for a particular purpose.

[0157] In one embodiment of the invention, a flexible linker is further attached to one or both ends of each protease cleavage site. The flexible linker attached to one end of the first protease cleavage site is referred to as the "first flexible linker," and the flexible linker attached to the other end is referred to as the "second flexible linker." Similarly, when a fusion protein of the invention contains two or more protease cleavage sites, the flexible linkers attached to the second protease cleavage site are referred to as the "third flexible linker" and "fourth flexible linker," the flexible linkers attached to the third protease cleavage site are referred to as the "fifth flexible linker" and "sixth flexible linker," and so on. The following description is provided to describe the first and second flexible linkers attached to the first protease cleavage site, but also applies to the third and subsequent flexible linkers attached to the second and subsequent protease cleavage sites. In certain embodiments, the protease cleavage site and flexible linker comprise any of the following formulas: (protease cleavage sequence), (first flexible linker)-(protease cleavage site), (protease cleavage site)-(second flexible linker), and (first flexible linker)-(protease cleavage site)-(second flexible linker). The flexible linker according to this embodiment is preferably a peptide linker. The first and second flexible linkers are each independently and optionally present, and may be the same or different, each containing at least one flexible amino acid (such as Gly). The flexible linker contains, for example, a sufficient number of residues to provide the protease cleavage sequence with the desired protease accessibility (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, and Ala, especially Gly and Ser, particularly Gly, etc.).

[0158] Flexible linkers suitable for use at both ends of a protease cleavage sequence are typically those that improve protease access to the protease cleavage sequence and increase the cleavage efficiency of the protease. Suitable flexible linkers can be easily selected and preferably can be selected from a variety of 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 present invention, the flexible linker is a peptide linker of 1 to 7 amino acids.

[0159] Examples of flexible linkers include, but are not limited to, glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n, (GSGGS: SEQ ID NO:145)n, and (GGGS: SEQ ID NO:136)n, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Among these, glycine and glycine-serine polymers have attracted attention because these amino acids are relatively unstructured and readily function as neutral tethers between components. Examples of flexible linkers comprised of glycine-serine polymers include: Ser Gly Ser(GS) Ser Gly(SG) 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: 136) Gly Gly Ser Gly (GGSG, SEQ ID NO: 137) Gly Ser Gly Gly (GSGG, SEQ ID NO: 138) Ser Gly Gly Gly (SGGG, SEQ ID NO: 139) Gly Ser Ser Gly (GSSG, SEQ ID NO: 140) Gly Gly Gly Gly Ser (GGGGS, SEQ ID NO: 141) Gly Gly Gly Ser Gly (GGGSG, SEQ ID NO: 142) Gly Gly Ser Gly Gly (GGSGG, SEQ ID NO: 143) Gly Ser Gly Gly Gly (GSGGG, SEQ ID NO: 144) Gly Ser Gly Gly Ser (GSGGS, SEQ ID NO: 145) Ser Gly Gly Gly Gly (SGGGG, SEQ ID NO: 146) Gly Ser Ser Gly Gly (GSSGG, SEQ ID NO: 147) Gly Ser Gly Ser Gly (GSGSG, SEQ ID NO: 148) Ser Gly Gly Ser Gly (SGGSG, SEQ ID NO: 149) Gly Ser Ser Ser Gly (GSSSG, SEQ ID NO: 150) Gly Gly Gly Gly Gly Ser (GGGGGS, SEQ ID NO: 151) Ser Gly Gly Gly Gly Gly (SGGGGG, SEQ ID NO: 152) Gly Gly Gly Gly Gly Gly Ser (GGGGGGS, SEQ ID NO: 153) Ser Gly Gly Gly Gly Gly Gly (SGGGGGG, SEQ ID NO: 154) (Gly Gly Gly Gly Ser (GGGGS, SEQ ID NO: 141)) (Ser Gly Gly Gly Gly (SGGGG, SEQ ID NO: 146)) (n is an integer greater than or equal to 1) may include, but are not limited to: However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0160] In some embodiments of the present invention, the ligand-binding moiety or molecule comprises a ligand-binding domain comprising an antibody VH and an antibody VL. Examples of ligand-binding moieties / molecules comprising a VH and a VL include, but are not limited to, Fv, scFv, Fab, Fab', Fab'-SH, F(ab')2, and full-length antibodies.

[0161] In some embodiments of the present invention, the ligand-binding moiety or molecule contains an Fc region. When an Fc region of an IgG antibody is used, the type is not limited, and for example, the Fc region of IgG1, IgG2, IgG3, or IgG4 may be used. When an Fc region of an IgG antibody is used, the type is not limited, and for example, the Fc region of IgG1, IgG2, or IgG4 may be used. For example, an Fc region containing one of the amino acid sequences represented by SEQ ID NOs: 155, 156, 157, and 158, or an Fc region variant prepared by modifying the Fc region, may be used. In some embodiments of the present invention, the ligand-binding moiety / molecule comprises an antibody constant region. For example, the heavy chain constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 are shown in SEQ ID NOs: 153 to 158, respectively. For example, the Fc regions of human IgG1, human IgG2, human IgG3, and human IgG4 are shown as partial sequences of SEQ ID NOs: 153 to 158.

[0162] In some more specific embodiments of the present invention, the ligand-binding moiety or molecule is an antibody. When an antibody is used as the ligand-binding moiety / molecule, binding to the ligand is achieved through the variable region. In some further specific embodiments, the ligand-binding moiety / molecule is an IgG antibody. When an IgG antibody is used as the ligand-binding moiety / molecule, the type is not limited, and IgG1, IgG2, IgG3, IgG4, etc. can be used. When an IgG antibody is used as the ligand-binding moiety / molecule, the type is not limited, and IgG1, IgG2, IgG4, etc. can be used. When an IgG antibody is used as the ligand-binding moiety / molecule, binding to the ligand is also achieved through the variable region. One or both of the two variable regions of an IgG antibody can achieve binding to the ligand. In the above-mentioned embodiments, the fusion protein of the present invention preferably contains one ligand moiety (monovalent) or two ligand moieties (bivalent) connected to the C-terminal region of the antibody moiety via one or two peptide linkers. In some embodiments, the antibody is a bispecific antibody in which only one of the two variable regions binds to the ligand of interest, the fusion protein preferably contains only one ligand moiety.

[0163] In some embodiments of the present invention, cleavage of a protease cleavage site or sequence in a ligand-binding moiety / molecule separates the domain having ligand-binding activity from the ligand-binding moiety / molecule, thereby attenuating binding to the ligand. In embodiments using an IgG antibody as the ligand-binding moiety / molecule, for example, one of the variable regions of the antibody is provided with a protease cleavage site or sequence, such that in the cleaved state the antibody is unable to form a full-length antibody variable region, thereby attenuating binding to the ligand.

[0164] As used herein, "association" can refer, for example, to a state in which two or more polypeptide regions interact with each other. Generally, hydrophobic bonds, hydrogen bonds, ionic bonds, etc. are formed between the intended polypeptide regions to form an association. As an example of a general association, it is known that antibodies, typified by natural antibodies, maintain a paired structure between a heavy chain variable region (VH) and a light chain variable region (VL) through non-covalent bonds between them.

[0165] In some embodiments of the present invention, the VH and VL contained in the ligand-binding domain associate with each other. The association between the antibody VH and the antibody VL can be disrupted, for example, by cleavage of a cleavage site or a protease cleavage sequence. Disruption of the association can be used interchangeably with, for example, complete or partial disruption of the interaction between two or more polypeptide regions. Disruption of the association between the VH and the VL may completely disrupt the interaction between the VH and the VL, or may partially disrupt the interaction between the VH and the VL. The ligand-binding domain of the present invention includes a ligand-binding portion or molecule in which the association between an antibody VL or a portion thereof and an antibody VH or a portion thereof is abolished by cleavage of a protease cleavage site or a protease cleavage sequence.

[0166] In some embodiments of the present invention, the ligand-binding moiety or molecule comprises a ligand-binding domain comprising an antibody VH and an antibody VL, and the antibody VH and VL in the ligand-binding moiety / molecule associate with each other when the protease cleavage site or protease cleavage sequence of the ligand-binding moiety / molecule is uncleaved, but the association between the antibody VH and VL in the ligand-binding moiety / molecule is abolished upon cleavage of the cleavage site or protease cleavage sequence. The cleavage site or protease cleavage sequence in the ligand-binding moiety / molecule may be located at any position in the ligand-binding moiety / molecule, as long as cleavage of the cleavage site or protease cleavage sequence can attenuate ligand binding of the ligand-binding moiety / molecule.

[0167] In some embodiments of the present invention, the ligand binding moiety or molecule comprises a ligand binding domain comprising an antibody VH, an antibody VL, and an antibody constant region. As described by Rothlisberger et al. (J Mol Biol. 2005 Apr 8; 347 (4): 773-89), it is known that the VH domain and VL domain, or the CH domain and CL domain of an antibody, interact with each other via many amino acid side chains. It is known that VH-CH1 and VL-CL can form a stable structure as a Fab domain. As previously reported, amino acid side chains generally interact between VH and VL, with a dissociation constant of 10 -5 M~10 -8 When only the VH domain and the VL domain are present, only a small proportion can form an associated state.

[0168] In some embodiments of the invention, a protease cleavage site or protease cleavage sequence is provided in a ligand-binding moiety or molecule comprising a ligand-binding domain comprising an antibody VH and an antibody VL, and the fusion protein is designed such that the two peptides in the Fab structure have an overall heavy chain-light chain interaction with each other before cleavage, but cleavage of the protease cleavage site or protease cleavage sequence results in a weakening of the interaction between the peptide containing the VH (or a portion of the VH) and the peptide containing the VL (or a portion of the VL), eliminating the association between the VH and VL.

[0169] In one embodiment of the present invention, the protease cleavage site or protease cleavage sequence is located in the antibody constant region. In a more specific embodiment, the protease cleavage site or protease cleavage sequence is located on the variable region side of amino acid position 140 (EU numbering) in the antibody heavy chain constant region, preferably on the variable region side of amino acid position 122 (EU numbering) in the antibody heavy chain constant region. In some specific embodiments, the cleavage site or protease cleavage sequence is introduced at any position in the sequence from amino acid position 118 (EU numbering) to amino acid position 140 (EU numbering) in the antibody heavy chain constant region. In another more specific embodiment, the cleavage site or protease cleavage sequence is located on the variable region side of amino acid position 130 (EU numbering) (Kabat numbering position 130) in the antibody light chain constant region, preferably on the variable region side of amino acid position 113 (EU numbering) (Kabat numbering position 113) in the antibody light chain constant region, or on the variable region side of amino acid position 112 (EU numbering) (Kabat numbering position 112) in the antibody light chain constant region. In some specific embodiments, the cleavage site or protease cleavage sequence is introduced at any position in the sequence from amino acid position 108 (EU numbering) (Kabat numbering position 108) to amino acid position 131 (EU numbering) (Kabat numbering position 131) of the antibody light chain constant region.

[0170] In one embodiment of the present invention, the protease cleavage site or protease cleavage sequence is located in the antibody VH or VL. In a more specific embodiment, the cleavage site or protease cleavage sequence is located in the antibody constant region relative to amino acid position 7 (Kabat numbering) of the antibody VH, preferably relative to amino acid position 40 (Kabat numbering) of the antibody VH, more preferably relative to amino acid position 101 (Kabat numbering) of the antibody VH, even more preferably relative to amino acid position 109 (Kabat numbering) of the antibody VH, or relative to amino acid position 111 (Kabat numbering) of the antibody VH. In more specific embodiments, the cleavage site or protease cleavage sequence is located in the antibody constant region relative to amino acid position 7 (Kabat numbering) of the antibody VL, preferably relative to amino acid position 39 (Kabat numbering) of the antibody VL, more preferably relative to amino acid position 96 (Kabat numbering) of the antibody VL, even more preferably relative to amino acid position 104 (Kabat numbering) of the antibody VL, or relative to amino acid position 105 (Kabat numbering) of the antibody VL. In some more specific embodiments, a protease cleavage site or protease cleavage sequence is introduced into an antibody VH or VL at a residue that constitutes a loop structure or at a residue close to the loop structure. The loop structure in an antibody VH or VL refers to a portion of the antibody VH or VL that does not form a secondary structure such as an α-helix or β-sheet. Specifically, the positions of the residues that constitute the loop structure and the residues close to the loop structure include amino acid positions 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), and 95 (Kabat numbering) of the antibody VH. It can refer to the range from amino acid position 7 (Kabat numbering) to amino acid position 99 (Kabat numbering), or amino acid position 101 (Kabat numbering) to amino acid position 113 (Kabat numbering) of the antibody VL, or the range from amino acid position 7 (Kabat numbering) to amino acid position 19 (Kabat numbering), amino acid position 39 (Kabat numbering) to amino acid position 46 (Kabat numbering), amino acid position 49 (Kabat numbering) to amino acid position 62 (Kabat numbering), or amino acid position 96 (Kabat numbering) to amino acid position 107 (Kabat numbering) of the antibody VL. In some more specific embodiments, the cleavage site or protease cleavage sequence is introduced at any position in the antibody VH sequence between amino acid position 7 (Kabat numbering) and amino acid position 16 (Kabat numbering), between amino acid position 40 (Kabat numbering) and amino acid position 47 (Kabat numbering), between amino acid position 55 (Kabat numbering) and amino acid position 69 (Kabat numbering), between amino acid position 73 (Kabat numbering) and amino acid position 79 (Kabat numbering), between amino acid position 83 (Kabat numbering) and amino acid position 89 (Kabat numbering), between amino acid position 95 (Kabat numbering) and amino acid position 99 (Kabat numbering), or between amino acid position 101 (Kabat numbering) and amino acid position 113 (Kabat numbering). In some more specific embodiments, the cleavage site or protease cleavage sequence is introduced at any position in the antibody VL sequence from amino acid position 7 (Kabat numbering) to amino acid position 19 (Kabat numbering), from amino acid position 39 (Kabat numbering) to amino acid position 46 (Kabat numbering), from amino acid position 49 (Kabat numbering) to amino acid position 62 (Kabat numbering), or from amino acid position 96 (Kabat numbering) to amino acid position 107 (Kabat numbering).

[0171] In one embodiment of the present invention, the protease cleavage site or protease cleavage sequence is located near the boundary between the antibody VH and the antibody constant region. The phrase "near the boundary between the antibody VH and the antibody heavy chain constant region" can refer to the region between amino acid position 101 (Kabat numbering) of the antibody VH and amino acid position 140 (EU numbering) of the antibody heavy chain constant region, preferably between amino acid position 109 (Kabat numbering) of the antibody VH and amino acid position 122 (EU numbering) of the antibody heavy chain constant region, or between amino acid position 111 (Kabat numbering) of the antibody VH and amino acid position 122 (EU numbering) of the antibody heavy chain constant region. When the antibody VH is fused to an antibody light chain constant region, the phrase "near the interface between the antibody VH and the antibody light chain constant region" can refer to the region between amino acid position 101 (Kabat numbering) of the antibody VH and amino acid position 130 (EU numbering) of the antibody light chain constant region (Kabat numbering position 130), and preferably between amino acid position 109 (Kabat numbering) of the antibody VH and amino acid position 113 (EU numbering) of the antibody light chain constant region (Kabat numbering position 113), or between amino acid position 111 (Kabat numbering) of the antibody VH and amino acid position 112 (EU numbering) of the antibody light chain constant region (Kabat numbering position 112).

[0172] In one embodiment, the cleavage site or protease cleavage sequence is located near the boundary between the antibody VL and the antibody constant region. The phrase "near the boundary between the antibody VL and the antibody light chain constant region" can refer to between amino acid position 96 (Kabat numbering) of the antibody VL and amino acid position 130 (EU numbering) of the antibody light chain constant region (Kabat numbering position 130), preferably between amino acid position 104 (Kabat numbering) of the antibody VL and amino acid position 113 (EU numbering) of the antibody light chain constant region (Kabat numbering position 113), or between amino acid position 105 (Kabat numbering) of the antibody VL and amino acid position 112 (EU numbering) of the antibody light chain constant region (Kabat numbering position 112). When the antibody VL is fused to an antibody heavy chain constant region, the phrase "near the interface between the antibody VL and the antibody heavy chain constant region" can refer to the region between amino acid position 96 (Kabat numbering) of the antibody VL and amino acid position 140 (EU numbering) of the antibody heavy chain constant region, preferably between amino acid position 104 (Kabat numbering) of the antibody VL and amino acid position 122 (EU numbering) of the antibody heavy chain constant region, or between amino acid position 105 (Kabat numbering) of the antibody VL and amino acid position 122 (EU numbering) of the antibody heavy chain constant region.

[0173] The ligand-binding moiety / molecule may be provided with protease cleavage sites or protease cleavage sequences at multiple positions selected from, for example, the antibody constant region, the antibody VH, 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. Those skilled in the art can change the form of the molecule comprising the antibody VH, the antibody VL, and the antibody constant region, for example, by swapping the antibody VH and the antibody VL. Such molecular forms are within the scope of the present invention.

[0174] As used herein, the term "ligand moiety" or "ligand molecule" refers to a moiety or molecule having biological activity. As used herein, "ligand moiety" and "ligand molecule" may simply be referred to as "ligand." Biologically active molecules typically function by interacting with receptors on the cell surface, thereby stimulating, inhibiting, or otherwise modulating biological functions. These functions are typically thought to be involved in intracellular signaling pathways in cells bearing the receptor.

[0175] As used herein, the term "ligand" encompasses a desired molecule that exerts biological activity through interaction with a biomolecule. For example, the term "ligand" not only refers to a molecule that interacts with a receptor, but also includes a molecule that exerts biological activity through interaction with a molecule, such as a receptor that interacts with a molecule, or a binding fragment thereof. For example, a protein containing the ligand-binding site of a protein known as a receptor and the receptor's interaction site with another molecule is included in the ligand according to the present invention. Specifically, for example, soluble receptors, soluble fragments of receptors, the extracellular domain of a transmembrane receptor, and polypeptides containing them are included in the ligand according to the present invention.

[0176] The ligands of the present invention can usually exert their desired biological activity by binding to one or more binding partners. The binding partners of the ligands can be extracellular, intracellular, or transmembrane proteins. In one embodiment, the binding partners of the ligands are extracellular proteins, such as soluble receptors. In another embodiment, the binding partners of the ligands are membrane-bound receptors. The ligands of the invention can specifically bind to a binding partner with a dissociation constant (KD) of 10 micromolar (μM), 1 micromolar, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 500 pM, 400 pM, 350 pM, 300 pM, 250 pM, 200 pM, 150 pM, 100 pM, 50 pM, 25 pM, 10 pM, 5 pM, 1 pM, 0.5 pM, or 0.1 pM, or smaller.

[0177] Examples of biologically active molecules include, but are not limited to, cytokines, chemokines, polypeptide hormones, growth factors, apoptosis inducers, PAMPs, DAMPs, nucleic acids, and fragments thereof. In specific embodiments, interleukins, interferons, hematopoietic factors, members of the TNF superfamily, chemokines, cell growth factors, members of the TGF-β family, myokines, adipokines, or neurotrophic factors can be used as ligands. In more specific embodiments, CXCL9, CXCL10, CXCL11, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-22, IFN-α, IFN-β, IFN-g, MIG, I-TAC, RANTES, MIP-1a, MIP-1b, IL-1R1 (interleukin-1 receptor, type I), IL-1R2 (interleukin-1 receptor, type II), IL-1RAcP (interleukin-1 receptor accessory protein), or IL-1Ra (protein accession number NP_776214, mRNA accession number NM_173842.2) can be used as a ligand. There are no limitations on the ligand used in the present disclosure. In some embodiments, the ligand may be a wild-type (or naturally occurring) ligand, or a mutant ligand having any mutation. In the case of IL-12, which is a heterodimeric cytokine, in some embodiments, the ligand IL-12 may be wild-type (or naturally occurring) IL-12 or a mutant IL-12 having any mutation. In some embodiments, IL-12 may be a single-chain IL-12 in which p35 and p40 are linked and contained in a single chain.

[0178] 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. These cytokines are secreted by glial cells of the nervous system and by many cells of the immune system. Cytokines can be classified into proteins, peptides, and glycoproteins, and comprise a large and diverse family of regulatory factors. Cytokines induce intracellular signaling through binding to their cell surface receptors, which can result in the regulation of enzyme activity, up- or down-regulation of several genes and their transcription factors, or feedback inhibition. In some embodiments, cytokines of the present invention include immune modulators such as interleukins (IL) and interferons (IFN). Suitable cytokines may include proteins from one or more of the following types: the four α-helical bundle family (including the IL-2 subfamily, the IFN subfamily, and the IL-10 subfamily); the IL-1 family (including IL-1 and IL-8); and the IL-17 family. Cytokines may also include those classified as type 1 cytokines (e.g., IFN-γ and TGF-β), which enhance cellular immune responses, or type 2 cytokines (e.g., IL-4, IL-10, and IL-13), which favor antibody responses.

[0179] Interleukin-12 (IL-12) is a heterodimeric cytokine consisting of disulfide-linked glycosylated polypeptide chains of 30 kD and 40 kD. The cytokine is synthesized and secreted by antigen-presenting cells, including dendritic cells, monocytes, macrophages, B cells, Langerhans cells, and keratinocytes, as well as natural killer (NK) cells. IL-12 mediates various biological processes and has been described as an NK cell-stimulating factor (NKSF), a T cell-stimulating factor, a cytotoxic T lymphocyte maturation factor, and an EBV-transformed B cell line factor.

[0180] Interleukin-12 binds to IL-12 receptors expressed on the cytoplasmic membrane of cells (e.g., T cells and NK cells), thereby altering (e.g., initiating or blocking) biological processes. For example, binding of IL-12 to the IL-12 receptor stimulates the proliferation of preactivated T cells and NK cells, promotes the cytolytic activity of cytotoxic T cells (CTLs), NK cells, and lymphokine-activated killer (LAK) cells, induces the production of gamma interferon (IFNγ) by T cells and NK cells, and induces the differentiation of naive Th0 cells into Th1 cells that produce IFNγ and IL-2. In particular, IL-12 is absolutely necessary for the production of cytolytic cells (e.g., NK cells and CTLs) and for initiating cellular immune responses (e.g., Th1 cell-mediated immune responses). Therefore, IL-12 is absolutely necessary for generating and regulating both protective immunity (e.g., eradication of infectious diseases) and pathological immune responses (e.g., autoimmunity).

[0181] Examples of methods for measuring the physiological activity of IL-12 include methods for measuring the cell proliferation activity of IL-12, STAT4 reporter assays, methods for measuring cell activation by IL-12 (cell surface marker expression, cytokine production, etc.), and methods for measuring the promotion of cell differentiation by IL-12.

[0182] Interleukin-22 (IL-22) is a member of the IL-10 family of cytokines. It is secreted by immune cells such as T cells, NKT cells, type 3 innate lymphoid cells (ILC3s), and, to a lesser extent, by neutrophils and macrophages. IL-22 binds to its receptor, IL-22R, a heterodimer composed of IL-22R1 and IL-10R2. IL-22R is primarily expressed on nonhematopoietic cells, such as epithelial and stromal cells. IL-22 activity is regulated by IL-22-binding protein (IL-22BP; also known as IL-22RA2), a secreted protein with high structural homology to IL-22R1. IL-22BP binds IL-22 with high affinity and blocks IL-22 from interacting with IL-22R1.

[0183] Binding of IL-22 to the IL-22 receptor results in the activation of JAK1 and TYK2 kinases, which in turn activates STAT3 signaling. IL-22 plays an important role in epithelial cell function. For example, in the intestinal tract, IL-22 promotes the integrity of the intestinal barrier by stimulating intestinal epithelial cell proliferation, mucus secretion, and antimicrobial peptide secretion. In the liver, IL-22 acts as a survival factor for hepatocytes during liver injury and also stimulates hepatocytes to proliferate for liver regeneration.

[0184] Examples of methods for measuring the physiological activity of IL-22 include methods for measuring the cell proliferation activity of IL-22, STAT3 reporter assays, and methods for measuring cell activation by IL-22 (cell surface marker expression, cytokine production, etc.).

[0185] Interleukin-2 (IL-2) is a monomeric cytokine secreted primarily by activated CD4 T cells and CD8 T cells. IL-2 binds to its receptor (IL-2R), which consists of three subunits: α, β, and γ. IL-2Rβ and γ are involved in signal transduction, while IL-2Rα and β are involved in binding. All three subunits are important for the high-affinity cytokine-receptor complex. IL-2 binds to and activates both effector and regulatory T cells, making it essential for both promoting and modulating immune responses. Examples of methods for measuring the physiological activity of IL-2 include methods for measuring the cell proliferation activity of IL-2, methods for measuring cell activation by IL-2 (cell surface marker expression, cytokine production, etc.), and methods for measuring the promotion of cell differentiation by IL-2. In some embodiments of the present invention, the ligand is a chemokine. Chemokines generally act as chemoattractants, recruiting immune effector cells to the site of chemokine expression. This may be beneficial for expressing 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 also have chemoattractant effects and will recognize that such cytokines can be classified under the term "chemokine."

[0186] Chemokines are a family of homogeneous serum proteins ranging from 7 to 16 kDa, originally characterized by their ability to induce leukocyte migration. Most chemokines contain four characteristic cysteines (Cys) and are classified into the CXC (alpha), CC (beta), C (gamma), and CX3C (delta) chemokine classes according to the motif formed by the first two cysteines. Two disulfide bonds are formed between the first and third cysteines and between the second and fourth cysteines. Disulfide bridges are generally considered necessary. Clark-Lewis and coworkers reported that disulfide bonds are crucial for the chemokine activity of at least CXCL10 (Clark-Lewis et al., J. Biol. Chem. 269: 16075-16081, 1994). The only exception to this is lymphotactin, which has only two cysteine ​​residues, and thus manages to maintain its functional structure through only one disulfide bond. The CXC or alpha subfamily is further divided into two groups: ELR-CXC chemokines and non-ELR-CXC chemokines, according to 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).

[0187] Interferon-inducible protein-10 (IP-10 or CXCL10) is induced by interferon-γ and tumor necrosis factor-α and is produced by keratinocytes, endothelial cells, fibroblasts, and monocytes. IP-10 is thought to play a role in recruiting activated T cells to sites of tissue inflammation (Dufour, et al., "IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking," J Immunol., 168: 3195-204, 2002). Furthermore, IP-10 may play a role in hypersensitivity reactions. IP-10 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).

[0188] 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; and Hattori et al., Blood 97: 3354-59, 2001), and anti-tumor hyperimmunity (Nomura et al., Int. J. Cancer 91: 597-606, 2001; and Mach and Dranoff, Curr. Opin. Immunol. 12:571-75, 2000). For example, previous reports known to those skilled in the art have discussed the biological activities of chemokines (Bruce, L. et al., "Radiolabeled Chemokine Binding Assays," Methods in Molecular Biology (2000) vol. 138, pp. 129-134; Raphaele, B. et al., "Calcium Mobilization," Methods in Molecular Biology (2000) vol. 138, pp. 143-148; and Paul D. Ponath et al., "Transwell Chemotaxis," Methods in Molecular Biology (2000) vol. 138, pp. 113-120, Humana Press, Totowa, New Jersey).

[0189] Examples of the biological activities of CXCL10 include binding to the CXCL10 receptor (CXCR3), CXCL10-induced calcium flux, CXCL10-induced cell chemotaxis, CXCL10 binding to glycosaminoglycans, and CXCL10 oligomerization. Examples of methods for measuring the physiological activity of CXCL10 include a method for measuring the cell chemotactic activity of CXCL10, a reporter assay using a cell line stably expressing CXCR3 (see PLoS One. 2010 Sep 13; 5(9): e12700), and the PathHunter™ β-arrestin recruitment assay, which uses β-arrestin recruitment induced early in GPCR signaling.

[0190] The programmed cell death 1 (PD-1) protein is an inhibitory member of the CD28 family of receptors. The CD28 family 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; and Bennett et al., (2003) J Immunol 170: 711-8). The first members of the family, CD28 and ICOS, were discovered based on their functional effects on increasing T cell proliferation after the addition of monoclonal antibodies (Hutloff et al., (1999) Nature 397: 263-266; and 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 family members, 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 possess unpaired cysteine ​​residues that allow homodimerization. In contrast, PD-1 is thought to exist as a monomer and lacks the unpaired cysteine ​​residue characteristic of other members of the CD28 family.

[0191] The PD-1 gene encodes a 55 kDa type I transmembrane protein that is part of the Ig superfamily. PD-1 contains a membrane-proximal immunoreceptor tyrosine-based inhibitory motif (ITIM) and a membrane-distal tyrosine-based switch motif (ITSM). PD-1 is structurally similar to CTLA-4, but lacks the MYPPPY motif (SEQ ID NO: 159), which is important for B7-1 and B7-2 binding. Two ligands for PD-1, PD-L1 and PD-L2, have been identified and shown to negatively regulate T cell activation upon binding to PD-1 (Freeman et al., (2000) J Exp Med 192: 1027-34; Latchman et al., (2001) Nat Immunol 2: 261-8; and 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 members of the CD28 family. PD-L1, one of the PD-1 ligands, is abundant in various human cancers (Dong et al., (2002) Nat. Med. 8: 787-9). The interaction between PD-1 and PD-L1 results in a reduction of tumor-infiltrating lymphocytes, reduced T cell receptor-mediated proliferation, and immune evasion by cancerous cells (Dong et al., (2003) J. Mol. Med. 81: 281-7; Blank et al., (2005) Cancer Immunol. Immunother. 54: 307-314; and Konishi et al., (2004) Clin. Cancer Res. 10: 5094-100). Immune suppression can be reversed by inhibiting the local interaction between PD-1 and PD-L1, and this effect is additive when the interaction between PD-2 and PD-L2 is also inhibited (Iwai et al., (2002) Proc. Natl. Acad. Sci. USA 99: 12293-7; and Brown et al., (2003) J. Immunol. 170: 1257-66).

[0192] 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; and Nishimura et al., (2001) Science 291: 319-22). PD-1 has also been found to play an important role in autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease (GVHD), type 1 diabetes mellitus, and rheumatoid arthritis (Salama et al., (2003) J Exp Med 198: 71-78; Prokunia and Alarcon-Riquelme (2004) Hum Mol Genet 13: R143; and Nielsen et al., (2004) Lupus 13: 510). In mouse B-cell tumor lines, the ITSM of PD-1 inhibits BCR-mediated Ca2+ expression. 2+ It has been shown to be essential for inhibiting the tyrosine phosphorylation of downstream effector molecules and downstream effector molecules (Okazaki et al., (2001) PNAS 98: 13866-71).

[0193] In some embodiments of the present invention, modified cytokines, modified chemokines, etc. (e.g., Annu Rev Immunol. 2015; 33: 139-67), or fusion proteins containing the modified cytokines (e.g., Stem Cells Transl Med. 2015 Jan; 4 (1): 66-73) can be used as ligands.

[0194] In some embodiments of the invention, the ligand is selected from CXCL9, CXCL10, CXCL11, PD-1, IL-2, IL-12, IL-22, IL-6R, IL-1R1, IL-1R2, IL-1RAcP, and IL-1Ra. CXCL10, PD-1, IL-2, IL-12, IL-22, IL-6R, IL-1R1, IL-1R2, IL-1RAcP, and IL-1Ra may have the same sequence as naturally occurring CXCL10, PD-1, IL-2, IL-12, IL-22, IL-6R, IL-1R1, IL-1R2, IL-1RAcP, and IL-1Ra, respectively. Alternatively, they may be modified ligands that differ in sequence from naturally occurring CXCL9, CXCL10, CXCL11, PD-1, IL-2, IL-12, IL-22, IL-6R, IL-1R1, IL-1R2, IL-1RAcP, and IL-1Ra but retain the physiological activity of the corresponding natural ligands. To obtain the modified ligands, artificial modifications may be made to the ligand sequences for various purposes. Preferably, the modified ligands are modified to be resistant to protease cleavage (protease-resistant modifications).

[0195] In some embodiments of the present invention, the biological activity of a ligand moiety or molecule is inhibited by binding to the ligand-binding domain of an uncleaved ligand-binding moiety or molecule. Examples of embodiments in which the biological activity of a ligand is inhibited include, but are not limited to, embodiments in which binding of the ligand moiety / molecule to the ligand-binding domain of an uncleaved ligand-binding moiety / molecule substantially or significantly interferes with or competes with binding of the ligand to its binding partner. When an antibody or fragment thereof having ligand-neutralizing activity is used as the ligand-binding moiety / molecule, the ligand-binding moiety / molecule bound to the ligand can inhibit the biological activity of the ligand by exerting its neutralizing activity.

[0196] In one embodiment of the present invention, the uncleaved ligand-binding moiety or molecule is preferably capable of sufficiently neutralizing the biological activity of the ligand moiety or molecule upon binding to the ligand moiety / molecule. Specifically, the biological activity of the ligand moiety / molecule bound to the uncleaved ligand-binding moiety / molecule is preferably lower than the biological activity of the ligand moiety / molecule not bound to the uncleaved ligand-binding moiety / molecule. The biological activity of the ligand bound to the uncleaved ligand-binding molecule can be, but is not limited to, for example, 90% or less, preferably 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, or 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 of the biological activity of the ligand not bound to the uncleaved ligand-binding molecule. Administration of a ligand-binding molecule that sufficiently neutralizes the biological activity of the ligand can be expected to prevent the ligand from exerting its biological activity before it reaches the target tissue. Alternatively, the present invention provides a method for neutralizing the biological activity of a ligand. The method comprises the steps of contacting a ligand-binding molecule of the present invention with a ligand whose biological activity is to be neutralized and collecting the binding product of the two molecules. The biological activity of the neutralized ligand can be restored by cleaving the ligand-binding molecule in the collected binding product. Therefore, the method for neutralizing the biological activity of a ligand according to the present invention may further comprise the step of restoring the biological activity of the ligand by cleaving the ligand-binding molecule in the binding product consisting of the ligand and the ligand-binding molecule (in other words, eliminating the neutralizing activity of the ligand-binding molecule).

[0197] In one embodiment of the present invention, the binding activity of the cleaved ligand-binding moiety or molecule to the ligand moiety or molecule is preferably lower than the binding activity of the natural ligand-binding partner (e.g., the natural receptor for the ligand) to the ligand in vivo. The binding activity of the cleaved ligand-binding moiety / molecule to the ligand moiety / molecule is, but is not limited to, for example, 90% or less, preferably 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, or 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 of the amount of ligand bound to the natural binding partner in vivo (per unit binding partner). Any desired index may be used as an index of binding activity. For example, the dissociation constant (KD) may be used. When using the dissociation constant (KD) as an index for evaluating binding activity, a dissociation constant (KD) of the cleaved ligand-binding moiety / molecule for the ligand that is greater than that of the natural in vivo ligand-binding partner means that the cleaved ligand-binding molecule has weaker binding activity for the ligand than that of the natural in vivo binding partner. The dissociation constant (KD) of the cleaved ligand-binding molecule for the ligand is, for example, at least 1.1 times, preferably at least 1.5 times, at least 2 times, at least 5 times, or at least 10 times, particularly preferably at least 100 times, the dissociation constant (KD) of the natural in vivo ligand-binding partner for the ligand. A ligand-binding molecule that has only low or almost no binding activity for the ligand after cleavage can be expected to ensure that the ligand is released by cleavage of the ligand-binding molecule and prevent it from rebinding to another ligand molecule.

[0198] The ligand desirably restores the inhibited biological activity after cleavage of the ligand-binding molecule. Desirably, the ligand binding of the cleaved ligand-binding molecule 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, methods that detect the binding of the ligand to its binding partner.

[0199] 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 through a non-covalent bond between the ligand-binding molecule and the ligand, e.g., antigen-antibody binding.

[0200] In the present invention, an uncleaved ligand-binding molecule is fused with a ligand molecule to form a fusion protein. The ligand-binding domain of the ligand-binding moiety and the ligand moiety 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 peptide linker. Even when the ligand-binding molecule and the ligand in the fusion protein are fused via a peptide linker, a non-covalent bond still exists between the ligand-binding domain of the ligand-binding moiety and the ligand moiety. In other words, even in an embodiment in which the ligand-binding molecule is fused with the ligand, the non-covalent bond between the ligand-binding domain of the ligand-binding moiety and the ligand moiety is similar to that when the ligand-binding molecule is not fused with the ligand. The non-covalent bond is weakened by cleavage of the ligand-binding moiety / molecule. In other words, the ligand binding of the ligand-binding moiety / molecule is weakened. In the present invention, the ligand-binding moiety or molecule and the ligand moiety or molecule are fused via a peptide linker. For example, any peptide linker that can be introduced by genetic engineering or a linker disclosed as a synthetic compound linker (see, for example, Protein Engineering, 9 (3), 299-305, 1996) can be used as a linker for fusing the ligand-binding molecule and the ligand. The length of the peptide linker is not particularly limited and may be appropriately selected by those skilled in the art depending on the purpose. Examples of peptide linkers include: 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: 136) Gly Gly Ser Gly (GGSG, SEQ ID NO: 137) Gly Ser Gly Gly (GSGG, SEQ ID NO: 138) Ser Gly Gly Gly (SGGG, SEQ ID NO: 139) Gly Ser Ser Gly (GSSG, SEQ ID NO: 140) Gly Gly Gly Gly Ser (GGGGS, SEQ ID NO: 141) Gly Gly Gly Ser Gly (GGGSG, SEQ ID NO: 142) Gly Gly Ser Gly Gly (GGSGG, SEQ ID NO: 143) Gly Ser Gly Gly Gly (GSGGG, SEQ ID NO: 144) Gly Ser Gly Gly Ser (GSGGS, SEQ ID NO: 145) Ser Gly Gly Gly Gly (SGGGG, SEQ ID NO: 146) Gly Ser Ser Gly Gly (GSSGG, SEQ ID NO: 147) Gly Ser Gly Ser Gly (GSGSG, SEQ ID NO: 148) Ser Gly Gly Ser Gly (SGGSG, SEQ ID NO: 149) Gly Ser Ser Ser Gly (GSSSG, SEQ ID NO: 150) Gly Gly Gly Gly Gly Ser (GGGGGS, SEQ ID NO: 151) Ser Gly Gly Gly Gly Gly (SGGGGG, SEQ ID NO: 152) Gly Gly Gly Gly Gly Gly Ser (GGGGGGS, SEQ ID NO: 153) Ser Gly Gly Gly Gly Gly Gly (SGGGGGG, SEQ ID NO: 154) (Gly Gly Gly Gly Ser (GGGGS, SEQ ID NO: 141)) (Ser Gly Gly Gly Gly (SGGGG, SEQ ID NO: 146)) (n is an integer greater than or equal to 1) may include, but are not limited to: However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0201] The synthetic compound linker (chemical crosslinker) is a crosslinker commonly used for peptide crosslinking, 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), or bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES). These crosslinkers are commercially available.

[0202] In some embodiments of the present application, the ligand portion comprises IL-12 and the ligand binding portion (or fusion protein) comprises an antibody heavy chain and a light chain selected from the group consisting of: (a) a light chain comprising the amino acid sequence of SEQ ID NO: 874, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 875; (b) a light chain comprising the amino acid sequence of SEQ ID NO: 876, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 880; (c) a light chain comprising the amino acid sequence of SEQ ID NO: 876, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 881; (d) a light chain comprising the amino acid sequence of SEQ ID NO: 874, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 884; (e) a light chain comprising the amino acid sequence of SEQ ID NO: 876, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 885; (f) a light chain comprising the amino acid sequence of SEQ ID NO: 876, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 886; (g) a light chain comprising the amino acid sequence of SEQ ID NO: 887, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 888; (h) a light chain comprising the amino acid sequence of SEQ ID NO: 890, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 891; (i) a light chain comprising the amino acid sequence of SEQ ID NO: 876, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 904; (j) a light chain comprising the amino acid sequence of SEQ ID NO: 906, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 907; (k) a light chain comprising the amino acid sequence of SEQ ID NO: 876, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 909; and (l) An antibody heavy chain and a light chain that compete with the antibody heavy chain and the antibody light chain described in (a) to (k).

[0203] In some embodiments of the present application, the ligand moiety comprises IL-12, and the ligand binding moiety comprises an antibody variable region comprising any one of the combinations of H chain CDR1, CDR2, and CDR3 and L chain CDR1, CDR2, and CDR3 selected from the following (a) to (l), or an antibody variable region comprising any one of the combinations of H chain CDR1, CDR2, and CDR3 and L chain CDR1, CDR2, and CDR3 of antibody variable regions functionally equivalent thereto: (a) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 875; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 874; (b) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 880; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 876; (c) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 881; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 876; (d) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 884; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 874; (e) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 885; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 876; (f) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 886; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 876; (g) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 888; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 887; (h) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 891; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 890; (i) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 904; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 876; (j) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 907; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 906; (k) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 909; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 876; and (l) H-chain and L-chain CDR1, CDR2, and CDR3 contained in antibody variable regions that compete with the antibody heavy chain variable region and antibody light chain variable region described in (a) to (k).

[0204] In some embodiments of the present application, the ligand portion comprises IL-12, and the ligand binding portion comprises any one of the following combinations of heavy chain variable region (VH) and light chain variable region (VL) selected from (a) to (l): (a) VH comprised in SEQ ID NO: 875; and VL comprised in SEQ ID NO: 874; (b) VH comprised in SEQ ID NO: 880; and VL comprised in SEQ ID NO: 876; (c) VH comprised in SEQ ID NO: 881; and VL comprised in SEQ ID NO: 876; (d) VH comprised in SEQ ID NO: 884; and VL comprised in SEQ ID NO: 874; (e) VH comprised in SEQ ID NO: 885; and VL comprised in SEQ ID NO: 876; (f) VH comprised in SEQ ID NO: 886; and VL comprised in SEQ ID NO: 876; (g) VH comprised in SEQ ID NO: 888; and VL comprised in SEQ ID NO: 887; (h) VH comprised in SEQ ID NO: 891; and VL comprised in SEQ ID NO: 890; (i) VH comprised in SEQ ID NO: 904; and VL comprised in SEQ ID NO: 876; (j) VH comprised in SEQ ID NO: 907; and VL comprised in SEQ ID NO: 906; (k) VH comprised in SEQ ID NO: 909; and VL comprised in SEQ ID NO: 876; and (l) VH and VL that compete with the VH and VL described in (a) to (k).

[0205] In some embodiments, as shown in Tables 2 and 3 herein, the ligand portion comprises IL-12, and the fusion protein comprises any one of the following combinations selected from (a)-(d): (a) a first light chain comprising the sequence of SEQ ID NO: 876, a first heavy chain comprising the sequence of SEQ ID NO: 881, a second light chain comprising the sequence of SEQ ID NO: 882, and a second heavy chain comprising the sequence of SEQ ID NO: 883; (b) a first light chain comprising the sequence of SEQ ID NO: 876, a first heavy chain comprising the sequence of SEQ ID NO: 886, a second light chain comprising the sequence of SEQ ID NO: 882, and a second heavy chain comprising the sequence of SEQ ID NO: 883; (c) a first light chain comprising the sequence of SEQ ID NO: 887, a first heavy chain comprising the sequence of SEQ ID NO: 888, a second light chain comprising the sequence of SEQ ID NO: 882, and a second heavy chain comprising the sequence of SEQ ID NO: 883; and (d) a first light chain comprising the sequence of SEQ ID NO: 890, a first heavy chain comprising the sequence of SEQ ID NO: 891, a second light chain comprising the sequence of SEQ ID NO: 882, and a second heavy chain comprising the sequence of SEQ ID NO: 883.

[0206] In some embodiments of the present application, the ligand portion comprises IL-22, and the ligand binding portion (or fusion protein) comprises an antibody heavy chain and a light chain selected from the group consisting of: (a) a light chain comprising the amino acid sequence of SEQ ID NO: 912, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 913; (b) a light chain comprising the amino acid sequence of SEQ ID NO: 915, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 916; and (c) a light chain comprising the amino acid sequence of SEQ ID NO: 912, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 929; (d) a light chain comprising the amino acid sequence of SEQ ID NO: 912, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 930; and (e) An antibody heavy chain and a light chain that compete with the antibody heavy chain and the antibody light chain described in (a) to (d).

[0207] In some embodiments of the present application, the ligand moiety comprises IL-22, and the ligand binding moiety comprises an antibody variable region comprising any one of the combinations of H chain CDR1, CDR2, and CDR3 and L chain CDR1, CDR2, and CDR3 selected from the following (a) to (e), or an antibody variable region comprising any one of the combinations of H chain CDR1, CDR2, and CDR3 and L chain CDR1, CDR2, and CDR3 of antibody variable regions functionally equivalent thereto: (a) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 913; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 912; (b) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 916; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 915; (c) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 929; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 912; (d) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 930; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 912; and (e) H-chain and L-chain CDR1, CDR2, and CDR3 contained in antibody variable regions that compete with the antibody heavy chain variable region and antibody light chain variable region described in (a) to (d).

[0208] In some embodiments of the present application, the ligand portion comprises IL-22, and the ligand binding portion comprises any one of the following combinations of heavy chain variable region (VH) and light chain variable region (VL) selected from (a) to (e): (a) VH comprised in SEQ ID NO: 913; and VL comprised in SEQ ID NO: 912; (b) VH comprised in SEQ ID NO: 916; and VL comprised in SEQ ID NO: 915; (c) VH comprised in SEQ ID NO: 929; and VL comprised in SEQ ID NO: 912; (d) a VH comprised in SEQ ID NO: 930; and a VL comprised in SEQ ID NO: 912; and (e) VH and VL that compete with the VH and VL described in (a) to (d).

[0209] In some embodiments of the present application, the ligand moiety comprises IL-2 and the ligand binding moiety (or fusion protein) comprises an antibody heavy and light chain selected from the group consisting of: (a) a light chain comprising the amino acid sequence of SEQ ID NO: 920, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 919; (b) a light chain comprising the amino acid sequence of SEQ ID NO: 923, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 922; and (c) An antibody heavy chain and a light chain that compete with the antibody heavy chain and the antibody light chain described in (a) to (b).

[0210] In some embodiments of the present application, the ligand moiety comprises IL-2, and the ligand binding moiety comprises an antibody variable region comprising any one of the combinations of H chain CDR1, CDR2, and CDR3 and L chain CDR1, CDR2, and CDR3 selected from the following (a) to (c), or an antibody variable region comprising any one of the combinations of H chain CDR1, CDR2, and CDR3 and L chain CDR1, CDR2, and CDR3 of an antibody variable region functionally equivalent thereto: (a) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 919; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 920; (b) the heavy chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 922; and the light chain CDR1, CDR2, and CDR3 contained in the antibody variable region are identical to the amino acid sequences of the CDR1, CDR2, and CDR3 regions contained in SEQ ID NO: 923; and (c) H-chain and L-chain CDR1, CDR2, and CDR3 contained in antibody variable regions that compete with the antibody heavy chain variable region and antibody light chain variable region described in (a) to (b).

[0211] In some embodiments of the present application, the ligand portion comprises IL-2, and the ligand binding portion comprises any one of the following combinations of heavy chain variable region (VH) and light chain variable region (VL) selected from (a) to (c): (a) VH comprised in SEQ ID NO: 919; and VL comprised in SEQ ID NO: 920; (b) VH comprised in SEQ ID NO: 922; and VL comprised in SEQ ID NO: 923; and (c) VH and VL that compete with the VH and VL described in (a)-(b).

[0212] The present invention also relates to a pharmaceutical composition (medicament) comprising the fusion protein of the present invention and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition of the present disclosure is a cytostatic agent. In certain embodiments, the pharmaceutical composition of the present disclosure is a pharmaceutical composition used for the treatment and / or prevention of cancer or malignant tumors. In certain embodiments, the pharmaceutical composition of the present disclosure is a pharmaceutical composition used for the treatment and / or prevention of inflammatory disease.In certain embodiments, the pharmaceutical composition of the present disclosure is a pharmaceutical composition used for the treatment and / or prevention of intestinal and liver inflammatory disease.In certain embodiments, the pharmaceutical composition of the present disclosure is a pharmaceutical composition used for the treatment and / or prevention of inflammatory bowel disease, alcoholic fatty liver disease, or non-alcoholic fatty liver disease.In certain embodiments, the pharmaceutical composition of the present disclosure is a pharmaceutical composition used for the treatment and / or prevention of ulcerative colitis or Crohn's disease. In certain embodiments, the pharmaceutical composition of the present disclosure is used for the treatment and / or prevention of autoimmune diseases.In certain embodiments, the pharmaceutical composition of the present disclosure is used for the treatment and / or prevention of rheumatoid arthritis, type 1 diabetes, and SLE.

[0213] As used herein, "treatment" (and its grammatical derivatives, e.g., "treat" and "treating") refers to a clinical intervention intended to alter the natural course of the individual being treated and can be performed both prophylactically and during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological effects of the disease, preventing metastasis, reducing the rate of disease progression, recovering from or alleviating the disease state, and remission or improved prognosis. In some embodiments, the ligand-binding molecules of the present invention can regulate the biological activity of the ligand and are used to delay the onset of disease or slow the progression of disease.

[0214] In the present invention, a pharmaceutical composition generally refers to a pharmaceutical product for treating or preventing a disease, or for testing or diagnosing a disease. In the present invention, the term "pharmaceutical composition comprising a fusion protein" may be used interchangeably with "a method for treating a disease, comprising administering a fusion protein to a subject to be treated," and may also be used interchangeably with "use of a fusion protein for the manufacture of a medicament for treating a disease." Also, the term "pharmaceutical composition comprising a fusion protein" may be used interchangeably with "use of a fusion protein for treating a disease."

[0215] In some embodiments of the present invention, the fusion protein of the present invention can be administered to an individual. A non-covalent bond still exists between the ligand-binding domain of the ligand-binding moiety and the ligand moiety. When the fusion protein of the present invention is administered to an individual, the fusion protein is delivered in vivo. The ligand-binding moiety in the fusion protein is cleaved in the target tissue, thereby weakening the non-covalent binding of the ligand-binding domain of the ligand-binding molecule moiety to the ligand and releasing the ligand and a portion of the ligand-binding molecule from the fusion protein. The released ligand and a portion of the released ligand-binding molecule can exert the biological activity of the ligand in the target tissue and treat a disease caused by the target tissue. When the ligand-binding domain is bound to the ligand, the ligand-binding moiety suppresses the biological activity of the ligand moiety. In embodiments where the ligand-binding moiety is specifically cleaved in the target tissue, the ligand in the fusion protein does not exert biological activity during delivery and only exerts biological activity when the fusion protein is cleaved in the target tissue. As a result, diseases can be treated with fewer systemic adverse reactions.

[0216] The pharmaceutical compositions of the present invention can be formulated by methods known to those skilled in the art. For example, the pharmaceutical compositions can be used parenterally in the form of an injection of a sterile solution or suspension with water or any other pharmaceutically acceptable liquid. Pharmaceutical compositions can be formulated, for example, by appropriately combining the peptide with a pharmacologically acceptable carrier or vehicle, specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc., and mixing them to form a unit dosage form required by generally accepted pharmaceutical practice. The amount of the active ingredient in these preparations is set to provide an appropriate volume within the prescribed range.

[0217] Sterile compositions for injection can be formulated according to common pharmaceutical practice using vehicles such as distilled water for injection.Examples of aqueous solutions for injection include isotonic solutions containing physiological saline, glucose, or other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, and sodium chloride).Aqueous solutions can be used in combination with suitable solubilizers, such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), or nonionic surfactants (e.g., Polysorbate 80™, HCO-50).

[0218] Examples of oils include sesame oil and soybean oil. Oils can also be used in combination with benzyl benzoate and / or benzyl alcohol as a solubilizer. Oils can be supplemented with buffers (e.g., phosphate buffer and sodium acetate buffer), soothing agents (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants. The prepared injection solution is usually filled into suitable ampoules.

[0219] The pharmaceutical composition of the present invention is preferably administered parenterally. For example, the composition may be administered in the form of an injection, a nasal dosage form, a pulmonary dosage form, or a transdermal dosage form. The pharmaceutical composition may be administered systemically or locally, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection.

[0220] The administration method can be selected appropriately depending on the patient's age and symptoms. The dosage of a pharmaceutical composition containing a ligand-binding molecule can be set, for example, in the range of 0.0001 mg to 1,000 mg per kg of body weight per dose. Alternatively, the dosage of a pharmaceutical composition containing a polypeptide can be set, for example, in the range of 0.001 to 100,000 mg per patient. However, the present invention is not necessarily limited by these numerical values. The dosage and administration method vary depending on the patient's body weight, age, symptoms, etc., but those skilled in the art can determine an appropriate dosage and administration method taking these conditions into consideration.

[0221] The present invention also relates to a method for producing the fusion protein of the present invention. (a) a ligand-binding molecule comprising a ligand-binding domain and at least one first protease cleavage site; (b) at least one ligand molecule, and (c) at least one peptide linker providing connecting at least one ligand molecule to the C-terminal region of the ligand-binding molecule via at least one peptide linker; The present invention provides a method for producing a fusion protein, comprising:

[0222] Examples of methods for introducing a protease cleavage sequence into a molecule capable of binding to a ligand include inserting a protease cleavage sequence into the amino acid sequence of a polypeptide capable of binding to a ligand, and replacing a portion of the amino acid sequence of a polypeptide capable of binding to a ligand with a protease cleavage sequence.

[0223] "Inserting" amino acid sequence A into amino acid sequence B means dividing amino acid sequence B into two parts without any deletion and linking the two parts with amino acid sequence A (i.e., producing an amino acid sequence such as "first half of amino acid sequence B-amino acid sequence A-second half of amino acid sequence B"). "Introducing" amino acid sequence A into amino acid sequence B means dividing amino acid sequence B into two parts and linking the two parts with amino acid sequence A. This not only refers to "inserting" amino acid sequence A into amino acid sequence B as described above, but also encompasses deleting one or more amino acid residues in amino acid sequence B, including those adjacent to amino acid sequence A, and then linking the two parts with amino acid sequence A (i.e., replacing a portion of amino acid sequence B with amino acid sequence A).

[0224] Examples of methods for obtaining a molecule capable of binding to a ligand include methods for obtaining a ligand-binding region capable of binding to a ligand, for example, by methods using antibody preparation methods known in the art. The antibody obtained by the preparation method may be used directly in a fusion protein, or only the Fv region of the obtained antibody may be used. If the Fv region in a single-chain (also called "sc") form can recognize the antigen, only the single chain may be used. Alternatively, a Fab region containing the Fv region may be used.

[0225] Specific antibody preparation methods are well known to those skilled in the art. For example, monoclonal antibodies can be produced by hybridoma methods (Kohler and Milstein, Nature 256: 495 (1975)) or recombinant methods (U.S. Patent No. 4,816,567). Alternatively, monoclonal antibodies can be isolated from phage display antibody libraries (Clackson et al., Nature 352: 624-628 (1991); and Marks et al., J. Mol. Biol. 222: 581-597 (1991)). Monoclonal antibodies can also be isolated from single B cell clones (N. Biotechnol. 28(5): 253-457 (2011)).

[0226] Humanized antibodies are also called reshaped human antibodies. Specifically, for example, humanized antibodies consisting of human antibodies to which CDRs of non-human animal (e.g., mouse) antibodies have been grafted are known in the art. General genetic recombination approaches for obtaining humanized antibodies are also known. Specifically, overlap extension PCR, for example, is known as a method for grafting CDRs of mouse antibodies onto human FRs.

[0227] A humanized antibody expression vector can be prepared by inserting DNA encoding an antibody variable region containing three linked CDRs and four FRs and DNA encoding a human antibody constant region into an expression vector so that these DNAs are fused in frame. The vector containing the insert is transfected into a host to establish recombinant cells. The recombinant cells are then cultured to express the DNA encoding the humanized antibody, and the humanized antibody is produced in the cultured cell (see European Patent Publication No. 239400 and International Publication No. WO1996 / 002576).

[0228] If necessary, amino acid residues in the FR may be substituted so that the CDRs of the reshaped human antibody form an appropriate antigen-binding site. For example, mutations can be introduced into the amino acid sequence of the FR by applying the PCR method used to graft mouse CDRs onto human FRs.

[0229] Desired human antibodies can be obtained by DNA immunization using transgenic animals carrying the full repertoire of human antibody genes (see International Publication Nos. WO1993 / 012227, WO1992 / 003918, WO1994 / 002602, WO1994 / 025585, WO1996 / 034096, and WO1996 / 033735).

[0230] In addition, techniques for obtaining human antibodies by panning using a human antibody library are also known. For example, human antibody Fv regions are expressed on the surface of phages as single-chain antibodies (also referred to as "scFvs") by phage display. Phages expressing antigen-binding scFvs can be selected. The genes of the selected phages can be analyzed to determine the DNA sequence encoding the Fv region of the antigen-binding human antibody. After determining the DNA sequence of the antigen-binding scFv, the Fv region sequence can be fused in frame with the sequence of the C region of the desired human antibody and then inserted into an appropriate expression vector to prepare an expression vector. To express the gene encoding the human antibody, the expression vector is transfected into the preferred expression cells listed above to obtain the human antibody. These methods are known in the art (see International Publication Nos. WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, and WO1995 / 015388).

[0231] Molecules that contain a protease cleavage sequence within a molecule capable of binding to a ligand serve as ligand-binding moieties or molecules in the present invention. Whether a ligand-binding moiety / molecule is cleaved by treatment with a protease appropriate for the protease cleavage sequence can be determined. For example, cleavage of the protease cleavage sequence can be determined by contacting a molecule containing a protease cleavage sequence within a molecule capable of binding to a ligand with a protease and determining the molecular weight of the protease-treated product by electrophoresis, such as SDS-PAGE.

[0232] Furthermore, to evaluate the protease activity and cleavage rate of a molecule into which a protease cleavage sequence has been introduced, the cleaved fragments after protease treatment can be separated and quantified by electrophoresis such as SDS-PAGE. Non-limiting examples of methods for evaluating the cleavage rate of a molecule into which a protease cleavage sequence has been introduced include the following. For example, when the cleavage rate of a modified antibody into which a protease cleavage sequence has been introduced is evaluated using recombinant human u-plasminogen activator / urokinase (human uPA, huPA) (R&D Systems; 1310-SE-010) or recombinant human matriptase / ST14 catalytic domain (human MT-SP1, hMT-SP1) (R&D Systems; 3946-SE-010), 100 micrograms / mL of the modified antibody is reacted with 40 nM huPA or 3 nM hMT-SP1 in PBS at 37°C for 1 hour, followed by capillary electrophoresis immunoassay. Capillary electrophoresis immunoassay can be performed using Wes (Protein Simple), but the method is not limited thereto. As an alternative to capillary electrophoresis immunoassay, SDS-PAGE or the like can be used for separation, followed by detection by Western blotting. The method is not limited thereto. Before and after cleavage, the light chain can be detected using an anti-human λ chain HRP-labeled antibody (abcam; ab9007), but any antibody capable of detecting cleaved fragments can also be used. The area of ​​each peak obtained after protease treatment can be output using software for Wes (Compass for SW; Protein Simple), and the cleavage rate (%) of the antibody variant can be determined using the following formula: (peak area of ​​cleaved light chain) × 100 / (peak area of ​​cleaved light chain + peak area of ​​uncleaved light chain) The cleavage rate can be determined by detecting protein fragments before and after protease treatment. Thus, the cleavage rate can be determined not only for antibody variants but also for various protein molecules into which a protease cleavage sequence has been introduced.

[0233] The in vivo cleavage rate of a molecule incorporating a protease cleavage sequence can be determined by administering the molecule to an animal and detecting the administered molecule in a blood sample. For example, an antibody variant incorporating a protease cleavage sequence is administered to mice, and plasma is collected from the blood samples. The antibody is purified from the plasma using Dynabeads Protein A (Thermo; 10001D) by methods known to those skilled in the art, and then subjected to capillary electrophoresis immunoassay to evaluate the protease cleavage rate of the antibody variant. Capillary electrophoresis immunoassay can be performed using Wes (Protein Simple), but this method is not limited thereto. As an alternative to capillary electrophoresis immunoassay, SDS-PAGE or the like can be used for separation, followed by detection by Western blotting. This method is not limited thereto. An anti-human λ chain HRP-labeled antibody (abcam; ab9007) can be used to detect the light chain of the antibody variant collected from mice, but any antibody capable of detecting cleavage fragments can be used. Once the area of ​​each peak obtained by capillary electrophoresis immunoassay was output using software for Wes (Compass for SW; Protein Simple), the proportion of remaining light chains could be calculated as [light chain peak area] / [heavy chain peak area], thereby determining the proportion of full-length light chains remaining uncleaved in the mouse body. In vivo cleavage efficiency can be determined if protein fragments collected from the living body are detectable. Therefore, the cleavage rate can be determined not only for antibody variants but also for various protein molecules into which protease cleavage sequences have been introduced. Calculating the cleavage rate using the above method makes it possible, for example, to compare the in vivo cleavage rates of antibody variants into which different cleavage sequences have been introduced, and to compare the cleavage rates of a single antibody variant between different animal models, such as normal mouse models and tumor-bearing mouse models.

[0234] The present invention also relates to polynucleotides encoding the fusion proteins of the present invention.

[0235] The polynucleotide according to the present invention is usually carried by (inserted into) an appropriate vector and transfected into a host cell. The vector is not particularly limited, as long as it can stably maintain the inserted nucleic acid. For example, when Escherichia coli is used as the host, the pBluescript vector (Stratagene Corp.) is a preferred cloning vector. Various commercially available vectors can be used. When a vector is used for the purpose of producing the fusion protein of the present invention, an expression vector is particularly useful. The expression vector is not particularly limited, as long as it enables the expression of the fusion protein in vitro, in Escherichia coli, in cultured cells, or in an individual organism. Preferred expression vectors include the pBEST vector (Promega Corp.) for in vitro expression, the pET vector (Invitrogen Corp.) for E. coli, the pME18S-FL3 vector (GenBank accession number AB009864) for cultured cells, and the pME18S vector (Mol Cell Biol. 8: 466-472 (1988)) for individual organisms. The DNA of the present invention can be inserted into the vector by a conventional method, for example, a ligase reaction using a restriction enzyme site (Current protocols in Molecular Biology, ed., Ausubel et al. (1987) Publish. John Wiley & Sons. Sections 11.4-11.11).

[0236] The host cell is not particularly limited, and various host cells can be used depending on the purpose. Examples of cells for expressing the fusion protein include bacterial cells (e.g., Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, and Bacillus subtilis), fungal cells (e.g., yeast and Aspergillus), insect cells (e.g., Drosophila S2 and Spodoptera SF9), animal cells (e.g., CHO, COS, HeLa, C127, 3T3, BHK, HEK293, and Bowes melanoma cells), and plant cells. Transfection of the vector into the host cell may be carried out by methods known in the art, such as calcium phosphate precipitation, electroporation (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), Lipofectamine method (GIBCO-BRL / Thermo Fisher Scientific Inc.), or microinjection.

[0237] An appropriate secretion signal can be incorporated into the fusion protein of interest to secrete the fusion protein expressed in the host cell into the lumen of the endoplasmic reticulum, the periplasmic space, or the extracellular environment. The signal can be endogenous to the fusion protein of interest or a heterologous signal.

[0238] When the fusion protein of the present invention is secreted into the medium, the fusion protein in the production method is recovered by collecting the medium. When the fusion protein of the present invention is produced in cells, the cells are first lysed, and then the fusion protein is recovered.

[0239] Fusion proteins of the invention can be recovered and purified from recombinant cell culture using methods known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography.

[0240] It should be understood by those skilled in the art that any combination of one or more aspects described herein is also included in the present invention, unless there is a technical contradiction based on the common general knowledge of those skilled in the art. Furthermore, the present invention excluding any combination of one or more aspects described herein should also be considered as the invention contemplated and described in this specification, unless there is a technical contradiction based on the common general knowledge of those skilled in the art. [Example]

[0241] Example 1. Preparation of IL-12 fusion antibodies with protease-cleavable linkers IL-12 is a proinflammatory cytokine that activates various cells, such as T, NK, and B cells. IL-12 is known to exhibit antitumor efficacy, but its systemic exposure causes severe toxicity, which prevents sufficient dosing for efficacy. To overcome this limitation, we developed an IL-12-releasing antibody that can release IL-12 only around the tumor by utilizing tumor-specific proteases.

[0242] 1-1. Preparation of free IL-12 Various IL-12 free antibodies were constructed by fusing the IL-12 molecule, consisting of p40 (SEQ ID NO: 871) and p35 (SEQ ID NO: 872), to an anti-IL-12 antibody via a protease-cleavable linker (SEQ ID NO: 873) in various ways. The anti-IL-12 antibody, Mab80 (WO2010017598), was used. Unless otherwise noted, the Fc region is a modified IgG1 Fc region containing mutations (L235R / G236R in EU numbering) to abolish FcγR binding.

[0243] The F2 bivalent IL-12-releasing Mab80 is a homodimer of a light chain (SEQ ID NO: 874) and a heavy chain (SEQ ID NO: 875). In the light chain, the p40 subunit was attached to the N-terminus of Mab80VL-k0 (SEQ ID NO: 876) via a cleavable linker (SEQ ID NO: 877). In the heavy chain, a cleavable linker (SEQ ID NO: 873) was introduced into the elbow hinge region between Mab80VH (SEQ ID NO: 878) and the CH1 domain. A GS linker was inserted into the hinge region, and the p35 subunit was attached to the C-terminus of the Fc domain via a cleavable linker (SEQ ID NO: 879). Once these cleavable linkers are digested by a protease, active IL-12 molecules are released (Figure 1A).

[0244] F4 bivalent IL-12-releasing Mab80 (Figure 1B) is a homodimer of a light chain (SEQ ID NO: 876) and a heavy chain (SEQ ID NO: 880). Mab80VL-k0 (SEQ ID NO: 876) was used as the light chain without any modifications. In the heavy chain, a cleavable linker was introduced into the elbow hinge region between Mab80VH (SEQ ID NO: 878) and the CH1 domain. A GS linker was inserted into the hinge region, and single-chain IL-12 was added to the C-terminus of the Fc domain via a cleavable linker (SEQ ID NO: 879). Once these cleavable linkers were digested by a protease, active IL-12 molecules were released (Figure 1B).

[0245] F4 monovalent IL-12-releasing Mab80 (Figure 1C) is a heterodimer of the light chain 1 (SEQ ID NO: 876) / heavy chain 1 (SEQ ID NO: 881) and light chain 2 (SEQ ID NO: 882) / heavy chain 2 (SEQ ID NO: 883) pairs. In heavy chain 1 (SEQ ID NO: 881), a cleavable linker was introduced into the elbow hinge region between the Mab80VH (SEQ ID NO: 878) and CH1 domains. Anti-KLH antibody was used as the variable region in heavy and light chains 2. To promote heterodimerization and correct association of the heavy and light chains, knob-into-hole mutations (Nat. Biotechnol. 1998, 16, 677-681) were introduced into the heavy chain CH3 domain, and heavy and light chains 2 were purified using CrossMab technology (PNAS, 2011, 108, 11187-11192). Mab80VL-k0 (SEQ ID NO: 876) was used as light chain 1 without any modifications. Heavy chain 1 and heavy chain 2 contain knob mutations (Y349C / T366W) and hole mutations (E356C / T366S / L368A / Y407V), respectively. Light chain 2 consisted of the VH domain of anti-KLH and a human kappa constant region. Heavy chain 2 consisted of the VL domain of anti-KLH and a modified IgG1 Fc region.

[0246] Expression vectors for each chain were prepared by methods known to those skilled in the art, and expressed using Expi293 (Life Technologies Corp.) by combining the chains as shown in Table 2. Antibody purification was performed using affinity purification with MabSelect SuRe (Cat. No.: 17-5438-01, GE Healthcare), followed by size-exclusion chromatography using a Superdex 200 gel filtration column (Cat. No.: 28-9893-35, GE Healthcare). Any aggregates present in the eluate from affinity chromatography were removed using size-exclusion chromatography.

[0247] Table 2: IL-12 free antibodies and sequence IDs of each chain TIFF2026010015000013.tif77157

[0248] 1-2. Preparation of IL-12 fusion protein An IL-12 fusion antibody was constructed by fusing an IL-12 molecule composed of p40 (SEQ ID NO: 871) and p35 (SEQ ID NO: 872) to an anti-IL-12 antibody via a GS linker. Mab80 (WO2010017598), ustekinumab (WO2002012500), and J695 (WO2000056772) were used as anti-IL-12 antibodies. Unless otherwise noted, the Fc region was a modified IgG1 Fc region containing mutations (L235R / G236R in EU numbering) to abolish FcγR binding.

[0249] The F2 bivalent IL-12 fusion Mab80 is a homodimer of a light chain (SEQ ID NO: 874) and a heavy chain (SEQ ID NO: 884). In the light chain, the p40 subunit was attached to the N-terminus of Mab80VL-k0 (SEQ ID NO: 876) via a cleavable linker (SEQ ID NO: 877). In the heavy chain, a cleavable linker was introduced into the elbow hinge region between Mab80VH (SEQ ID NO: 878) and the CH1 domain. A GS linker was inserted into the hinge region, and the p35 subunit was attached to the C-terminus of the Fc domain via a GS linker. Once these cleavable linkers are digested by a protease, the active IL-12 molecule-fused Fc is released (Figure 2A).

[0250] The F4 bivalent IL-12 fusion Mab80 (Figure 2B) is a homodimer of a light chain (SEQ ID NO: 876) and a heavy chain (SEQ ID NO: 885). Mab80VL-k0 (SEQ ID NO: 876) was used as the light chain without any modifications. In the heavy chain, a cleavable linker was introduced into the elbow hinge region between Mab80VH (SEQ ID NO: 878) and the CH1 domain. A GS linker was inserted into the hinge region, and single-chain IL-12 was attached to the C-terminus of the Fc domain via the GS linker. Once these cleavable linkers were digested by a protease, the active IL-12 molecule fused to the Fc molecule was released (Figure 2B).

[0251] F4 monovalent IL-12 fusion Mab80 (Figure 2C) is a heterodimer of the light chain 1 (SEQ ID NO: 876) / heavy chain 1 (SEQ ID NO: 886) and light chain 2 (SEQ ID NO: 882) / heavy chain 2 (SEQ ID NO: 883) pairs. In heavy chain 1 (SEQ ID NO: 886), a cleavable linker was introduced into the elbow hinge region between Mab80VH (SEQ ID NO: 878) and the CH1 domain. Anti-KLH antibody was used as the variable region in heavy chain 2 and light chain 2. To promote heterodimerization and correct association of the heavy and light chains, knob-into-hole mutations were introduced into the heavy chain CH3 domain, and CrossMab technology was used in heavy chain 2 and light chain 2. Mab80VL-k0 (SEQ ID NO: 876) was used as light chain 1 without any modifications.

[0252] Heavy chain 1 and heavy chain 2 contained knob mutations (Y349C / T366W) and hole mutations (E356C / T366S / L368A / Y407V), respectively. Light chain 2 consisted of the anti-KLH VH domain and a human kappa constant region. Heavy chain 2 consisted of the anti-KLH VL domain and a modified IgG1 Fc region.

[0253] The F4 monovalent IL-12 fusion UstK (Figure 2D) is a heterodimer of the light chain 1 (SEQ ID NO: 887) / heavy chain 1 (SEQ ID NO: 888) and light chain 2 (SEQ ID NO: 882) / heavy chain 2 (SEQ ID NO: 883) pairs. In heavy chain 1 (SEQ ID NO: 888), a cleavable linker was introduced into the elbow hinge region between UstKVH (SEQ ID NO: 889) and the CH1 domain. Anti-KLH antibody was used as the variable region in heavy chain 2 and light chain 2. To promote heterodimerization and correct association of the heavy and light chains, knob-into-hole mutations were introduced into the heavy chain CH3 domain, and CrossMab technology was used in heavy chain 2 and light chain 2. UstkVL (SEQ ID NO: 887) was used as light chain 1 without any modifications.

[0254] Heavy chain 1 and heavy chain 2 contained knob mutations (Y349C / T366W) and hole mutations (E356C / T366S / L368A / Y407V), respectively. Light chain 2 consisted of the anti-KLH VH domain and a human kappa constant region. Heavy chain 2 consisted of the anti-KLH VL domain and a modified IgG1 Fc region.

[0255] The F4 monovalent IL-12 fusion J695 (Figure 2E) is a heterodimer of the light chain 1 (SEQ ID NO: 890) / heavy chain 1 (SEQ ID NO: 891) and light chain 2 (SEQ ID NO: 882) / heavy chain 2 (SEQ ID NO: 883) pairs. In heavy chain 1 (SEQ ID NO: 891), a cleavable linker was introduced into the elbow hinge region between the J695VH (SEQ ID NO: 892) and CH1 domains. Anti-KLH antibody was used as the variable region in heavy chain 2 and light chain 2. To promote heterodimerization and correct association of the heavy and light chains, knob-into-hole mutations were introduced into the heavy chain CH3 domain, and CrossMab technology was used in heavy chain 2 and light chain 2. J695VL (SEQ ID NO: 890) was used as light chain 1 without any modifications.

[0256] Heavy chain 1 and heavy chain 2 contained knob mutations (Y349C / T366W) and hole mutations (E356C / T366S / L368A / Y407V), respectively. Light chain 2 consisted of the anti-KLH VH domain and a human kappa constant region. Heavy chain 2 consisted of the anti-KLH VL domain and a modified IgG1 Fc region.

[0257] Expression vectors for each chain were prepared by methods known to those skilled in the art, and expressed using Expi293 (Life Technologies Corp.) by combining the chains as shown in Table 3. Antibody purification was performed using affinity purification with MabSelect SuRe (Cat. No.: 17-5438-01, GE Healthcare), followed by size-exclusion chromatography using a Superdex 200 gel filtration column (Cat. No.: 28-9893-35, GE Healthcare). Any aggregates present in the eluate from affinity chromatography were removed using size-exclusion chromatography.

[0258] Table 3. IL-12 free antibodies with Fc fusions and sequence IDs for each chain TIFF2026010015000014.tif119170

[0259] Example 2. Evaluation of protease cleavage of IL-12-releasing antibodies having protease cleavage sequences and flexible linker sequences We examined whether the antibodies prepared in Example 1 could be cleaved by proteases. Recombinant ...

Claims

1. (a) a ligand-binding moiety comprising a ligand-binding domain and at least one first protease cleavage site; (b) at least one ligand moiety; and (c) at least one peptide linker connecting at least one ligand moiety to the C-terminal region of the ligand-binding moiety; A fusion protein comprising: the ligand-binding domain is capable of binding to the at least one ligand moiety and releasing the at least one ligand moiety in the presence of a protease; Fusion proteins.

2. The fusion protein of claim 1 , wherein the at least one peptide linker does not contain any protease cleavage sites.

3. The fusion protein of claim 1 , wherein the at least one peptide linker comprises a second protease cleavage site.

4. The fusion protein of any one of claims 1 to 3, wherein the ligand-binding domain comprises an antibody variable region.

5. The fusion protein of claim 4, wherein the ligand-binding domain comprises a VH region and a VL region that associate with each other.

6. The fusion protein of claim 4 or 5, wherein the ligand-binding portion further comprises a CH1 domain and a CL domain.

7. 7. The fusion protein of claim 6, wherein at least one of the at least one first protease cleavage site is located near the boundary between the VH or VL domain and the CH1 or CL domain.

8. The fusion protein of any one of claims 4 to 7, wherein the ligand-binding portion further comprises an Fc region.

9. The fusion protein of claim 8 , wherein the ligand-binding portion comprises a full-length antibody.

10. The fusion protein of claim 9, wherein the full-length antibody is an IgG antibody.

11. 11. The fusion protein of claim 9 or 10, wherein the at least one ligand moiety comprises two ligand moieties and the at least one peptide linker comprises two peptide linkers, each ligand moiety being connected to the C-terminal region of the ligand-binding moiety via a respective peptide linker.

12. 12. The fusion protein of claim 8, wherein the at least one ligand moiety is connected to an amino acid residue exposed on the surface of the CH3 region of the Fc region via the at least one peptide linker.

13. 13. The fusion protein of any one of claims 1 to 12, wherein the at least one ligand moiety is connected to the C-terminal amino acid residue of the ligand-binding moiety via the at least one peptide linker.

14. further comprising a cleavable linker comprising a third protease cleavage site; the at least one ligand moiety is connected to the N-terminal region of the ligand binding moiety via the cleavable linker; A fusion protein according to any one of claims 1 to 13.

15. further comprising a cleavable linker comprising a third protease cleavage site; the at least one ligand moiety is connected via the cleavable linker to one of amino acid residues 1 to 230 from the N-terminus of the ligand binding moiety; A fusion protein according to any one of claims 8 to 13.

16. 16. The fusion protein of claim 14 or 15, wherein the at least one ligand moiety is connected to the N-terminal amino acid residue of the ligand-binding moiety via the cleavable linker.

17. 17. The fusion protein of any one of claims 1 to 16, wherein the at least one ligand moiety has biological activity and binding of the ligand binding domain to the ligand moiety inhibits the biological activity of the ligand moiety.

18. 18. The fusion protein of claim 17, wherein the at least one ligand moiety comprises a protein or polypeptide having biological activity.

19. 19. The fusion protein of claim 18, wherein the at least one ligand moiety comprises a cytokine or a chemokine.

20. 19. The fusion protein of claim 18, wherein the at least one ligand moiety comprises a ligand protein or polypeptide selected from the group consisting of CXCL10, IL-2, IL-12, IL-22, PD-1, or IL-6R.

21. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 20.

22. (a) a ligand-binding molecule comprising a ligand-binding domain and at least one first protease cleavage site; (b) at least one ligand molecule; and (c) at least one peptide linker providing connecting at least one ligand molecule to the C-terminal region of the ligand-binding molecule via at least one peptide linker; A method for producing a fusion protein according to any one of claims 1 to 20, comprising: the ligand-binding domain binds to the ligand molecule; the ligand-binding domain is capable of releasing the ligand molecule in the presence of a protease; method.

23. A polynucleotide encoding the fusion protein of any one of claims 1 to 20.

24. A vector comprising the polynucleotide of claim 23.

25. 25. A host cell comprising the polynucleotide of claim 23 or the vector of claim 24.

26. A method for producing a fusion protein according to any one of claims 1 to 20, comprising culturing a host cell according to claim 25.

27. comprising a ligand-binding moiety and having the general formula (I): [Ligand-binding domain]-[Lx]-[Cx]-[Ly]-[ligand moiety] (I) A fusion protein represented by During the ceremony, Lx represents a first peptide linker optionally containing a first protease cleavage site, or Lx is absent; Cx represents a constant region comprising a second peptide linker and, optionally, one or more amino acid residues that are modified to or from cysteine; Ly represents a third peptide linker; the ligand-binding domain is capable of binding to the ligand moiety and releasing the ligand moiety in the presence of a protease; Fusion proteins.

28. 28. The fusion protein of claim 27, comprising two sets of ligand-binding domains, a ligand moiety, a first peptide linker, a constant region, and a third peptide linker.

29. General formula (II): [ligand-binding domain]-[Lx]-[Cx]-[Ly]-[ligand moiety] / / [non-ligand-binding domain]-[Lz]-[Cz] (II) is represented by During the ceremony, Lx, Cx, and Ly are as defined in claim 1; Lz represents a fourth peptide linker optionally containing a first protease cleavage site, or Lz is absent; Cz represents a second constant region, optionally including a fifth peptide linker and, optionally, one or more amino acid residues that are modified to or from cysteine; 28. The fusion protein of claim 27.

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