Ligand-binding molecule containing a single-domain antibody

Abstract

The present invention relates to single-domain antibody-containing ligand-binding molecules whose binding activity to a ligand is attenuated by cleavage of the cleavage site, methods for producing the same, complexes formed between the ligand-binding molecules and ligands, fusion proteins comprising the ligand-binding molecules and ligands, and pharmaceutical compositions comprising the ligand-binding molecules or fusion proteins of the ligand-binding molecules and ligands.

Description

Technical Field

[0001] The present invention provides a single-domain antibody-containing ligand-binding molecule, a method for producing the ligand-binding molecule, a pharmaceutical composition containing the ligand-binding molecule, a fusion protein containing the ligand-binding molecule, a method for producing the fusion protein, and a pharmaceutical composition containing the fusion protein.

Background Art

[0002] Antibodies have attracted attention as pharmaceuticals because of their high stability in plasma and few side effects. Among them, many IgG-type antibody drugs have been launched, and currently, numerous antibody drugs are being developed (Non-Patent Document 1 and Non-Patent Document 2).

[0003] As cancer therapeutic drugs using antibody drugs, Rituxan against CD20 antigen, Cetuximab against EGFR antigen, Herceptin against HER2 antigen, etc. have been approved so far (Non-Patent Document 3). These antibody molecules bind to antigens expressed on cancer cells and exhibit cytotoxic activity against cancer cells by means of ADCC, signal inhibition, etc.

[0004] In addition, a method of delivering a ligand to solid cancer by an immunocytokine in which a ligand having biological activity such as a cytokine is fused to an antibody molecule that binds to a cancer antigen highly expressed on cancer cells is known. The cytokine delivered to solid cancer by the immunocytokine activates the immune system to exert an antitumor effect. Since cytokines such as IL-2, IL-12, and TNF are highly toxic, it is expected to reduce side effects and enhance the effect by delivering these cytokines to the cancer site by an antibody in order to make them work at the cancer site (Non-Patent Documents 4, 5, and 6). However, all of these have problems such as not showing sufficient clinical effects with systemic administration, having a narrow therapeutic window, being highly toxic and unable to be administered systemically, and have not yet been approved as pharmaceuticals.

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

[0006] [Non-Patent Document 1] Monoclonal antibody successes in the clinic. Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Nat. Biotechnol. (2005) 23, 1073-1078 [Non-Patent Document 2] The therapeutic antibodies market to 2008. Pavlou AK, Belsey MJ., Eur. J. Pharm. Biopharm. (2005) 59 (3), 389-396 [Non-Patent Document 3] Monoclonal antibodies: versatile platforms for cancer immunotherapy. Weiner LM, Surana R, Wang S., Nat. Rev. Immunol. (2010) 10 (5), 317-327 [Non-Patent Document 4] Cyclophosphamide and tucotuzumab (huKS-IL2) following first-line chemotherapy in responding patients with extensive-disease small-cell lung cancer. Gladkov O, Ramlau R, Serwatowski P, Milanowski J, Tomeczko J, Komarnitsky PB, Kramer D, Krzakowski MJ. Anticancer Drugs. 2015 Nov;26(10):1061-8.

Direct Environment 5

Outdoor Configuration6

Non - Patent Document 7

Summary of the Invention

Problems to be Solved by the Invention

[0007] The present invention has been made in view of the above circumstances, and one of its objectives is to provide a single-domain antibody-containing ligand-binding molecule that selectively activates ligands such as cytokines or chemokines in target tissue, a complex of the ligand-binding molecule and the ligand, a fusion protein containing the ligand-binding molecule, and methods for producing the same. Another objective of the present invention is to provide a pharmaceutical composition containing the ligand-binding molecule, the complex of the ligand-binding molecule and the ligand, or a fusion protein containing the ligand-binding molecule as an active ingredient, and a method for producing the pharmaceutical composition. [Means for solving the problem]

[0008] The inventors diligently conducted research to achieve the above objectives and created a ligand-binding molecule containing a single-domain antibody with a cleavage site and a fusion protein containing the ligand-binding molecule. The inventors also found that the ligand-binding molecule, the complex of the ligand-binding molecule and the ligand, the fusion protein containing the ligand-binding molecule, or pharmaceutical compositions containing them are useful for treating diseases using the ligand, and are also useful for treating diseases by administering the ligand-binding molecule, the complex of the ligand-binding molecule and the ligand, or the fusion protein containing the ligand-binding molecule, and that the ligand-binding molecule or the fusion protein containing the ligand-binding molecule is useful in the manufacture of pharmaceuticals for the treatment of diseases. Furthermore, the inventors completed the present invention by creating a method for producing the ligand-binding molecule, the complex of the ligand-binding molecule and the ligand, or the fusion protein containing the ligand-binding molecule.

[0009] The present invention is based on these findings and specifically includes the embodiments described below as illustrative examples. (A1) A ligand-binding molecule comprising a single-domain antibody, wherein the single-domain antibody is capable of binding to a ligand and has at least one cleavage site introduced thereon, and the binding of the ligand-binding molecule to the ligand when the cleavage site is cleaved is attenuated compared to the binding of the ligand-binding molecule to the ligand when the cleavage site is not cleaved. (A2) The ligand-binding molecule according to (A1), wherein the ligand is released from the ligand-binding molecule when the cleavage site is cleaved. (A3) The ligand-binding molecule according to (A1) or (A2), wherein the cleavage site includes a protease cleavage sequence. (A4) The ligand-binding molecule described in (A3), wherein the protease is a target tissue-specific protease. (A5) The ligand-binding molecule according to (A4), wherein the target tissue is cancer tissue and the target tissue-specific protease is cancer tissue-specific protease. (A6) The ligand-binding molecule according to (A4), wherein the target tissue is inflammatory tissue and the target tissue-specific protease is an inflammatory tissue-specific protease. (A7) The ligand-binding molecule according to any one of (A3) to (A6), wherein the protease is at least one protease selected from matryptase, urokinase (uPA), and metalloproteinase. (A8) The ligand-binding molecule according to any one of (A3) to (A7), wherein the protease cleavage sequence is a sequence containing one or more sequences selected from the sequences shown in SEQ ID NOs: 2-82, 788-827, and the sequences listed in Table 1. (A9) A ligand-binding molecule according to any one of (A3) to (A8), wherein a first movable linker is further added to one end of the protease cleavage sequence. (A10) The ligand-binding molecule according to (A9), wherein the first movable linker is a movable linker made of a glycine-serine polymer. (A11) The ligand-binding molecule according to (A9) or (A10), wherein a second movable linker is further added to the other end of the protease cleavage sequence. (A12) The ligand-binding molecule according to (A11), wherein the second movable linker is a movable linker made of a glycine-serine polymer. (A13) The ligand-binding molecule described in any one of (A1) to (A12), wherein the monodomain antibody is VHH, a monodomain VH antibody, or a monodomain VL antibody. (A14) The monodomain antibody is a VHH or monodomain VH antibody, and the cleavage site, or the protease cleavage sequence, or the protease cleavage sequence and the first mobile linker, or the protease cleavage sequence, the first mobile linker and the second mobile linker are introduced at one or more positions in one or more sequences selected from the following sequences of the monodomain antibody, the ligand-binding molecule as described in (A13): The sequences of single-domain antibodies from amino acid 7 (Kabat numbering) to amino acid 17 (Kabat numbering), the sequences of single-domain antibodies from amino acid 12 (Kabat numbering) to amino acid 17 (Kabat numbering), the sequences of single-domain antibodies from amino acid 31 (Kabat numbering) to amino acid 35b (Kabat numbering), the sequences of single-domain antibodies from amino acid 40 (Kabat numbering) to amino acid 47 (Kabat numbering), the sequences of single-domain antibodies from amino acid 50 (Kabat numbering) to amino acid 65 (Kabat numbering), and from amino acid 55 (Kabat numbering) Sequences up to amino acid 69 (Kabat numbering), sequences from amino acid 73 (Kabat numbering) to amino acid 79 (Kabat numbering) of single-domain antibodies, sequences from amino acid 83 (Kabat numbering) to amino acid 89 (Kabat numbering) of single-domain antibodies, sequences from amino acid 95 (Kabat numbering) to amino acid 99 (Kabat numbering) of single-domain antibodies, sequences from amino acid 95 (Kabat numbering) to amino acid 102 (Kabat numbering) of single-domain antibodies, sequences from amino acid 101 (Kabat numbering) to amino acid 113 (Kabat numbering) of single-domain antibodies. (A15) The monodomain antibody is a monodomain VL antibody, and the cleavage site, or the protease cleavage sequence, or the protease cleavage sequence and the first mobile linker, or the protease cleavage sequence, the first mobile linker and the second mobile linker are introduced at one or more positions in one or more sequences selected from the following sequences of the monodomain antibody, the ligand-binding molecule as described in (A13): Sequences of single-domain antibodies from amino acid 7 (Kabat numbering) to amino acid 19 (Kabat numbering), sequences of single-domain antibodies from amino acid 24 (Kabat numbering) to amino acid 34 (Kabat numbering), sequences of single-domain antibodies from amino acid 39 (Kabat numbering) to amino acid 46 (Kabat numbering), sequences of single-domain antibodies from amino acid 49 (Kabat numbering) to amino acid 62 (Kabat numbering), sequences of single-domain antibodies from amino acid 50 (Kabat numbering) to amino acid 56 (Kabat numbering), sequences of single-domain antibodies from amino acid 89 (Kabat numbering) to amino acid 97 (Kabat numbering), and sequences of single-domain antibodies from amino acid 96 (Kabat numbering) to amino acid 107 (Kabat numbering). (A16) The ligand-binding molecule according to any one of (A1) to (A15), wherein the ligand is a biologically active molecule, and the single-domain antibody inhibits the biological activity of the ligand by binding to it. (A17) The ligand-binding molecule according to any one of (A1) to (A16), wherein the ligand is a biologically active molecule and the single-domain antibody has neutralizing activity against the ligand. (A18) The ligand-binding molecule according to any one of (A1) to (A17), wherein the ligand-binding molecule comprises only a single-domain antibody containing the cleavage site or protease cleavage sequence. (A19) The ligand-binding molecule further comprises an antibody Fc region, as described in any one of (A1) to (A18). (A20) The ligand-binding molecule according to any one of (A1) to (A19), wherein the ligand-binding molecule comprises a series of peptide chains consisting of a single-domain antibody-antibody Fc region from the N-terminus to the C-terminus. (A21) The ligand-binding molecule described in (A1) to (A19), wherein the ligand-binding molecule is a dimer containing two peptide chains consisting of a single-domain antibody-antibody hinge region-antibody Fc region. (A22) The ligand-binding molecule described in (A19) to (A21), wherein the antibody Fc region is an Fc region containing one sequence selected from the amino acid sequences shown in SEQ ID NOs: 103 to 106, or an Fc region variant obtained by modifying these Fc regions. (A23) The ligand-binding molecule according to any one of (A1) to (A22), wherein the ligand is a cytokine or chemokine. (A24) The ligand-binding molecule described in any one of (A1) to (A23), wherein the ligand is selected from interleukins, interferons, hematopoietic factors, the TNF superfamily, chemokines, cell growth factors, and the TGF-β family. (A25) The ligand-binding molecule according to any one of (A1) to (A24), wherein the ligand is CXCL10, IL-12, PD-1, IL-6R, or IL-1Ra. (A26) A ligand-binding molecule according to any one of (A1) to (A25) that is bound to the ligand. (A27) A ligand-binding molecule according to any one of (A1) to (A25), which is fused with the ligand. (A28) The ligand-binding molecule as described in (A27), wherein when the ligand-binding molecule is fused with a ligand, the single-domain antibody contained in the ligand-binding molecule does not further bind to another ligand. (A29) The ligand-binding molecule according to (A27) or (A28), wherein the ligand-binding molecule is fused with the ligand via a linker. (A30) The ligand-binding molecule described in (A29), wherein the linker does not contain a protease cleavage sequence. (A31) A complex formed by the ligand and a ligand-binding molecule described in any one of (A1) to (A25). (A32) A fusion protein in which the ligand and a ligand-binding molecule described in any one of (A1) to (A25) are fused. (A33) The fusion protein according to (A32), wherein when the ligand-binding molecule is fused with the ligand, the single-domain antibody does not further bind to another ligand. (A34) The fusion protein according to (A32) or (A33), wherein the ligand-binding molecule is fused with the ligand via a linker. (A35) The fusion protein according to (A34), wherein the linker does not contain a protease cleavage sequence. (A36) The fusion protein according to (A34) or (A35), which is fused in the order of ligand-linker-ligand-binding molecule from the N-terminus to the C-terminus. (B1) A ligand-binding molecule, which contains a single-domain antibody, the single-domain antibody can bind to a ligand and contains at least one protease cleavage sequence, the protease cleavage sequence can be cleaved by a target tissue-specific protease, and the binding of the ligand-binding molecule to the ligand in the state where the protease cleavage sequence is cleaved is attenuated compared to the binding of the ligand-binding molecule to the ligand in the state where the cleavage site is not cleaved. (B2) The ligand-binding molecule according to (B1), wherein in the state where the protease cleavage sequence is cleaved, the ligand is released from the ligand-binding molecule. (B3) The ligand-binding molecule according to any one of (B1) to (B2), wherein the target tissue is a cancer tissue and the target tissue-specific protease is a cancer tissue-specific protease. (B4) The ligand-binding molecule according to (B1) to (B2), wherein the target tissue is an inflammatory tissue and the target tissue-specific protease is an inflammatory tissue-specific protease. (B5) The ligand-binding molecule according to any one of (B1) to (B4), wherein the protease is at least one protease selected from matryptase, urokinase (uPA), and metalloproteinase. (B6) The ligand-binding molecule according to any one of (B1) to (B5), wherein the protease cleavage sequence is a sequence containing one or more sequences selected from the sequences shown in SEQ ID NOs: 2-82, 788-827, and the sequences listed in Table 1. (B7) A ligand-binding molecule according to any one of (B1) to (B6), wherein a first movable linker is further added to one end of the protease cleavage sequence. (B8) The ligand-binding molecule according to (B7), wherein the first movable linker is a movable linker made of a glycine-serine polymer. (B9) The ligand-binding molecule according to (B7) or (B8), wherein a second movable linker is further added to the other end of the protease cleavage sequence. (B10) The ligand-binding molecule according to (B9), wherein the second movable linker is a movable linker made of a glycine-serine polymer. (B11) The ligand-binding molecule according to any one of (B1) to (B10), wherein the monodomain antibody is VHH, or a monodomain VH antibody, or a monodomain VL antibody. (B12) The monodomain antibody is a VHH or monodomain VH antibody, and the cleavage site, or the protease cleavage sequence, or the protease cleavage sequence and the first mobile linker, or the protease cleavage sequence, the first mobile linker and the second mobile linker are introduced at one or more positions in one or more sequences selected from the following sequences of the monodomain antibody, the ligand-binding molecule as described in (B11): The sequences of single-domain antibodies from amino acid 7 (Kabat numbering) to amino acid 17 (Kabat numbering), the sequences of single-domain antibodies from amino acid 12 (Kabat numbering) to amino acid 17 (Kabat numbering), the sequences of single-domain antibodies from amino acid 31 (Kabat numbering) to amino acid 35b (Kabat numbering), the sequences of single-domain antibodies from amino acid 40 (Kabat numbering) to amino acid 47 (Kabat numbering), the sequences of single-domain antibodies from amino acid 50 (Kabat numbering) to amino acid 65 (Kabat numbering), and from amino acid 55 (Kabat numbering) Sequences up to amino acid 69 (Kabat numbering), sequences from amino acid 73 (Kabat numbering) to amino acid 79 (Kabat numbering) of single-domain antibodies, sequences from amino acid 83 (Kabat numbering) to amino acid 89 (Kabat numbering) of single-domain antibodies, sequences from amino acid 95 (Kabat numbering) to amino acid 99 (Kabat numbering) of single-domain antibodies, sequences from amino acid 95 (Kabat numbering) to amino acid 102 (Kabat numbering) of single-domain antibodies, sequences from amino acid 101 (Kabat numbering) to amino acid 113 (Kabat numbering) of single-domain antibodies. (B13) The monodomain antibody is a monodomain VL antibody, and the cleavage site, or the protease cleavage sequence, or the protease cleavage sequence and the first mobile linker, or the protease cleavage sequence, the first mobile linker and the second mobile linker are introduced at one or more positions in one or more sequences selected from the following sequences of the monodomain antibody, the ligand-binding molecule as described in (B11): Sequences of single-domain antibodies from amino acid 7 (Kabat numbering) to amino acid 19 (Kabat numbering), sequences of single-domain antibodies from amino acid 24 (Kabat numbering) to amino acid 34 (Kabat numbering), sequences of single-domain antibodies from amino acid 39 (Kabat numbering) to amino acid 46 (Kabat numbering), sequences of single-domain antibodies from amino acid 49 (Kabat numbering) to amino acid 62 (Kabat numbering), sequences of single-domain antibodies from amino acid 50 (Kabat numbering) to amino acid 56 (Kabat numbering), sequences of single-domain antibodies from amino acid 89 (Kabat numbering) to amino acid 97 (Kabat numbering), and sequences of single-domain antibodies from amino acid 96 (Kabat numbering) to amino acid 107 (Kabat numbering). (B14) The ligand-binding molecule according to any one of (B1) to (B13), wherein the ligand is a biologically active molecule, and the single-domain antibody inhibits the biological activity of the ligand by binding to it. (B15) The ligand-binding molecule according to any one of (B1) to (B14), wherein the ligand is a biologically active molecule and the single-domain antibody has neutralizing activity against the ligand. (B16) The ligand-binding molecule according to any one of (B1) to (B15), wherein the ligand-binding molecule comprises only a single-domain antibody containing the cleavage site or protease cleavage sequence. (B17) The ligand-binding molecule further comprises an antibody Fc region, as described in any one of (B1) to (B16). (B18) The ligand-binding molecule according to any one of (B1) to (B17), wherein the ligand-binding molecule comprises a series of peptide chains consisting of a single-domain antibody-antibody Fc region from the N-terminus to the C-terminus. (B19) The ligand-binding molecule is a dimer containing two peptide chains consisting of a single-domain antibody-antibody hinge region-antibody Fc region, as described in (B1) to (B17). (B20) The ligand-binding molecule according to (B17) to (B19), wherein the antibody Fc region is an Fc region containing one sequence selected from the amino acid sequences shown in SEQ ID NOs: 103 to 106, or an Fc region variant obtained by modifying these Fc regions. (B21) The ligand-binding molecule according to any one of (B1) to (B20), wherein the ligand is a cytokine or chemokine. (B22) The ligand-binding molecule according to any one of (B1) to (B21), wherein the ligand is selected from interleukins, interferons, hematopoietic factors, the TNF superfamily, chemokines, cell growth factors, and the TGF-β family. (B23) The ligand-binding molecule according to any one of (B1) to (B22), wherein the ligand is CXCL10, IL-12, PD-1, IL-6R, or IL-1Ra. (B24) A ligand-binding molecule according to any one of (B1) to (B23) that is bound to the ligand. (B25) A ligand-binding molecule according to any one of (B1) to (B23), which is fused with the ligand. (B26) The ligand-binding molecule as described in (B25), wherein when the ligand-binding molecule is fused with a ligand, the single-domain antibody contained in the ligand-binding molecule does not further bind to another ligand. (B27) The ligand-binding molecule according to (B25) or (B26), wherein the ligand-binding molecule is fused with the ligand via a linker. (B28) The ligand-binding molecule described in (B27), wherein the linker does not contain a protease cleavage sequence. (B29) A complex formed of the ligand and any one of the ligand-binding molecules described in (B1) to (B23). (B30) A fusion protein in which the ligand is fused with any one of the ligand-binding molecules described in (B1) to (B23). (B31) The fusion protein according to (B30), wherein the single-domain antibody does not further bind to another ligand when the ligand-binding molecule is fused with the ligand. (B32) The fusion protein according to (B30) or (B31), wherein the ligand-binding molecule is fused with the ligand via a linker. (B33) The fusion protein according to (B32), wherein the linker does not contain a protease cleavage sequence. (B34) A fusion protein as described in (B32) or (B33), wherein the ligand-linker-ligand binding molecule is fused in the order from the N-terminus to the C-terminus. (C1) A ligand-binding molecule containing a monodomain antibody, wherein the monodomain antibody is a VHH or monodomain VH antibody, and the monodomain antibody has cleavage sites at one or more positions in one or more sequences selected from the following sequences: monodomain antibody sequence from amino acid 7 (Kabat numbering) to amino acid 17 (Kabat numbering), monodomain antibody sequence from amino acid 12 (Kabat numbering) to amino acid 17 (Kabat numbering), monodomain antibody sequence from amino acid 31 (Kabat numbering) to amino acid 35b (Kabat numbering), monodomain antibody sequence from amino acid 40 (Kabat numbering) to amino acid 47 (Kabat numbering), monodomain antibody sequence from amino acid 50 (Kabat numbering) to amino acid 65 (Kabat numbering), monodomain antibody from amino acid 55 (Kabat numbering) Sequences up to amino acid 69 (Kabat numbering), sequences from amino acid 73 (Kabat numbering) to amino acid 79 (Kabat numbering) of single-domain antibodies, sequences from amino acid 83 (Kabat numbering) to amino acid 89 (Kabat numbering) of single-domain antibodies, sequences from amino acid 95 (Kabat numbering) to amino acid 99 (Kabat numbering) of single-domain antibodies, sequences from amino acid 95 (Kabat numbering) to amino acid 102 (Kabat numbering) of single-domain antibodies, sequences from amino acid 101 (Kabat numbering) to amino acid 113 (Kabat numbering) of single-domain antibodies. (C2) A ligand-binding molecule containing a monodomain antibody, wherein the monodomain antibody is a monodomain VL antibody, and the monodomain antibody has cleavage sites at one or more positions in one or more sequences selected from the following sequences: Sequences of single-domain antibodies from amino acid 7 (Kabat numbering) to amino acid 19 (Kabat numbering), sequences of single-domain antibodies from amino acid 24 (Kabat numbering) to amino acid 34 (Kabat numbering), sequences of single-domain antibodies from amino acid 39 (Kabat numbering) to amino acid 46 (Kabat numbering), sequences of single-domain antibodies from amino acid 49 (Kabat numbering) to amino acid 62 (Kabat numbering), sequences of single-domain antibodies from amino acid 50 (Kabat numbering) to amino acid 56 (Kabat numbering), sequences of single-domain antibodies from amino acid 89 (Kabat numbering) to amino acid 97 (Kabat numbering), and sequences of single-domain antibodies from amino acid 96 (Kabat numbering) to amino acid 107 (Kabat numbering). (C3) The ligand-binding molecule according to (C1) or (C2), wherein the single-domain antibody is capable of binding to the ligand, and the binding of the ligand-binding molecule to the ligand is attenuated when the cleavage site is cleaved compared to the binding of the ligand-binding molecule to the ligand when the cleavage site is not cleaved. (C4) A ligand-binding molecule according to any one of (C1) to (C3), wherein the ligand is released from the ligand-binding molecule when the cleavage site is cleaved. (C5) The ligand-binding molecule according to any one of (C1) to (C4), wherein the cleavage site includes a protease cleavage sequence. (C6) The ligand-binding molecule described in (C5), wherein the protease is a target tissue-specific protease. (C7) The ligand-binding molecule according to (C6), wherein the target tissue is cancer tissue and the target tissue-specific protease is cancer tissue-specific protease. (C8) The ligand-binding molecule according to (C6), wherein the target tissue is inflammatory tissue and the target tissue-specific protease is an inflammatory tissue-specific protease. (C9) The ligand-binding molecule according to any one of (C5) to (C8), wherein the protease is at least one protease selected from matryptase, urokinase (uPA), and metalloproteinase. (C10) The ligand-binding molecule according to any one of (C5) to (C9), wherein the protease cleavage sequence is a sequence containing one or more sequences selected from the sequences shown in SEQ ID NOs: 2-82, 788-827, and the sequences listed in Table 1. (C11) A ligand-binding molecule according to any one of (C5) to (C10), wherein a first movable linker is further added to one end of the protease cleavage sequence. (C12) The ligand-binding molecule according to (C11), wherein the first movable linker is a movable linker made of a glycine-serine polymer. (C13) The ligand-binding molecule according to (C11) or (C12), wherein a second movable linker is further added to the other end of the protease cleavage sequence. (C14) The ligand-binding molecule described in (C13), wherein the second movable linker is a movable linker made of a glycine-serine polymer. (C15) The ligand-binding molecule according to any one of (C1) to (C14), wherein the ligand is a biologically active molecule, and the single-domain antibody inhibits the biological activity of the ligand by binding to it. (C16) The ligand-binding molecule according to any one of (C1) to (C15), wherein the ligand is a biologically active molecule and the single-domain antibody has neutralizing activity against the ligand. (C17) The ligand-binding molecule according to any one of (C1) to (C16), wherein the ligand-binding molecule comprises only a single domain including the cleavage site or protease cleavage sequence. (C18) The ligand-binding molecule further comprises an antibody Fc region, as described in any one of (C1) to (C17). (C19) The ligand-binding molecule according to any one of (C1) to (C18), wherein the ligand-binding molecule comprises a series of peptide chains consisting of a single-domain antibody-antibody Fc region from the N-terminus to the C-terminus. (C20) The ligand-binding molecule is a dimer containing two peptide chains consisting of a single-domain antibody-antibody hinge region-antibody Fc region, as described in (C1) to (C18). (C21) The ligand-binding molecule described in (C18) to (C20), wherein the antibody Fc region is an Fc region containing one sequence selected from the amino acid sequences shown in SEQ ID NOs: 103 to 106, or an Fc region variant obtained by modifying these Fc regions. (C22) The ligand-binding molecule according to any one of (C1) to (C21), wherein the ligand is a cytokine or chemokine. (C23) The ligand-binding molecule according to any one of (C1) to (C22), wherein the ligand is selected from interleukins, interferons, hematopoietic factors, the TNF superfamily, chemokines, cell growth factors, and the TGF-β family. (C24) The ligand-binding molecule according to any one of (C1) to (C23), wherein the ligand is CXCL10, IL-12, PD-1, IL-6R, or IL-1Ra. (C25) A ligand-binding molecule according to any one of (C1) to (C24) that is bound to the ligand. (C26) A ligand-binding molecule according to any one of (C1) to (C24), which is fused with the ligand. (C27) The ligand-binding molecule as described in (C26), wherein when the ligand-binding molecule is fused with a ligand, the single-domain antibody contained in the ligand-binding molecule does not further bind to another ligand. (C28) The ligand-binding molecule according to (C26) or (C27), wherein the ligand-binding molecule is fused with the ligand via a linker. (C29) The ligand-binding molecule described in (C28), wherein the linker does not contain a protease cleavage sequence. (C30) A complex formed of the ligand and any one of the ligand-binding molecules described in (C1) to (C24). (C31) A fusion protein in which the ligand is fused with one of the ligand-binding molecules described in (C1) to (C24). (C32) The fusion protein according to (C31), wherein the single-domain antibody does not further bind to another ligand when the ligand-binding molecule is fused with the ligand. (C33) The fusion protein according to (C31) or (C32), wherein the ligand-binding molecule is fused with the ligand via a linker. (C34) The fusion protein described in (C33), wherein the linker does not contain a protease cleavage sequence. (C35) A fusion protein as described in (C33) or (C34), in which a ligand-linker-ligand binding molecule is fused from the N-terminus to the C-terminus. A pharmaceutical composition comprising a ligand-binding molecule described in any one of (D1)(A1) to (A27), (B1) to (B25), or (C1) to (C26). A pharmaceutical composition comprising a ligand-binding molecule and a ligand as described in any one of (D2)(A1) to (A26), (B1) to (B24), or (C1) to (C25). A pharmaceutical composition comprising the complex described in (D3)(A31) or (B29) or (C30). A pharmaceutical composition comprising a fusion protein described in any one of (D4)(A32) to (A36), (B30) to (B34), or (C31) to (C35). A method for producing a ligand-binding molecule described in any one of (E1)(A1) to (A25), (B1) to (B24), or (C1) to (C25). (E2) The method for producing the monodomain antibody in a ligand-binding molecule containing a monodomain antibody, comprising introducing a protease cleavage sequence into the monodomain antibody in (E1). (E3) A method for producing a fusion protein according to any one of (A32) to (A36), (B30) to (B34), or (C31) to (C35), comprising fusing a ligand-binding molecule containing a monodomain antibody into which a protease cleavage sequence has been introduced with a ligand capable of binding to the monodomain antibody. (E4) A polynucleotide encoding a ligand-binding molecule as described in any one of (A1) to (A25), (B1) to (B23), or (C1) to (C24). A vector containing the polynucleotides described in (E5)(E4). A host cell containing the polynucleotide described in (E6)(E4) or the vector described in (E5). A method for producing a ligand-binding molecule according to any one of (A1) to (A25), (B1) to (B23), or (C1) to (C24), comprising the step of culturing the host cells described in (E7) and (E6). A polynucleotide encoding a fusion protein as described in any one of (E8)(A32) to (A36), (B30) to (B34), or (C31) to (C35). A vector containing the polynucleotides described in (E9)(E8). A host cell containing the polynucleotides described in (E10)(E8) or the vector described in (E9). A method for producing a fusion protein according to any one of (A32) to (A36), (B30) to (B34), or (C31) to (C35), comprising the step of culturing the host cells described in (E11) and (E10). [Brief explanation of the drawing]

[0010] [Figure 1] This figure shows an example of a ligand-binding molecule of the present invention. When the cleavage site contained in the single-domain antibody is uncleaved, the single-domain antibody can bind to the ligand. When the cleavage site is cleaved, the single-domain antibody is cleaved and cannot bind to the ligand, and the ligand is released. [Figure 2]This figure shows an example of a fusion protein of the ligand-binding molecule and ligand of the present invention. When the cleavage site in the single-domain antibody is uncleaved, the single-domain antibody in the fusion protein can bind to the ligand in the fusion protein. When the cleavage site is cleaved, the single-domain antibody is cleaved and cannot bind to the ligand, and a portion of the fusion protein containing the ligand is released. [Figure 3] This figure shows another example of the ligand-binding molecule of the present invention. In this example, the ligand-binding molecule is a dimeric protein containing a single-domain antibody-antibody hinge region-antibody Fc region. [Figure 4] This figure shows another example of a ligand-binding molecule and ligand fusion protein of the present invention. In this example, the ligand-binding molecule is a dimer protein containing a single-domain antibody-antibody hinge region-antibody Fc region. [Figure 5] This figure shows the SDS-PAGE results for protease-treated ligand-binding molecules and untreated ligand-binding molecules. While control molecules without protease cleavage sequences (Lanes 12 and 13) have the same band position regardless of whether they are protease-treated or untreated, ligand-binding molecules into which each protease cleavage sequence has been introduced show new bands that appear only after protease treatment. This indicates that the single-domain antibody-containing ligand-binding molecules into which each protease cleavage sequence has been introduced were cleaved by protease treatment. [Figure 6] This is a real-time binding graph evaluating the binding of protease-treated ligand-binding molecules and protease-untreated ligand-binding molecules to IL-6R. The title of each figure is the name of the measurement sample, the vertical axis shows the relative binding between the ligand-binding molecule and IL-6R, and the horizontal axis shows time (s). The gray line shows data for the protease-untreated sample, and the black line shows data for the protease-treated sample. [Figure 7-1]This figure shows the SDS-PAGE results for protease-treated and untreated fusion proteins. While the control molecule, which does not contain a protease cleavage sequence, has the same band position regardless of whether it is protease-treated or untreated, each fusion protein containing a single-domain antibody-containing ligand-binding molecule into which a protease cleavage sequence has been introduced has a new band that appears only after protease treatment. This indicates that each fusion protein containing a single-domain antibody-containing ligand-binding molecule into which a protease cleavage sequence has been introduced was cleaved by protease treatment. [Figure 7-2] This figure is a continuation of Figure 7-1. [Figure 7-3] This figure is a continuation of Figure 7-2. [Figure 7-4] This figure is a continuation of Figure 7-3. [Figure 8-1] This is a real-time graph evaluating the binding of free IL-6R to IL6R90-bio in solutions of protease-treated and untreated fusion proteins. The title of each figure is the name of the measurement sample, the vertical axis shows the relative binding between IL-6R and the biotinylated anti-IL-6R monodomain antibody-containing molecule (IL6R90-bio), and the horizontal axis shows time (s). The gray line shows data from the untreated sample, and the black line shows data from the protease-treated sample. [Figure 8-2] This figure is a continuation of Figure 8-1. [Figure 9] This figure shows the SDS-PAGE results for protease-treated and untreated fusion proteins. [Figure 10] This is a real-time graph showing the binding of free human PD-1, present in solutions of protease-treated and untreated fusion proteins, to a biotinylated anti-PD-1 monodomain antibody-containing molecule (PD1-bio). The title of each figure is the name of the measurement sample, the vertical axis shows the relative binding of PD-1 and PD1-bio, and the horizontal axis shows time (s). The gray line shows data from the untreated sample, and the black line shows data from the protease-treated sample. [Figure 11] This figure shows the results of protease cleavage of IgG into which various protease cleavage sequences have been introduced. [Figure 12] This figure shows the protease cleavage results of IgG into which various protease cleavage sequences have been introduced. [Figure 13] This figure shows the results of protease cleavage of IgG into which various protease cleavage sequences have been introduced. [Modes for carrying out the invention]

[0011] Polypeptide In the present invention, polypeptides typically refer to peptides and proteins having a length of about four amino acids or more. When a series of amino acids linked by peptide bonds from the N-terminus to the C-terminus is considered a peptide chain, the polypeptides of the present invention may also be complex proteins formed by interactions such as disulfide bonds, hydrophobic interactions, and ionic bonds between multiple series of peptide chains. Furthermore, while polypeptides in the present invention are typically polypeptides consisting of artificially designed sequences, they are not particularly limited and may, for example, be polypeptides of biological origin. They may also be natural polypeptides, synthetic polypeptides, recombinant polypeptides, etc. Moreover, fragments of the above polypeptides are also included in the polypeptides of the present invention.

[0012] amino acid In this specification, amino acids are represented by one-letter codes, three-letter codes, or both, for example, Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, Val / V.

[0013] Amino acid modification For modifying amino acids in the amino acid sequence of polypeptides, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately employed. In addition, several known methods can be used for modifying amino acids by substituting them with amino acids other than natural ones (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing tRNA in which a non-natural amino acid is bound to the complementary amber suppressor tRNA of the UAG codon (amber codon), one of the stop codons, is also suitably used.

[0014] In this specification, the term "and / or" used to describe amino acid modification sites includes any combination of "and" and "or" as appropriate. Specifically, for example, "amino acids 37, 45, and / or 47 are substituted" includes the following variations of amino acid modification: (a) No. 37, (b) No. 45, (c) No. 47, (d) No. 37 and No. 45, (e) No. 37 and No. 47, (f) No. 45 and No. 47, (g) No. 37, No. 45 and No. 47.

[0015] In this specification, expressions that include the one-letter or three-letter codes of the original and modified amino acids before and after a number representing a specific position may be used as appropriate to indicate amino acid modifications. For example, the modification F37V or Phe37Val, used when making amino acid substitutions in the antibody variable region, represents the substitution of Phe at position 37, as represented by Kabat numbering, to Val. That is, the number represents the position of the amino acid as represented by Kabat numbering, the one-letter or three-letter code of the amino acid listed before it represents the original amino acid, and the one-letter or three-letter code of the amino acid listed after it represents the substituted amino acid. Similarly, the modification P238A or Pro238Ala, used when making amino acid substitutions in the Fc region included in the antibody constant region, represents the substitution of Pro at position 238, as represented by EU numbering, to Ala. That is, the number represents the position of the amino acid as represented by EU numbering, the one-letter or three-letter code of the amino acid listed before it represents the original amino acid, and the one-letter or three-letter code of the amino acid listed after it represents the substituted amino acid.

[0016] Antibodies and antibody fragments In this specification, the term “antibody” is used in its broadest sense and encompasses a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0017] An "antibody fragment" refers to a molecule other than the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments are not limited to these, but include Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0018] The terms "full-length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a native antibody, or having a heavy chain containing an Fc region as defined herein.

[0019] Variable region The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of an antibody (VH and VL, respectively) typically have a similar structure, with each domain containing four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0020] CDR As used herein, the terms “complementarity-determining region” or “CDR” refer to a region in a sequence that is hypervariable and / or forms a structurally defined loop ("hypervariable loop") and / or an antigen contact residue ("antigen contact"), a variable domain of an antibody, or a region of a monodomain antibody. Typically, an antibody contains six CDRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Illustrative antibody CDRs as used herein include: (a) Hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contact occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and, (d) A combination of (a), (b), and / or (c) containing CDR 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), or 94-102 (H3).

[0021] Typically, a single-domain antibody contains three CDRs: CDR1, CDR2, and CDR3. If the single-domain antibody is a VHH antibody or a single-domain VH antibody, the CDRs of the single-domain antibody may include, exemplarily, the following: (a) Hypervariable loops occurring at amino acid residues 26-32 (CDR1), 53-55 (CDR2), and 96-101 (CDR3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 31-35b (CDR1), 50-65 (CDR2), and 95-102 (CDR3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contact occurring at amino acid residues 30-35b (CDR1), 47-58 (CDR2), and 93-101 (CDR3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and, (d) A combination of (a), (b), and / or (c) containing CDR amino acid residues 26-35 (CDR1), 26-35b (CDR1), 49-65 (CDR2), 93-102 (CDR3), or 94-102 (CDR3).

[0022] If a single-domain antibody is a single-domain VL antibody, the CDR of the single-domain antibody may include, as an example, the following: (a) Hypervariable loops occurring at amino acid residues 26-32 (CDR1), 50-52 (CDR2), and 91-96 (CDR3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contact occurring at amino acid residues 27c-36 (CDR1), 46-55 (CDR2), and 89-96 (CDR3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and, (d) A combination of (a), (b), and / or (c) containing CDR amino acid residues 46-56 (CDR2), 47-56 (CDR2), 48-56 (CDR2), or 49-56 (CDR2).

[0023] Unless otherwise indicated, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein in accordance with Kabat et al.

[0024] FR "Framework" or "FR" refers to variable domain residues or single-domain antibody residues other than complementarity-determining region (CDR) residues. The FR of a variable domain or single-domain antibody typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the sequences of the CDR and FR usually appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. In single-domain antibodies, the sequences of the CDR and FR usually appear in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0025] The "Human Consensus Framework" is a framework that shows the most commonly occurring amino acid residues in selected human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from subgroups of variable domain sequences. Typically, the sequence subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI by Kabat et al. As described above. In another embodiment, for VH, the subgroup is subgroup III by Kabat et al. As described above.

[0026] Steady-state region In this specification, the term “constant region” or “constant domain” refers to the portion of an antibody other than the variable region. For example, an IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons, composed of two identical disulfide-linked light chains and two identical heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by a heavy chain constant region (CH) containing the CH1 domain, hinge region, CH2 domain, and CH3 domain. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chains of native antibodies may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains. The term “contains the constant region” may include the entire constant region or a portion of the constant region.

[0027] The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0028] Fc area In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes the Fc region of the native sequence and mutant Fc regions. In one embodiment, in the case of human IgG1, the heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain, provided that the lysine (Lys447) or glycine-lysine (Gly446-Lys447) at the C-terminus of the Fc region is present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0029] Hinge area The terms "hinge region" or "antibody hinge region" can refer to the region in the antibody heavy chain consisting of amino acids from EU numbering 216 to 230, or a portion of that region.

[0030] Single-domain antibody In this specification, the term "monodomain antibody" refers to an antibody that can exhibit antigen-binding activity with its domain alone. The structure of a monodomain antibody is not limited as long as its domain alone can exhibit antigen-binding activity. While conventional antibodies, such as IgG antibodies, exhibit antigen-binding activity when a variable region is formed by the pairing of VH and VL, monodomain antibodies are known to exhibit antigen-binding activity with their own domain structure alone, without pairing with other domains. Monodomain antibodies usually have a relatively low molecular weight and exist in monomeric form. In some embodiments, the morphology of a monodomain antibody is similar to that of the antibody heavy chain variable region or the antibody light chain variable region. Examples of single-domain antibodies, though not limited to them, include the variable region (VHH) of heavy chain antibodies in camelid animals and V in sharks. NAR Examples include antigen-binding molecules that congenitally lack a light chain, or antibody fragments that contain all or part of the VH domain or all or part of the VL domain of an antibody. Examples of monodomain antibodies that are antibody fragments containing all or part of the VH / VL domain of an antibody include, but are not limited to, monodomain antibodies artificially produced starting from human VH or human VL antibodies, such as those described in U.S. Patent No. 6,248,516B1, etc. (hereinafter referred to as monodomain VH antibodies and monodomain VL antibodies).

[0031] Monodomain antibodies can be obtained from animals capable of producing monodomain antibodies, or by immunizing animals capable of producing monodomain antibodies. Examples of animals capable of producing monodomain antibodies include, but are not limited to, camelids and transgenic animals into which a gene capable of producing monodomain antibodies has been introduced. Camelids include camels, llamas, alpacas, dromedaries, and guanacos. Examples of transgenic animals into which a gene capable of producing monodomain antibodies has been introduced include, but are not limited to, the transgenic animals described in International Publication WO2015 / 143414 and U.S. Patent Publication US2011 / 0123527A1. Humanized monodomain antibodies can also be obtained by using a human germline sequence or a similar sequence as the framework sequence of a monodomain antibody obtained from an animal. Humanized monodomain antibodies (e.g., humanized VHH) are also one embodiment of the monodomain antibody of the present invention.

[0032] Monodomain antibodies can be obtained from animals capable of producing monodomain antibodies, or by immunizing animals capable of producing monodomain antibodies. Examples of animals capable of producing monodomain antibodies include, but are not limited to, camelids and transgenic animals into which a gene capable of producing monodomain antibodies has been introduced. Camelids include camels, llamas, alpacas, dromedaries, and guanacos. Examples of transgenic animals into which a gene capable of producing monodomain antibodies has been introduced include, but are not limited to, the transgenic animals described in International Publication WO2015 / 143414 and U.S. Patent Publication US2011 / 0123527A1. Humanized monodomain antibodies can also be obtained by using a human germline sequence or a similar sequence as the framework sequence of a monodomain antibody obtained from an animal. Humanized monodomain antibodies (e.g., humanized VHH) are also one embodiment of the monodomain antibody of the present invention.

[0033] Furthermore, single-domain antibodies can be obtained from polypeptide libraries containing single-domain antibodies by methods such as ELISA and panning. Examples of polypeptide libraries containing single-domain antibodies include, but are not limited to, naive antibody libraries obtained from various animals or humans (e.g., Methods in Molecular Biology 2012 911 (65-78), Biochimica et Biophysica Acta - Proteins and Proteomics 2006 1764:8 (1307-1319)), antibody libraries obtained by immunizing various animals (e.g., Journal of Applied Microbiology 2014 117:2 (528-536)), or synthetic antibody libraries created from antibody genes of various animals or humans (e.g., Journal of Biomolecular Screening 2016 21:1 (35-43), Journal of Biological Chemistry 2016 291:24 (12641-12657), AIDS 2016 30:11). (1691-1701) is one example.

[0034] In the present invention, there are embodiments in which a cleavage site / protease cleavage sequence is introduced into a single-domain antibody, but regardless of whether or not a cleavage site / protease cleavage sequence is introduced, it can be described as a "single-domain antibody".

[0035] The single-domain antibody of the present invention, in some embodiments, generally, a) an amino acid sequence consisting of four framework regions / sequences with three complementarity-determining regions / sequences inserted between them (the amino acid residue at position 11 according to Kabat numbering is selected from the group consisting of L, M, S, V, and W, preferably L), and / or b) an amino acid sequence consisting of four framework regions / sequences with three complementarity-determining regions / sequences inserted between them (the amino acid residue at position 37 according to Kabat numbering is selected from the group consisting of F, Y, H, I, L, and V, preferably F or Y), and / or c) an amino acid sequence consisting of four framework regions / sequences with three complementarity-determining regions / sequences inserted between them (the amino acid residue at position 44 according to Kabat numbering is selected from the group consisting of G, E, A, D, Q, R, S, L, preferably G, E, or Q, more preferably G or E), and / or d) an amino acid sequence consisting of four framework regions / sequences with three complementarity-determining regions / sequences inserted between them (the amino acid residue at position 45 according to Kabat numbering is selected from the group consisting of L, R, C, I, L, P, Q, V, preferably L or R), and / or e) an amino acid sequence consisting of four framework regions / sequences with three complementarity-determining regions / sequences inserted between them (the amino acid residue at position 47 according to Kabat numbering is selected from the group consisting of W, L, F, A, G, I, M, R, S, V, Y, preferably W, L, F, or R), and / or f) an amino acid sequence consisting of four framework regions / sequences with three complementarity-determining regions / sequences inserted between them (the amino acid residue at position 83 according to Kabat numbering is selected from the group consisting of R, K, N, E, G, I, M, Q, T, preferably K or R, more preferably K), and / or g) an amino acid sequence consisting of four framework regions / sequences with three complementarity-determining regions / sequences inserted between them (the amino acid residue at position 84 according to Kabat numbering is selected from the group consisting of P, A, L, R, S, T, D, V, preferably P), and / or h) an amino acid sequence consisting of four framework regions / sequences with three complementarity-determining regions / sequences inserted between them (the amino acid residue at position 103 according to Kabat numbering is selected from the group consisting of W, P, R, and S, or more specifically, W), and / or i) an amino acid sequence consisting of four framework regions / sequences with three complementarity-determining regions / sequences inserted between them (the amino acid residue at position 104 according to Kabat numbering is G or D, preferably G), and / or j) A polypeptide can be defined as comprising an amino acid sequence consisting of four framework regions / sequences with three complementarity-determining regions / sequences inserted between them (the amino acid residue at position 108 according to Kabat numbering is selected from the group consisting of Q, L, and R, preferably Q or L).

[0036] More specifically, though not mutually exclusive, a monodomain antibody can be defined as a polypeptide containing one of four amino acid sequences consisting of four framework regions / sequences with the following three complementarity-determining regions / sequences inserted between them: k) Amino acid sequences in which the amino acid residues at positions 43-46 according to Kabat numbering are KERE or KQRE; l) The amino acid sequence in which amino acid residues from position 44 to 47 according to Kabat numbering are GLEW; m) An amino acid sequence in which the amino acid residues at positions 83-84 according to Kabat numbering are either KP or EP.

[0037] Chimeric antibodies The term "chimeric" refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remaining heavy chain and / or light chain originates from a different source or species. A "chimeric monodomain antibody" refers to a monodomain antibody in which a portion of the monodomain antibody originates from a specific source or species, while the remaining monodomain antibody originates from a different source or species.

[0038] Humanized antibodies A "humanized" antibody is a chimeric antibody that contains amino acid residues from a non-human CDR and amino acid residues from a human FR. In some embodiments, a humanized antibody contains substantially all of at least one, typically two, variable domains, in which all or substantially all CDRs correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. A "humanized single-domain antibody" is a chimeric single-domain antibody that contains amino acid residues from a non-human CDR and amino acid residues from a human FR. In some embodiments, a humanized single-domain antibody contains all or substantially all CDRs corresponding to those of a non-human antibody, and all or substantially all FRs corresponding to those of a human antibody. Even if some of the residues in the FR of a humanized antibody do not correspond to those of a human antibody, this can be considered an example where substantially all of the FRs correspond to those of a human antibody. For example, when humanizing VHH, a form of monodomain antibody, it is necessary to replace some of the residues in FR with residues that do not correspond to those in human antibodies (C Vincke et al., The Journal of Biological Chemistry 284, 3273-3284). Humanized antibodies may optionally contain at least a portion of the constant region of an antibody derived from a human antibody. The "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0039] Affinity "Affinity" refers to the strength of the combined non-covalent interactions between one binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" as used herein refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X to its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific examples and exemplary embodiments for measuring binding affinity are described below.

[0040] The same epitope-binding antibody An antibody that "binds to the same epitope as the reference antibody" is defined as an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay. Conversely, the reference antibody blocks the binding of the aforementioned antibody to its antigen by 50% or more in a competitive assay. An exemplary competitive assay is provided herein.

[0041] Isolated antibodies "Isolated" antibodies are those separated from the components of their original environment. In some embodiments, antibodies are purified to a purity of over 95% or 99% by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0042] Monoclonal antibodies As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies constituting that population are identical and / or bind to the same epitope, except for any possible mutant antibodies (e.g., mutant antibodies containing naturally occurring mutations, or mutant antibodies that arise during the production of a monoclonal antibody preparation; such variants are usually present in small amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. Therefore, the modifier “monoclonal” indicates a characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be prepared by a variety of methods, including, but are not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0043] Sequence identity "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage ratio of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after the sequences have been aligned to obtain the greatest possible percentage sequence identity and gaps have been introduced where necessary, and no conservative substitutions are considered part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved by using various methods within the scope of the art, such as publicly available computer software, including BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetics Co., Ltd.). A person skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve the greatest possible alignment over the entire length of the sequences being compared.

[0044] vector As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures, and vectors incorporated into the genome of a host cell into which they are introduced. Some vectors can result in the expression of the nucleic acid to which they are operationally ligated. Such vectors are also referred to herein as "expression vectors."

[0045] host cells etc. The terms “host cell,” “host cell line,” and “host cell culture” refer to cells (including their offspring) that are interchangeably used and into which foreign nucleic acids have been introduced. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their offspring, regardless of passage number. Offspring do not have to be completely identical to the parent cells in terms of nucleic acid content and may contain mutations. Mutant offspring that have the same function or biological activity as those used when the original transformed cells were screened or selected are also included herein.

[0046] protease In this specification, the term "protease" refers to an enzyme such as an endopeptidase or exopeptidase that hydrolyzes peptide bonds, usually an endopeptidase. The proteases used in the present invention are limited only by their ability to cleave protease cleavage sequences, and their type is not particularly limited. In some embodiments, target tissue-specific proteases are used. Target tissue-specific proteases are, for example, (1) Proteases that are expressed at higher levels in target tissue than in normal tissue, (2) Proteases that have higher activity in target tissue than in normal tissue, (3) Proteases expressed at higher levels in target cells than in normal cells, (4) Proteases that have higher activity in target cells than in normal cells, This can refer to either of the above. In a more specific embodiment, cancer tissue-specific proteases or inflammatory tissue-specific proteases are used.

[0047] target tissue In this specification, the term “target tissue” means tissue containing at least one target cell. In some embodiments of the present invention, the target tissue is cancerous tissue. In some embodiments of the present invention, the target tissue is inflammatory tissue.

[0048] The term "cancer tissue" means tissue containing at least one cancer cell. Therefore, it refers to all cell types that contribute to the formation of a tumor mass, including cancer cells and endothelial cells, such as cancer tissue containing cancer cells and blood vessels. In this specification, "tumor" means a foci of tumor tissue. The term "tumor" is generally used to mean either a benign or malignant neoplasm.

[0049] In this specification, "inflammatory tissue" includes, for example, the following: Joints in rheumatoid arthritis and osteoarthritis • Lungs (alveoli) in bronchial asthma and COPD • Digestive organs in inflammatory bowel disease, Crohn's disease, and ulcerative colitis • Fibrotic tissue in fibrosis of the liver, kidneys, and lungs • Tissues that are being rejected in organ transplants • Blood vessels and heart (myocardium) in arteriosclerosis and heart failure • Visceral fat in metabolic syndrome • Skin tissue in atopic dermatitis and other skin inflammations • Spinal nerves in herniated discs and chronic lower back pain

[0050] Target tissue-specific proteases In some types of target tissues, proteases that are specifically expressed or specifically activated, or proteases that are thought to be associated with the disease state of the target tissue (target tissue-specific proteases), are known. For example, proteases that are specifically expressed in cancer tissue are disclosed in international publications WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846, among others. Furthermore, proteases thought to be associated with inflammation have been disclosed in J Inflamm (Lond). 2010; 7: 45., Nat Rev Immunol. 2006 Jul;6(7):541-50., Nat Rev Drug Discov. 2014 Dec;13(12):904-27., Respir Res. 2016 Mar 4;17:23., Dis Model Mech. 2014 Feb;7(2):193-203., and Biochim Biophys Acta. 2012 Jan;1824(1):133-45.

[0051] In addition to proteases that are specifically expressed in target tissues, there are also proteases that are specifically activated in target tissues. For example, proteases may be expressed in an inactive form and then become active, and in many tissues, substances that inhibit the active protease exist, and the activity is controlled by the activation process and the presence of inhibitors (Nat Rev Cancer. 2003 Jul;3(7):489-501.). In target tissues, the active protease may escape inhibition and be specifically activated. Active proteases can be measured using methods that employ antibodies that recognize active proteases (PNAS 2013 Jan 2; 110(1): 93-98.) or by fluorescently labeling the peptide recognized by the protease, quenching it before cleavage, and then emitting light after cleavage (Nat Rev Drug Discov. 2010 Sep;9(9):690-701. doi: 10.1038 / nrd3053.).

[0052] While not meant to be interpreted restrictively, specific proteases include cysteine ​​proteases (including cathepsin family B, L, S, etc.), aspartyl proteases (cathepsin D, E, K, O, etc.), serine proteases (matryptase (including MT-SP1), cathepsin A and G), thrombin, plasmin, urokinase (uPA), tissue plasminogen activator (tPA), elastase, proteinase 3, thrombin, kallikrein, trip Metalloproteinases (including tase and chymase), metalloproteinases (including both membrane-bound (MMP14-17 and MMP24-25) and secreted (MMP1-13, MMP18-23, and MMP26-28) metalloproteinases (MMP1-28)), proteases A disintegrin and metalloproteinases (ADAM), metalloproteinases with A disintegrin or thrombospongin motifs (ADAMTS), meprin (meprin α (meprin alpha, meprin beta, CD10 (CALLA), prostate-specific antigen (PSA), regmine, TMPRSS3, TMPRSS4, neutrophil elastase (HNE), beta-secretase (BACE), fibroblast-activating protein alpha (FAP), granzyme B, guanidinobenzoate (GB), hepsin, neprilysin, NS3 / 4A, HCV-NS3 / 4, calpain, ADAMDEC1, renin, cathepsin C, cathepsin V / L2, cathepsin X / Z / P, Kurjipain, Otsubine 2, Kallikrein-related peptidases (KLKs (KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14)), Bone morphogenetic protein 1 (BMP-1), Activated protein C, Blood coagulation-related proteases (Factor VIIa, Factor IXa, Factor Xa, Factor XIa, Factor XIIa), HtrA1, lactoferrin, malapsin, PACE4, DESC1, dipeptidyl peptidase 4 (DPP-4), TMPRSS2, cathepsin F, cathepsin H, cathepsin L2, cathepsin O, cathepsin S, granzyme A, gepsin calpain 2, glutamate carboxypeptidase 2, AMSH-LikeExamples include proteases, AMSH, gamma secretase, anti-plasmin cleavage enzyme (APCE), Decysin 1, N-Acetylated Alpha-Linked Acidic Dipeptidase-Like 1 (NAALADL1), and furin.

[0053] From another perspective, target tissue-specific proteases can refer to cancer tissue-specific proteases or inflammatory tissue-specific proteases.

[0054] Cancer tissue-specific proteases Examples of cancer tissue-specific proteases include those disclosed in international publications WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846, which are specifically expressed in cancer tissue.

[0055] The type of cancer tissue-specific protease that exhibits high specificity in the target cancer tissue yields a greater reduction in side effects. It is preferable that the concentration of the cancer tissue-specific protease in cancer tissue is at least five times higher than that in normal tissue, more preferably at least ten times higher, even more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. Furthermore, it is preferable that the activity of the cancer tissue-specific protease in cancer tissue is at least twice as high as that in normal tissue, more preferably at least three times higher, four times higher, five times higher, ten times higher, even more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. Furthermore, cancer tissue-specific proteases may be bound to the cell membrane of cancer cells, or they may not be bound to the cell membrane and may be secreted extracellularly. If cancer tissue-specific proteases are not bound to the cell membrane of cancer cells, it is preferable that the cancer tissue-specific protease is located inside or near the cancer tissue in order for the cytotoxicity by immune cells to be specific to cancer cells. In this specification, "near the cancer tissue" means within the range in which the cancer tissue-specific protease cleavage sequence is cleaved and exerts a ligand-binding activity reduction effect. However, it is preferable that this range does not damage normal cells as much as possible. From another perspective, cancer tissue-specific proteases are, (i) Proteases expressed at higher levels in cancer tissue than in normal tissue, (ii) Proteases that have higher activity in cancer tissue than in normal tissue, (iii) Proteases expressed at higher levels in cancer cells than in normal cells, (iv) Proteases that have higher activity in cancer cells than in normal cells, It is one of the following: Cancer tissue-specific proteases may be used individually or in combination of two or more. The number of types of cancer tissue-specific proteases can be appropriately determined by a person skilled in the art, taking into consideration the type of cancer being treated.

[0056] From the above viewpoint, among the proteases exemplified above, serine proteases and metalloproteases are preferred as cancer tissue-specific proteases, matryptase (including MT-SP1), urokinase (uPA), and metalloproteases are more preferred, and MT-SP1, uPA, MMP-2, and MMP-9 are even more preferred.

[0057] Inflammatory tissue-specific proteases The type of inflammation-specific protease that exhibits high specificity in the inflammatory tissue being treated is preferable to achieve a reduction in side effects. It is preferable that the concentration of the inflammation-specific protease in inflammatory tissue is at least five times higher than that in normal tissue, more preferably at least ten times higher, even more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. Furthermore, it is preferable that the activity of the inflammation-specific protease in inflammatory tissue is at least twice as high as that in normal tissue, more preferably at least three times, four times, five times, ten times, even more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. Furthermore, the inflammation tissue-specific protease may be bound to the cell membrane of inflammatory cells, or it may not be bound to the cell membrane and may be secreted extracellularly. If the inflammation tissue-specific protease is not bound to the cell membrane of inflammatory cells, it is preferable that the inflammation tissue-specific protease is located inside or near the inflammatory tissue in order for the cytotoxicity by immune cells to be specific to inflammatory cells. In this specification, "near the inflammatory tissue" means within the range in which the inflammation tissue-specific protease cleavage sequence is cleaved and exerts a ligand-binding activity reduction effect. However, it is preferable that this range does not damage normal cells as much as possible. From another perspective, inflammation-specific proteases are, (i) Proteases expressed at higher levels in inflammatory tissue than in normal tissue, (ii) Proteases that have higher activity in inflammatory tissue than in normal tissue, (iii) Proteases expressed at higher levels in inflammatory cells than in normal cells, (iv) Proteases that have higher activity in inflammatory cells than in normal cells, It is one of the following: Inflammatory tissue-specific proteases may be used individually or in combination of two or more types. The number of types of inflammatory tissue-specific proteases can be appropriately determined by those skilled in the art, taking into consideration the disease condition being treated.

[0058] From the above perspective, among the proteases exemplified above, metalloproteases are preferred as inflammatory tissue-specific proteases, and among metalloproteases, ADAMTS5, MMP-2, MMP-7, MMP-9, and MMP-13 are more preferred.

[0059] Method for producing antibodies Methods for producing antibodies with desired binding activity are known to those skilled in the art. The following is an example of a method for producing antibodies that bind to IL-6R (anti-IL-6R antibodies). Antibodies that bind to antigens other than IL-6R can also be produced as appropriate in accordance with the following examples. When producing single-domain antibodies, although there may be differences in the immunizing animals, they can also be produced as appropriate in accordance with the following examples.

[0060] Anti-IL-6R antibodies can be obtained as polyclonal or monoclonal antibodies using known methods. Mammalian-derived monoclonal antibodies are preferably produced as anti-IL-6R antibodies. Mammalian-derived monoclonal antibodies include those produced by hybridomas and those produced by host cells transformed with expression vectors containing antibody genes using genetic engineering techniques. Antibodies referred to in this application include "humanized antibodies" and "chimeric antibodies."

[0061] Monoclonal antibody-producing hybridomas can be produced, for example, by using known techniques as follows: Mammals are immunized according to a standard immunization method using the IL-6R protein as a sensitizing antigen. The resulting immune cells are fused with known parent cells by a standard cell fusion method. Next, hybridomas that produce anti-IL-6R antibodies can be selected by screening monoclonal antibody-producing cells using a standard screening method.

[0062] Specifically, the production of monoclonal antibodies is carried out as follows: First, the IL-6R protein, which can be used as a sensitizing antigen for antibody acquisition, can be obtained by expressing the IL-6R gene. That is, suitable host cells are transformed by inserting the gene sequence encoding IL-6R into a known expression vector. The desired human IL-6R protein is purified from the host cells or culture supernatant by a known method. To obtain soluble IL-6R from the culture supernatant, soluble IL-6R is expressed, for example, as described by Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968). Alternatively, purified native IL-6R protein can also be used as a sensitizing antigen.

[0063] The purified IL-6R protein can be used as a sensitizing antigen for immunization against mammals. A partial peptide of IL-6R can also be used as a sensitizing antigen. In this case, the partial peptide can be obtained by chemical synthesis from the amino acid sequence of human IL-6R. It can also be obtained by incorporating a part of the IL-6R gene into an expression vector and expressing it. Furthermore, it can be obtained by degrading the IL-6R protein using a protease, but the region and size of the IL-6R peptide used as a partial peptide are not particularly limited to any special form. Preferably, the number of amino acids constituting the peptide to be used as a sensitizing antigen is at least 5, for example, 6 or more, or 7 or more. More specifically, a peptide of 8 to 50 residues, preferably 10 to 30 residues, can be used as a sensitizing antigen.

[0064] Furthermore, fusion proteins obtained by fusing a desired partial polypeptide or peptide of the IL-6R protein with a different polypeptide can be used as sensitization antigens. For example, antibody Fc fragments or peptide tags can be suitably used to produce fusion proteins used as sensitization antigens. A vector expressing a fusion protein can be produced by fusing genes encoding two or more desired polypeptide fragments in-frame, and inserting the fusion gene into an expression vector as described above. The method for producing fusion proteins is described in Molecular Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58 (1989) Cold Spring Harbor Lab. press). Methods for obtaining IL-6R used as a sensitization antigen and immunization methods using it are also specifically described in WO2003 / 000883, WO2004 / 022754, WO2006 / 006693, etc.

[0065] While the mammals immunized with the sensitizing antigen are not limited to specific animals, it is preferable to select them considering their compatibility with the parent cells used for cell fusion. Generally, rodents such as mice, rats, hamsters, rabbits, and monkeys are preferred. When obtaining single-domain antibodies, camelids or genetically modified animals into which genes capable of producing single-domain antibodies have been introduced are preferred.

[0066] The animals described above are immunized with the sensitizing antigen according to known methods. For example, a common method is to administer the sensitizing antigen to mammals intraperitoneally or subcutaneously. Specifically, the sensitizing antigen, diluted to an appropriate dilution ratio with PBS (Phosphate-Buffered Saline) or physiological saline, is mixed with a conventional adjuvant, such as Freund's complete adjuvant, if desired, and emulsified. After emulsification, the sensitizing antigen is administered to mammals several times every 4 to 21 days. A suitable carrier may also be used during immunization with the sensitizing antigen. In particular, when a partial peptide with a small molecular weight is used as the sensitizing antigen, it may be desirable to immunize with the sensitizing antigen peptide bound to a carrier protein such as albumin or keyhole limpet hemocyanin.

[0067] Furthermore, hybridomas that produce the desired antibody can also be produced using DNA immunization as follows. DNA immunization is an immunization method in which a vector DNA constructed in such a manner that a gene encoding an antigen protein can be expressed in the immunized animal is administered, and the sensitized antigen is expressed in the immunized animal, thereby providing immune stimulation. Compared to general immunization methods in which protein antigens are administered to immunized animals, DNA immunization is expected to have the following advantages. - It is possible to maintain the structure of membrane proteins such as IL-6R and deliver immunostimulation. - There is no need to purify immune antigens.

[0068] To obtain the monoclonal antibody of the present invention by DNA immunization, first, DNA expressing the IL-6R protein is administered to an immunized animal. The DNA encoding IL-6R can be synthesized by known methods such as PCR. The obtained DNA is inserted into a suitable expression vector and administered to an immunized animal. Commercial expression vectors such as pcDNA3.1 can be suitably used as the expression vector. Commonly used methods can be used to administer the vector into a living organism. For example, DNA immunization is performed by introducing gold particles to which the expression vector is adsorbed into the cells of an immunized animal using a gene gun. Furthermore, antibodies that recognize IL-6R can also be produced using the method described in International Publication WO 2003 / 104453.

[0069] After the mammal is immunized in this manner and an increase in antibody titers binding to IL-6R in the serum is confirmed, immune cells are collected from the mammal and used for cell fusion. Splenocytes, in particular, may be used as preferred immune cells.

[0070] Mammalian myeloma cells are used as the cells fused with the aforementioned immune cells. It is preferable that the myeloma cells possess appropriate selection markers for screening. A selection marker refers to a trait that allows (or prevents) survival under specific culture conditions. Known selection markers include hypoxanthine-guanine-phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) or thymidine kinase deficiency (hereinafter abbreviated as TK deficiency). Cells lacking HGPRT or TK are hypoxanthine-aminopterin-thymidine sensitive (hereinafter abbreviated as HAT sensitive). HAT-sensitive cells cannot synthesize DNA in HAT-selective medium and die, but when fused with normal cells, they can continue DNA synthesis using the normal cell's salvage pathway and thus proliferate even in HAT-selective medium.

[0071] HGPRT-deficient and TK-deficient cells can be selected in media containing 6-thioguanine, 8-azaguanine (hereinafter abbreviated as 8AG), or 5'-bromodeoxyuridine, respectively. Normal cells that incorporate these pyrimidine analogs into their DNA will die. On the other hand, cells lacking these enzymes and unable to incorporate these pyrimidine analogs can survive in the selective medium. Another selection marker, known as G418 resistance, confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) via the neomycin resistance gene. Various myeloma cells suitable for cell fusion are known.

[0072] Examples of such myeloma cells include P3 (P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (C. Eur. J. Immunol. (1976) 6 (7), 511-519), MPC-11 (Cell (1976) 8 (3), 405-415), SP2 / 0 (Nature (1978) 276 (5685), 269-270), FO (J. Immunol. Methods (1980) 35 (1-2), 1-21), S194 / 5.XX0.BU.1 (J. Exp. Med.(1978)148 (1), 313-323), R210 (Nature(1979)277 (5692), 131-133), etc., can be suitably used.

[0073] Cell fusion between the immune cells and myeloma cells is basically performed according to known methods, such as the method of Köhler and Myrstein et al. (Methods Enzymol. (1981) 73, 3-46). More specifically, the cell fusion can be carried out, for example, in a normal nutrient culture medium in the presence of a cell fusion promoter. Examples of fusion promoters include polyethylene glycol (PEG) and Sendai virus (HVJ), and additional adjuvants such as dimethyl sulfoxide may be added as desired to further enhance fusion efficiency.

[0074] The ratio of immune cells to myeloma cells can be set arbitrarily. For example, it is preferable to use 1 to 10 times more immune cells than myeloma cells. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium, MEM culture medium, or other common culture mediums used for this type of cell culture can be used, and serum supplements such as fetal bovine serum (FCS) may be suitably added.

[0075] Cell fusion is performed by thoroughly mixing predetermined amounts of the immune cells and myeloma cells in the culture medium, and then adding a PEG solution (for example, with an average molecular weight of about 1000 to 6000) that has been preheated to about 37°C, usually at a concentration of 30 to 60% (w / v). The desired fused cells (hybridomas) are formed by the gradual mixing of the mixture. Subsequently, the appropriate culture medium mentioned above is added sequentially, and the process of centrifugation and removal of the supernatant is repeated, thereby removing cell fusion agents and other substances unfavorable to hybridoma growth.

[0076] The hybridomas obtained in this manner can be selected by culturing them in a standard selective culture medium, such as HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Culturing with the HAT culture medium can be continued for a sufficient time (usually several days to several weeks) to kill cells other than the desired hybridoma (non-fusion cells). Subsequently, screening and single cloning of hybridomas that produce the desired antibody is performed using a standard limiting dilution method.

[0077] The hybridomas obtained in this way can be selected by using a selective culture medium corresponding to the selection markers present in the myeloma used for cell fusion. For example, cells lacking HGPRT or TK can be selected by culturing them in HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells that successfully fuse with normal cells can be selectively proliferated in the HAT culture medium. Culturing with the HAT culture medium is continued for a sufficient amount of time for cells other than the desired hybridoma (non-fused cells) to die. Specifically, generally, the desired hybridoma can be selected by culturing for several days to several weeks. Subsequently, screening and single cloning of hybridomas that produce the desired antibody can be performed using the usual limiting dilution method.

[0078] Screening and single cloning of desired antibodies can be suitably carried out by known antigen-antibody reaction-based screening methods. For example, a monoclonal antibody that binds to IL-6R can bind to IL-6R expressed on the cell surface. Such monoclonal antibodies can be screened, for example, by FACS (fluorescence activated cell sorting). FACS is a system that allows for the measurement of antibody binding to the cell surface by analyzing cells contacted with a fluorescent antibody using laser light and measuring the fluorescence emitted by individual cells.

[0079] To screen for hybridomas that produce the monoclonal antibody of the present invention by FACS, cells expressing IL-6R are first prepared. Preferred cells for screening are mammalian cells that overexpress IL-6R. By using untransformed mammalian cells as the host cell as a control, the antibody binding activity to IL-6R on the cell surface can be selectively detected. That is, by selecting hybridomas that produce antibodies that do not bind to host cells but bind to IL-6R-overexpressing cells, hybridomas that produce IL-6R monoclonal antibodies can be obtained.

[0080] Alternatively, the binding activity of antibodies against immobilized IL-6R-expressing cells can be evaluated based on the principles of ELISA. For example, IL-6R-expressing cells are immobilized in the wells of an ELISA plate. The culture supernatant of hybridomas is brought into contact with the immobilized cells in the wells, and antibodies that bind to the immobilized cells are detected. If the monoclonal antibody is derived from a mouse, the antibody bound to the cells can be detected by an anti-mouse immunoglobulin antibody. Hybridomas that produce the desired antibody with antigen-binding ability, selected through these screenings, can be cloned by methods such as limiting dilution.

[0081] The hybridomas producing monoclonal antibodies thus created can be subcultured in a normal culture medium. Furthermore, these hybridomas can be stored for extended periods in liquid nitrogen.

[0082] The hybridoma can be cultured according to conventional methods, and the desired monoclonal antibody can be obtained from the culture supernatant. Alternatively, the hybridoma can be administered to a compatible mammal to proliferate, and the monoclonal antibody can be obtained from its ascites fluid. The former method is suitable for obtaining high-purity antibodies.

[0083] Antibodies encoded by antibody genes cloned from antibody-producing cells such as hybridomas can also be suitably utilized. By incorporating the cloned antibody gene into a suitable vector and introducing it into a host, the antibody encoded by the gene is expressed. Methods for isolating antibody genes, introducing them into vectors, and transforming host cells have already been established, for example, by Vandamme et al. (Eur.J. Biochem.(1990)192 (3), 767-775). Methods for producing recombinant antibodies are also known, as described below.

[0084] For example, cDNA encoding the variable region (V region) of the anti-IL-6R antibody can be obtained from hybridoma cells that produce anti-IL-6R antibodies. To do this, total RNA is usually extracted from the hybridoma first. Methods such as the following can be used to extract mRNA from cells. - Guanidine ultracentrifugation (Biochemistry (1979) 18 (24), 5294-5299) - AGPC method (Anal. Biochem. (1987) 162 (1), 156-159)

[0085] The extracted mRNA can be purified using an mRNA Purification Kit (GE Healthcare Biosciences), etc. Alternatively, kits for directly extracting total mRNA from cells are commercially available, such as the QuickPrep mRNA Purification Kit (GE Healthcare Biosciences). mRNA can be obtained from hybridomas using such kits. From the obtained mRNA, cDNA encoding the antibody V region can be synthesized using reverse transcriptase. cDNA can be synthesized using an AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation), etc. Furthermore, for cDNA synthesis and amplification, the SMART RACE cDNA amplification kit (Clontech) and the 5'-RACE method using PCR (Proc. Natl. Acad. Sci. USA (1988) 85 (23), 8998-9002, Nucleic Acids Res. (1989) 17 (8), 2919-2932) may be used as appropriate. Furthermore, during the process of synthesizing cDNA, appropriate restriction enzyme sites, as described later, can be introduced at both ends of the cDNA.

[0086] The target cDNA fragment is purified from the obtained PCR product and then ligated to vector DNA. A recombinant vector is thus prepared, introduced into E. coli or other organisms, and after colony selection, the desired recombinant vector can be prepared from the E. coli that formed the colonies. Then, whether or not the recombinant vector possesses the target cDNA base sequence is confirmed by known methods, such as dideoxynucleotide chain intermination.

[0087] To obtain genes encoding variable regions, the 5'-RACE method using primers for variable region gene amplification is a convenient approach. First, cDNA is synthesized using RNA extracted from hybridoma cells as a template, yielding a 5'-RACE cDNA library. Commercially available kits, such as the SMART RACE cDNA amplification kit, can be used as appropriate for synthesizing the 5'-RACE cDNA library.

[0088] The obtained 5'-RACE cDNA library is used as a template to amplify the antibody gene by PCR. Primers for mouse antibody gene amplification can be designed based on known antibody gene sequences. These primers have different nucleotide sequences for each immunoglobulin subclass. Therefore, it is desirable to determine the subclass in advance using a commercially available kit such as the Iso Strip mouse monoclonal antibody isotyping kit (Roche Diagnostics).

[0089] Specifically, for example, when the goal is to obtain genes encoding mouse IgG, primers capable of amplifying genes encoding γ1, γ2a, γ2b, and γ3 as heavy chains, and κ and λ chains as light chains, can be used. To amplify the variable region genes of IgG, a primer that anneals to the constant region close to the variable region is generally used for the 3' side. On the other hand, for the 5' side primer, the primers included with the 5' RACE cDNA library preparation kit are used.

[0090] Using the PCR product thus amplified, an immunoglobulin consisting of a combination of heavy and light chains can be reconstituted. The binding activity of the reconstituted immunoglobulin to IL-6R can be used as an indicator to screen for the desired antibody. For example, when the goal is to obtain an antibody against IL-6R, it is even more preferable that the antibody's binding to IL-6R is specific. Antibodies that bind to IL-6R can be screened, for example, as follows: (1) A step of contacting IL-6R expressing cells with an antibody containing a V region encoded by cDNA obtained from a hybridoma, (2) A step to detect the binding of IL-6R-expressing cells to an antibody, and (3) A step of selecting an antibody that binds to IL-6R expressing cells.

[0091] Methods for detecting the binding of antibodies (including monodomain antibodies) to IL-6R-expressing cells are known. Specifically, the binding of antibodies to IL-6R-expressing cells can be detected by methods such as FACS, as mentioned earlier. Fixed specimens of IL-6R-expressing cells can be used as appropriate to evaluate the binding activity of antibodies.

[0092] As a screening method for antibodies that use binding activity as an indicator, panning using phage vectors is also suitably employed. Even when screening single-domain antibodies, screening can be appropriately performed according to the examples below. When antibody genes are obtained as libraries of heavy and light chain subclasses from polyclonal antibody-expressing cell populations, a screening method using phage vectors is advantageous. Genes encoding the variable regions of the heavy and light chains can be linked with a suitable linker sequence to form a single-chain Fv (scFv). By inserting the scFv-encoding gene into a phage vector, a phage expressing scFv on its surface can be obtained. After contact between this phage and the desired antigen, the phage bound to the antigen can be recovered, thereby recovering the DNA encoding scFv with the desired binding activity. By repeating this operation as needed, scFv with the desired binding activity can be enriched.

[0093] After obtaining the cDNA encoding the V region of the target anti-IL-6R antibody, the cDNA is digested by restriction enzymes that recognize restriction enzyme sites inserted at both ends of the cDNA. Preferred restriction enzymes recognize and digest base sequences that appear infrequently in the base sequence constituting the antibody gene. Furthermore, to insert one copy of the digested fragment into the vector in the correct orientation, insertion of a restriction enzyme that provides an adhesive end is preferable. By inserting the cDNA encoding the V region of the anti-IL-6R antibody, digested as described above, into a suitable expression vector, an antibody expression vector can be obtained. At this time, if the gene encoding the antibody constant region (C region) and the gene encoding the V region are fused in-frame, a chimeric antibody is obtained. Here, a chimeric antibody means that the constant region and the variable region originate from different sources. Therefore, in addition to heterologous chimeric antibodies such as mouse-human, human-human allologous chimeric antibodies are also included in the chimeric antibodies of this invention. A chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector that already has a constant region. Specifically, for example, a restriction enzyme recognition sequence for a restriction enzyme that digests the V region gene can be appropriately placed on the 5' end of an expression vector containing DNA encoding the desired antibody constant region (C region). A chimeric antibody expression vector is constructed by in-frame fusion of the two, which have been digested with the same combination of restriction enzymes.

[0094] To produce an anti-IL-6R monoclonal antibody, the antibody gene is incorporated into an expression vector so that it is expressed under the control of an expression regulatory region. This regulatory region for antibody expression includes, for example, enhancers and promoters. Furthermore, an appropriate signal sequence may be added to the amino terminus so that the expressed antibody is secreted extracellularly. For example, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 1) may be used as the signal sequence, but other suitable signal sequences may also be added. The expressed polypeptide is cleaved at the carboxyl terminus of the above sequence, and the cleaved polypeptide can be secreted extracellularly as a mature polypeptide. Subsequently, recombinant cells expressing DNA encoding the anti-IL-6R antibody can be obtained by transforming a suitable host cell with this expression vector.

[0095] Ligand-binding molecules One aspect of the ligand-binding molecule of the present invention is a single-domain antibody contained within the ligand-binding molecule that is capable of binding to a ligand, particularly a molecule capable of binding to a ligand in an uncleaved state. Here, "binding" usually refers to binding by interactions mainly involving non-covalent bonds such as electrostatic forces, van der Waals forces, and hydrogen bonds. Preferred examples of ligand-binding modes of the ligand-binding molecule of the present invention, but not limited to these, include antigen-antibody reactions in which an antigen-binding region, antigen-binding molecule, antibody, and antibody fragments bind to an antigen.

[0096] Furthermore, "ligand-binding capability" means that the ligand-binding molecule can bind to the ligand even if the ligand and the ligand are separate molecules, and does not mean that the ligand-binding molecule and the ligand are linked by a covalent bond. For example, the fact that the ligand and the ligand-binding molecule are covalently bonded via a linker does not mean that it is ligand-binding capability. Also, "weakening of ligand binding" means that the ability to bind is weakened. For example, if the ligand and the ligand-binding molecule are covalently bonded via a linker, the cleavage of that linker does not mean that the binding to the ligand is weakened. In this invention, as long as the ligand-binding molecule is ligand-binding capability, the ligand-binding molecule may be linked to the ligand via a linker or the like.

[0097] The ligand-binding molecule of the present invention is limited only to the fact that it binds to the ligand with a single-domain antibody contained in the molecule in an uncleaved state; any molecule with any structure can be used as long as it can bind to the target ligand with a single-domain antibody contained in the molecule in an uncleaved state.

[0098] One aspect of the ligand-binding molecule of the present invention is a polypeptide containing a cleavage site. The cleavage site can be cleaved, for example, by an enzyme, reduced by a reducing agent, or photodegraded. The cleavage site may be located at any position in the polypeptide, as long as cleavage weakens the binding of the ligand-binding molecule to the ligand. Furthermore, the polypeptide may contain one or more cleavage sites.

[0099] Furthermore, the ligand-binding molecule of the present invention exhibits weaker ligand binding (i.e., attenuated) when cleaved compared to when it is uncleaved. More specifically, the binding of the single-domain antibody in the ligand-binding molecule of the present invention to the ligand is weaker (i.e., attenuated) when the ligand-binding molecule is cleaved compared to when the ligand-binding molecule is uncleaved. The attenuation of ligand binding can be evaluated by the ligand-binding activity of the ligand-binding molecule.

[0100] Evaluation of ligand binding activity The binding activity of ligand-binding molecules to ligands can be evaluated using well-known methods such as FACS, ELISA format, ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), BIACORE method utilizing surface plasmon resonance (SPR), and BLI (Bio-Layer Interferometry) method (Octet) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010). The ALPHA screen is performed using ALPHA technology, which employs two beads, a donor and an acceptor, based on the following principle: Molecules bound to the donor bead interact with molecules bound to the acceptor bead, and an emission signal is detected only when the two beads are in close proximity. A photosensitiver within the donor bead, excited by a laser, converts surrounding oxygen into excited singlet oxygen. The singlet oxygen diffuses around the donor bead and, upon reaching the nearby acceptor bead, triggers a chemiluminescent reaction within the bead, ultimately emitting light. When the molecules bound to the donor bead and the molecules bound to the acceptor bead do not interact, the singlet oxygen produced by the donor bead does not reach the acceptor bead, and therefore no chemiluminescent reaction occurs.

[0101] For example, a biotin-labeled ligand-binding molecule is bound to a donor bead, and a ligand tagged with glutathione S-transferase (GST) is bound to an acceptor bead. In the absence of competing untagged ligand-binding molecules, the ligand-binding molecule and the ligand interact, producing a signal in the 520-620 nm range. Untagged ligand-binding molecules compete with the interaction between the tagged ligand-binding molecule and the ligand. The relative binding affinity can be determined by quantifying the decrease in fluorescence that results from this competition. Biotinylation of ligand-binding molecules such as antibodies using sulfo-NHS-biotin is a well-known method. As a method for tagging ligands with GST, a GST-fusion ligand can be expressed in cells containing a vector capable of expressing a fusion gene in which a polynucleotide encoding the ligand and a polynucleotide encoding GST are fused in frame, and then purified using a glutathione column. The obtained signals can be suitably analyzed by fitting them to a one-site competition model that utilizes nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).

[0102] When one of the substances whose interaction is to be observed (ligand) is fixed onto a gold thin film on a sensor chip, and light is shone from the back of the sensor chip so as to cause total internal reflection at the interface between the gold thin film and glass, a region of reduced reflectance intensity (SPR signal) is formed in a part of the reflected light. When the other substance whose interaction is to be observed (analyte) is flowed onto the surface of the sensor chip and the ligand and analyte bind, the mass of the immobilized ligand molecule increases, and the refractive index of the solvent on the surface of the sensor chip changes. This change in refractive index causes the position of the SPR signal to shift (conversely, when the bond dissociates, the signal position returns to its original position). The Biacore system plots the amount of the above shift, i.e., the change in mass on the sensor chip surface, on the vertical axis and displays the change in mass over time as measurement data (sensorgram). From the sensorgram curve, kinetics: the binding rate constant (ka) and the dissociation rate constant (kd) can be determined, and the dissociation constant (KD) can be determined from the ratio of these constants. Inhibition measurement methods and equilibrium value analysis methods are also suitably used in the BIACORE method. Examples of inhibition assays are described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010, and examples of equilibrium value analysis methods are described in Methods Enzymol. 2000;323:325-40.

[0103] A reduced ability of a ligand-binding molecule to bind to a ligand means, for example, that, based on the measurement method described above, the amount of ligand bound per test ligand-binding molecule is 50% or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, 15% or less, particularly preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less compared to the control ligand-binding molecule. Any desired index may be used as an indicator of binding activity, for example, the dissociation constant (KD) may be used. When using the dissociation constant (KD) as an evaluation index for binding activity, a larger dissociation constant (KD) of the test ligand-binding molecule with respect to the ligand compared to the dissociation constant (KD) of the control ligand-binding molecule with respect to the ligand indicates that the binding activity of the test ligand-binding molecule with respect to the ligand is weaker than that of the control ligand-binding molecule. A weakened ability to bind to a ligand means, for example, that the keying factor (KD) of the test ligand-binding molecule relative to the ligand is 2 times or more, preferably 5 times or more, 10 times or more, and particularly preferably 100 times or more, compared to the keying factor (KD) of the control ligand-binding molecule relative to the ligand. Examples of control ligand-binding molecules include uncleaved ligand-binding molecules.

[0104] In one embodiment of the present invention, the ligand is released from the ligand-binding molecule when a cleavage site is cleaved. Here, if the ligand is fused to a portion of the ligand-binding molecule via a linker and there is no cleavage site on the linker, the ligand will be released while still connected to that portion of the ligand-binding molecule via the linker (see, for example, Figures 2 and 4). In this way, even when the ligand is released together with a portion of the ligand-binding molecule, as long as there is a portion of the ligand-binding molecule that can no longer stably interact with the ligand, it can be said that the ligand has been released from the ligand-binding molecule.

[0105] One method for detecting the release of a ligand from a ligand-binding molecule by cleavage at a cleavage site is to detect the ligand using a ligand-detection antibody that recognizes the ligand. When the ligand-binding molecule is an antibody fragment, it is preferable that the ligand-detection antibody binds to an epitope similar to that of the ligand-binding molecule. Ligand detection using ligand-detection antibodies can be confirmed by well-known methods such as FACS, ELISA format, ALPHA screen (Amplified Luminescent Proximity Homogeneous Assay), BIACORE method utilizing surface plasmon resonance (SPR), and BLI (Bio-Layer Interferometry) method (Octet) (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010). For example, when detecting ligand release using Octet, ligand release can be detected by biotinylating a ligand-recognizing antibody, contacting it with a biosensor, and then measuring its binding to the ligand in the sample. Specifically, ligand release can be detected by measuring the amount of ligand using a ligand-detection antibody in a sample containing ligand-binding molecules and ligand before or after protease treatment, and comparing the amount of ligand detected in the sample before and after protease treatment. Furthermore, ligand release can be detected by measuring the amount of ligand using a ligand-detection antibody in a sample containing protease, ligand-binding molecules, and ligand, and in a sample containing ligand-binding molecules and ligand without protease, and comparing the amount of ligand detected in the sample with and without protease. More specifically, ligand release can be detected by the method in the embodiment of this application. When a ligand-binding molecule is fused with a ligand to form a fusion protein, ligand release can be detected by measuring the amount of ligand using a ligand-detection antibody in a sample containing the fusion protein before or after protease treatment, and comparing the amount of ligand detected in the sample before and after protease treatment. Furthermore, ligand release can be detected by measuring the amount of ligand using a ligand detection antibody in samples containing a protease and a fusion protein, and in samples containing a fusion protein but without a protease, and comparing the amount of ligand detected in the samples with and without protease. More specifically, ligand release can be detected by the method described in the present invention.

[0106] Furthermore, in embodiments where the biological activity of a ligand is inhibited upon binding to a ligand-binding molecule, the release of the ligand from the ligand-binding molecule can be detected by measuring the biological activity of the ligand in the sample. Specifically, ligand release can be detected by measuring and comparing the biological activity of the ligand in a sample containing a ligand-binding molecule and a ligand before or after protease treatment. Ligand release can also be detected by measuring and comparing the biological activity of the ligand in a sample containing a protease, a ligand-binding molecule, and a ligand, and in a sample containing a ligand-binding molecule and a ligand without a protease. When a ligand-binding molecule is fused with a ligand to form a fusion protein, ligand release can be detected by measuring and comparing the biological activity of the ligand in a sample containing the fusion protein before or after protease treatment. Ligand release can also be detected by measuring and comparing the biological activity of the ligand in a sample containing a protease and a fusion protein, and in a sample containing a fusion protein without a protease.

[0107] In one embodiment of the present invention, the cleavage site includes a protease cleavage sequence and is cleaved by a protease.

[0108] Protease cleavage sequence A protease cleavage sequence is a specific amino acid sequence that is specifically recognized by a target tissue-specific protease when a polypeptide is hydrolyzed by that protease in an aqueous solution. From the standpoint of reducing side effects, the protease cleavage sequence is preferably an amino acid sequence that is hydrolyzed with high specificity by a target tissue-specific protease that is more specifically expressed in or more specifically activated in the target tissue / cells being treated. Specific examples of protease cleavage sequences include, for example, target sequences that are specifically hydrolyzed by proteases specifically expressed in cancer tissue, inflammatory tissue-specific proteases, etc., as disclosed in International Publications WO2013 / 128194, WO2010 / 081173, WO2009 / 025846, etc. Artificially modified sequences, such as those with amino acid mutations introduced into known target sequences that are specifically hydrolyzed by proteases, can also be used. Furthermore, protease cleavage sequences identified by methods known to those skilled in the art, as described in Nature Biotechnology 19, 661 - 667 (2001), may also be used. Furthermore, naturally occurring protease cleavage sequences may also be used. For example, sequences that undergo protease cleavage in proteins whose molecular shape changes upon protease cleavage can be used, such as TGFβ changing to its latent form upon protease cleavage.

[0109] Examples of protease cleavage sequences, but not limited to these, include International Publications WO2015 / 116933, WO2015 / 048329, WO2016 / 118629, WO2016 / 179257, WO2016 / 179285, WO2016 / 179335, WO2016 / 179003, WO2016 / 046778, WO2016 / 014974, U.S. Patent Publication US2016 / 0289324, U.S. Patent Publication US2016 / 0311903, PNAS (2000) 97: 7754-7759, Biochemical Journal (2010) 426: The sequences shown in pp. 219-228 and Beilstein J Nanotechnol. (2016) 7: 364-373 can be used. As described above, the protease cleavage sequence is more preferably an amino acid sequence that is specifically hydrolyzed by a suitable target tissue-specific protease. Among amino acid sequences that are specifically hydrolyzed by target tissue-specific proteases, the following amino acid sequences are preferred. LSGRSDNH (Sequence ID: 2, MT-SP1, can be cut by uPA) PLGLAG (Sequence ID: 3, can be cut by MMP-2 and MMP-9) VPLSLTMG (Sequence ID: 4, can be cut by MMP-7) The following sequences can also be used as protease cleavage sequences. TSTSGRSANPRG (Sequence ID: 5, MT-SP1, can be cut by uPA) ISSGLLSGRSDNH (Sequence number: 6, MT-SP1, can be cut by uPA) AVGLLAPPGGLSGRSDNH (Sequence ID: 7, MT-SP1, can be cut by uPA) GAGVPMSMRGGAG (Sequence ID: 8, can be cut by MMP-1) GAGIPVSLRSGAG ​​(Sequence number: 9, can be cut by MMP-2) GPLGIAGQ (Sequence ID: 10, can be cut using MMP-2) GGPLGMLSQS (Sequence ID: 11, can be cut by MMP-2) PLGLWA (Sequence ID: 12, can be disconnected by MMP-2) GAGRPFSMIMGAG (Sequence ID: 13, can be cut by MMP-3) GAGVPLSLTMGAG (Sequence number: 14, can be cut by MMP-7) GAGVPLSLYSGAG (Sequence number: 15, can be cut by MMP-9) AANLRN (Sequence ID: 16, can be cleaved by MMP-11) AQAYVK (Sequence ID: 17, can be disconnected by MMP-11) AANYMR (Sequence ID: 18, can be cleaved by MMP-11) AAALTR (Sequence ID: 19, can be cleaved by MMP-11) AQNLMR (Sequence ID: 20, can be cleaved by MMP-11) AANYTK (Sequence ID: 21, can be cut by MMP-11) GAGPQGLAGQRGIVAG (Sequence ID: 22, can be cleaved by MMP-13) PRFKIIGG (Sequence ID: 23, cleavable by pro-urokinase) PRFRIIGG (SEQ ID NO: 24, cleavable by pro-urokinase) GAGSGRSAG (Sequence ID: 25, can be cut by uPA) SGRSA (Sequence ID: 26, can be cleaved by uPA) GSGRSA (Sequence ID: 27, can be cleaved by uPA) SGKSA (Sequence ID: 28, can be cut by uPA) SGRSS (Sequence ID: 29, can be cut by uPA) SGRRA (Sequence ID: 30, can be cut by uPA) SGRNA (Sequence ID: 31, cleavable by uPA) SGRKA (Sequence ID: 32, can be cut by uPA) QRGRSA (Sequence ID: 33, can be cleaved by tPA) GAGSLLKSRMVPNFNAG (Sequence ID: 34, cleavable by cathepsin B) TQGAAA (Sequence ID: 35, cleavable by cathepsin B) GAAAAA (Sequence ID: 36, cleavable by cathepsin B) GAGAAG (Sequence ID: 37, cleavable by cathepsin B) AAAAAG (Sequence ID: 38, cleavable by cathepsin B) LCGAAI (Sequence ID: 39, cleavable by cathepsin B) FAQALG (Sequence ID: 40, cleavable by cathepsin B) LLQANP (Sequence ID: 41, cleavable by cathepsin B) LAAANP (Sequence ID: 42, cleavable by cathepsin B) LYGAQF (Sequence ID: 43, cleavable by cathepsin B) LSQAQG (Sequence ID: 44, cleavable by cathepsin B) ASAASG (Sequence ID: 45, cleavable by cathepsin B) FLGASL (Sequence ID: 46, cleavable by cathepsin B) AYGATG (Sequence ID: 47, cleavable by cathepsin B) LAQATG (Sequence ID: 48, cleavable by cathepsin B) GAGSGVVIATVIVITAG (Sequence ID: 49, cleavable by cathepsin L) APMAEGGG (Sequence ID: 50, cleavable by meprin α and meprin β) EAQGDKII (Sequence ID: 51, cleavable by meprin α and meprin β) LAFSDAGP (Sequence ID: 52, cleavable by meprin α and meprin β) YVADAPK (Sequence ID: 53, cleavable by meprin α and meprin β) RRRRR (Sequence ID: 54, can be cleaved by Fuulin) RRRRRR (Sequence ID: 55, can be cleaved by Fuhrin) GQSSRHRRAL (Sequence ID: 56, cleavable by Fuhrin) SSRHRRALD (Sequence ID: 57) RKSSIIIRMRDVVL (Sequence ID: 58, cleavable by plasminogen) SSSFDKGKYKKGDDA (Sequence ID: 59, cleavable by staphylokinase) SSSFDKGKYKRGDDA (Sequence ID: 60, cleavable by staphylokinase) IEGR (Sequence ID: 61, can be cut by FactorIXa) IDGR (Sequence ID: 62, can be cut by FactorIXa) GGSIDGR (Sequence ID: 63, can be cut by FactorIXa) GPQGIAGQ (Sequence ID: 64, cleavable by collagenase) GPQGLLGA (Sequence ID: 65, can be cleaved by collagenase) GIAGQ (Sequence ID: 66, can be cleaved by collagenase) GPLGIAG (Sequence ID: 67, cleavable by collagenase) GPEGLRVG (Sequence ID: 68, can be cleaved by collagenase) YGAGLGVV (Sequence ID: 69, cleavable by collagenase) AGLGVVER (Sequence ID: 70, cleavable by collagenase) AGLGISST (Sequence ID: 71, can be cleaved by collagenase) EPQALAMS (Sequence ID: 72, cleavable by collagenase) QALAMSAI (Sequence ID: 73, can be cleaved by collagenase) AAYHLVSQ (Sequence ID: 74, can be cleaved by collagenase) MDAFLESS (Sequence ID: 75, can be cleaved by collagenase) ESLPVVAV (Sequence ID: 76, can be cleaved by collagenase) SAPAVESE (Sequence ID: 77, can be cleaved by collagenase) DVAQFVLT (Sequence ID: 78, can be cleaved by collagenase) VAQFVLTE (Sequence ID: 79, can be disconnected by Collagenase) AQFVLTEG (Sequence ID: 80, can be cleaved by collagenase) PVQPIGPQ (Sequence ID: 81, cleavable by collagenase) LVPRGS (Sequence ID: 82, can be cut by Thrombin) TSGSGRSANARG (Sequence ID: 170, can be cut by uPA and MT-SP1) TSQSGRSANQRG (Sequence ID: 171, can be cut using uPA and MT-SP1) TSPSGRSAYPRG (Sequence ID: 172, can be cut by uPA and MT-SP1) TSGSGRSATPRG (Sequence ID: 173, can be cleaved by uPA and MT-SP1) TSQSGRSATPRG (Sequence ID: 174, can be cleaved by uPA and MT-SP1) TSASGRSATPRG (Sequence ID: 175, can be cleaved by uPA and MT-SP1) TSYSGRSAVPRG (Sequence ID: 176, can be disconnected by uPA and MT-SP1) TSYSGRSANFRG (Sequence ID: 177, can be disconnected by uPA and MT-SP1) TSSSGRSATPRG (Sequence ID: 178, can be cut by uPA and MT-SP1) TSTTGRSASPRG (Sequence ID: 179, can be cut by uPA and MT-SP1)

[0110] The sequences shown in Table 1 can also be used as protease cleavage sequences.

[0111] [Table 1] TIFF0007909374000002.tif252150TIFF0007909374000003.tif252150TIFF00079093740 00004.tif252150TIFF0007909374000005.tif252150TIFF0007909374000006.tif252150 TIFF0007909374000007.tif252150TIFF0007909374000008.tif252150TIFF00079093740 00009.tif238151TIFF0007909374000010.tif252150TIFF0007909374000011.tif101150

[0112] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 788) In this case, X1 to X8 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0113] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 789) In this context, X1 to X8 each represent a single amino acid, where X1 is an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is A, X8 is an amino acid selected from D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0114] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 790) In this case, X1 to X8 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, F, L, M, P, Q, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is A, D, E, X8 is an amino acid selected from F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0115] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 791) In this context, X1 to X8 each represent a single amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is A, X8 is an amino acid selected from D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0116] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 792) In this context, X1 to X8 each represent a single amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, G, H, I, K, L, M, N, Q, R, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is A, X8 is an amino acid selected from D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0117] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 793) In this case, X1 to X8 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from E, F, K, M, N, P, Q, R, S, and W; X7 is A, D, E, F, G, X8 is an amino acid selected from H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0118] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 794) In this case, X1 to X8 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and X7 is an amino acid selected from Y; X8 is an amino acid selected from A, D, F, G, L, M, P, Q, V, and W; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0119] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 795) In this case, X1 to X8 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and X7 is an amino acid selected from Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, I, K, N, T and W.

[0120] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 796) In this sequence, X1 through X8 each represent a single amino acid. X1 is an amino acid selected from A, G, I, P, Q, S, and Y; X2 is an amino acid selected from K or T; X3 is G; X4 is R; X5 is S; X6 is A; X7 is an amino acid selected from H, I, and V; and X8 is an amino acid selected from H, V, and Y.

[0121] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 797) In this sequence, X1 through X8 each represent a single amino acid: X1 is Y; X2 is an amino acid selected from S and T; X3 is G; X4 is R; X5 is S; X6 is an amino acid selected from A and E; and X8 is an amino acid selected from H, P, V, and Y.

[0122] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 798) In this case, X1 to X9 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and X7 is an amino acid selected from Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0123] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 799) In this case, X1 to X9 each represent one amino acid, where X1 is an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is A, X8 is an amino acid selected from D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0124] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 800) In this case, X1 to X9 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, F, L, M, P, Q, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is A, D, E, X8 is an amino acid selected from F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0125] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 801) In this context, 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; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is A, X8 is an amino acid selected from D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0126] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 802) In this case, X1 to X9 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, G, H, I, K, L, M, N, Q, R, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is A, X8 is an amino acid selected from D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0127] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 803) In this case, X1 to X9 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from E, F, K, M, N, P, Q, R, S, and W; X7 is A, D, E, F, G, X8 is an amino acid selected from H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0128] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 804) In this case, X1 to X9 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and X7 is an amino acid selected from Y; X8 is an amino acid selected from A, D, F, G, L, M, P, Q, V, and W; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0129] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 805) In this case, X1 to X9 each represent one amino acid, where X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and X7 is an amino acid selected from Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, I, K, N, T, and W; X9 is an amino acid selected from A, G, H, I, L, and R.

[0130] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 806) In this sequence, X1 through X9 each represent a single amino acid. X1 is an amino acid selected from A, G, I, P, Q, S, and Y; X2 is an amino acid selected from K or T; X3 is G; X4 is R; X5 is S; X6 is A; X7 is an amino acid selected from H, I, and V; X8 is an amino acid selected from H, V, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0131] The following can also be used as protease cleavage sequences: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 807) In this sequence, X1 through X9 each represent a single amino acid: X1 is Y; X2 is an amino acid selected from S and T; X3 is G; X4 is R; X5 is S; X6 is an amino acid selected from A and E; 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.

[0132] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 808) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, X7 is an amino acid selected from M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0133] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 809) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, M, N, P, Q, X7 is an amino acid selected from R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0134] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 810) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, F, L, M, P, Q, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, M, N, P, Q, R, X7 is an amino acid selected from S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0135] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 811) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, M, N, P, X7 is an amino acid selected from Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0136] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 812) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, G, H, I, K, L, M, N, Q, R, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, M, N, P, X7 is an amino acid selected from Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0137] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 813) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being E, F, K, M, N, P, Q, R, X7 is an amino acid selected from S and W; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0138] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 814) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, X7 is an amino acid selected from M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, F, G, L, M, P, Q, V, and W; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0139] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 815) Among them, X1 to X11 each represent one amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, I, K, N, T, and W.

[0140] The following can also be used as the protease cleavage sequence: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (SEQ ID NO: 816) Among them, X1 to X11 each represent one amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, G, I, P, Q, S, and Y; X2 is an amino acid selected from K or T; X3 is G; X4 is R; X5 is S; X6 is A; X7 is an amino acid selected from H, I, and V; X8 is an amino acid selected from H, V, and Y.

[0141] The following can also be used as the protease cleavage sequence: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 817) In this sequence, X1 to X11 each represent a single amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being Y; X2 being an amino acid selected from S and T; X3 being G; X4 being R; X5 being S; X6 being an amino acid selected from A and E; X7 being an amino acid selected from N and V; and X8 being an amino acid selected from H, P, V, and Y.

[0142] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 818) Among them, X1 to X11 each represent one amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0143] As the protease cleavage sequence, the following can also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (SEQ ID NO: 819) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, M, N, P, X7 is an amino acid selected from Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0144] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 820) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, F, L, M, P, Q, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, M, N, P, Q, R, X7 is an amino acid selected from S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0145] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 821) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, M, N, P, X7 is an amino acid selected from Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0146] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 822) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, G, H, I, K, L, M, N, Q, R, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, M, N, P, X7 is an amino acid selected from Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0147] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 823) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being E, F, K, M, N, P, Q, R, X7 is an amino acid selected from S and W; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0148] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 824) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, X7 is an amino acid selected from M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, F, G, L, M, P, Q, V, and W; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, G, H, I, L, and R.

[0149] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 825) In this case, X1 to X11 each represent one amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 being an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 being R; X5 being an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 being A, D, E, F, H, I, K, L, X7 is an amino acid selected from M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X9 is an amino acid selected from A, D, E, F, G, I, K, N, T, and W; X9 is an amino acid selected from A, G, H, I, L, and R.

[0150] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 826) In this sequence, X1 to X11 each represent a single amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being an amino acid selected from A, G, I, P, Q, S, and Y; X2 being an amino acid selected from K or T; X3 being G; X4 being R; X5 being S; X6 being A; X7 being an amino acid selected from H, I, and V; X8 being an amino acid selected from H, V, and Y; and X9 being an amino acid selected from A, G, H, I, L, and R.

[0151] The following can also be used as protease cleavage sequences: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 827) In this sequence, X1 to X11 each represent a single amino acid, with X10 being an amino acid selected from I, T, and Y; X11 being S; X1 being Y; X2 being an amino acid selected from S and T; X3 being G; X4 being R; X5 being S; X6 being an amino acid selected from A and E; X7 being an amino acid selected from N and V; X8 being an amino acid selected from H, P, V, and Y; and X9 being an amino acid selected from A, G, H, I, L, and R.

[0152] In addition to using the above-mentioned protease cleavage sequences, new protease cleavage sequences may be obtained through screening. For example, new protease cleavage sequences can be searched for by changing the interaction between the cleavage sequence and the enzyme's active and recognized residues based on the results of crystal structure analysis of known protease cleavage sequences. Furthermore, new protease cleavage sequences can be searched for by modifying the amino acids in known protease cleavage sequences and confirming their interaction with the protease. As another example, sequences cleaved by proteases can be searched for by displaying a peptide library using in vitro display methods such as phage display or ribosome display, or by confirming the interaction with the protease using a peptide array immobilized on a chip or beads. The interaction between the protease cleavage sequence and the protease can be confirmed by observing protease cleavage in vitro or in vivo.

[0153] For example, the polypeptides containing protease cleavage sequences exemplified in Table 1 are all useful as protease substrates that are hydrolyzed by the action of proteases. The protease substrates shown in SEQ ID NOs: 788-827 and Table 1 can be used as a library for selecting substrates with properties appropriate for a given purpose, for example, when incorporating them into ligand-binding molecules. Specifically, their protease sensitivity can be evaluated in order to selectively cleave ligand-binding molecules with proteases localized in the lesion. Ligand-binding molecules bound to ligands may reach the lesion after being administered to the body and coming into contact with various proteases. Therefore, it is desirable to have sensitivity to proteases localized in the lesion while having as high a tolerance as possible to other proteases. In order to select a desirable protease cleavage sequence according to the purpose, protease resistance can be determined by comprehensively analyzing the sensitivity of each protease substrate to various proteases in advance. Based on the obtained protease resistance spectrum, protease cleavage sequences with the required sensitivity and resistance can be identified. Alternatively, ligand-binding molecules incorporating protease cleavage sequences reach the lesion not only through enzymatic action by proteases, but also through various environmental stresses such as pH changes, temperature, and redox stress. Even in response to such external factors, it is possible to select a protease cleavage sequence with desirable properties for the purpose based on information comparing the resistance of various protease substrates.

[0154] In one embodiment of the present invention, a movable linker is further added to either one or both ends of the protease cleavage sequence. The movable linker at one end of the protease cleavage sequence may be referred to as the first movable linker, and the movable linker at the other end may be referred to as the second movable linker. In a particular embodiment, the protease cleavage sequence and the movable linker include one of the following formulas. (Protease cleavage sequence) (First movable linker)-(Protease cleavage sequence) (Protease cleavage sequence)-(Second movable linker) (First movable linker)-(Protease cleavage sequence)-(Second movable linker) In this embodiment, the movable linker is preferably a peptide linker. The first movable linker and the second movable linker exist independently and optionally, and are the same or different movable linkers containing at least one flexible amino acid (such as Gly). For example, the protease cleavage sequence contains a sufficient number of residues (amino acids arbitrarily selected from Arg, Ile, Gln, Glu, Cys, Tyr, Trp, Thr, Val, His, Phe, Pro, Met, Lys, Gly, Ser, Asp, Asn, Ala, etc., especially Gly, Ser, Asp, Asn, Ala, more particularly Gly and Ser, especially Gly, etc.) so as to obtain the desired protease accessibility.

[0155] The movable linkers suitable for use at both ends of the protease cleavage sequence usually improve the protease access to the protease cleavage sequence and increase the protease cleavage efficiency. Suitable movable linkers can be easily selected, and can be selected from suitable ones of different lengths, such as from 1 amino acid (such as Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, or from 4 amino acids to 10 amino acids, from 5 amino acids to 9 amino acids, from 6 amino acids to 8 amino acids or from 7 amino acids to 8 amino acids, and also from 3 amino acids to 12 amino acids, etc. In some embodiments of the present invention, the movable linker is a peptide linker of 1 to 7 amino acids.

[0156] Examples of the movable linker include, but are not limited to, for example, glycine polymers (G)n, glycine-serine polymers (such as (GS)n, (GSGGS: SEQ ID NO: 92)n and (GGGS: SEQ ID NO: 83)n, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other movable linkers well-known in the prior art. Among these, glycine and glycine-serine polymers have attracted attention, because these amino acids are not relatively structured and are likely to function as neutral tethers between components. Examples of movable linkers made of glycine-serine polymers include, but are not limited to, the following: Ser Gly·Ser(GS) Ser·Gly(SG) Gly·Gly·Ser(GGS) Gly·Ser·Gly (GSG) Ser·Gly·Gly (SGG) Gly·Ser·Ser (GSS) Ser·Ser·Gly (SSG) Ser·Gly·Ser(SGS) Gly·Gly·Gly·Ser(GGGS, Sequence ID: 83) Gly·Gly·Ser·Gly (GGSG, Sequence ID: 84) Gly·Ser·Gly·Gly (GSGG, Sequence ID: 85) Ser·Gly·Gly·Gly (SGGG, Sequence ID: 86) Gly·Ser·Ser·Gly(GSSG, Sequence ID: 87) Gly·Gly·Gly·Gly·Ser(GGGGS, Sequence ID: 88) Gly·Gly·Gly·Ser·Gly (GGGSG, Sequence ID: 89) Gly·Gly·Ser·Gly·Gly(GGSGG, Sequence ID: 90) Gly·Ser·Gly·Gly·Gly(GSGGG, Sequence ID: 91) Gly·Ser·Gly·Gly·Ser(GSGGS, Sequence ID: 92) Ser·Gly·Gly·Gly·Gly (SGGGG, Sequence ID: 93) Gly·Ser·Ser·Gly·Gly (GSSGG, Sequence ID: 94) Gly·Ser·Gly·Ser·Gly(GSGSG, Sequence ID: 95) Ser·Gly·Gly·Ser·Gly (SGGSG, Sequence ID: 96) Gly·Ser·Ser·Ser·Gly(GSSSG, Sequence ID: 97) Gly·Gly·Gly·Gly·Gly·Ser(GGGGGS, Sequence ID: 98) Ser·Gly·Gly·Gly·Gly·Gly(SGGGGG, Sequence ID: 99) Gly·Gly·Gly·Gly·Gly·Gly·Ser(GGGGGGS, Sequence ID: 100) Ser·Gly·Gly·Gly·Gly·Gly·Gly(SGGGGGG, Sequence ID: 101) (Gly·Gly·Gly·Gly·Ser(GGGGS, Sequence ID: 88))n (Ser·Gly·Gly·Gly·Gly(SGGGG, Sequence ID: 93))n Examples include [n is an integer greater than or equal to 1]. However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0157] In some embodiments of the present invention, the cleavage site / protease cleavage sequence in the ligand-binding molecule is cleaved, thereby fragmenting the monodomain antibody in the ligand-binding molecule and reducing its binding to the ligand.

[0158] In one embodiment of the present invention, the cleavage site / protease cleavage sequence is introduced into a single-domain antibody in a ligand-binding molecule. In some more specific embodiments, the cleavage site / protease cleavage sequence is introduced at the position of residues forming a loop structure or residues close to a loop structure in the single-domain antibody. The loop structure in a single-domain antibody refers to a portion of the single-domain antibody that does not form secondary structures such as α-helices or β-sheets.

[0159] In embodiments where the monodomain antibody is VHH or a monodomain VH antibody, the positions of the residues forming the loop structure and the residues close to the loop structure are specifically: monodomain antibody amino acids 7 (Kabat numbering) to 17 (Kabat numbering), amino acids 12 (Kabat numbering) to 17 (Kabat numbering), amino acids 31 (Kabat numbering) to 35b (Kabat numbering), amino acids 40 (Kabat numbering) to 47 (Kabat numbering), amino acids 50 (Kabat numbering) to 65 (Kabat numbering) This can refer to the range from amino acid 55 (Kabat numbering) to amino acid 69 (Kabat numbering), from amino acid 73 (Kabat numbering) to amino acid 79 (Kabat numbering), from amino acid 83 (Kabat numbering) to amino acid 89 (Kabat numbering), from amino acid 95 (Kabat numbering) to amino acid 99 (Kabat numbering), from amino acid 95 (Kabat numbering) to amino acid 102 (Kabat numbering), and from amino acid 101 (Kabat numbering) to amino acid 113 (Kabat numbering).

[0160] In embodiments where the monodomain antibody is VHH or a monodomain VH antibody, the cleavage site / protease cleavage sequence is introduced at one or more positions within one or more sequences selected from the following sequences of the monodomain antibody: The sequences of single-domain antibodies from amino acid 7 (Kabat numbering) to amino acid 17 (Kabat numbering), the sequences of single-domain antibodies from amino acid 12 (Kabat numbering) to amino acid 17 (Kabat numbering), the sequences of single-domain antibodies from amino acid 31 (Kabat numbering) to amino acid 35b (Kabat numbering), the sequences of single-domain antibodies from amino acid 40 (Kabat numbering) to amino acid 47 (Kabat numbering), the sequences of single-domain antibodies from amino acid 50 (Kabat numbering) to amino acid 65 (Kabat numbering), and from amino acid 55 (Kabat numbering) Sequences up to amino acid 69 (Kabat numbering), sequences from amino acid 73 (Kabat numbering) to amino acid 79 (Kabat numbering) of single-domain antibodies, sequences from amino acid 83 (Kabat numbering) to amino acid 89 (Kabat numbering) of single-domain antibodies, sequences from amino acid 95 (Kabat numbering) to amino acid 99 (Kabat numbering) of single-domain antibodies, sequences from amino acid 95 (Kabat numbering) to amino acid 102 (Kabat numbering) of single-domain antibodies, sequences from amino acid 101 (Kabat numbering) to amino acid 113 (Kabat numbering) of single-domain antibodies.

[0161] In embodiments where the monodomain antibody is a monodomain VL antibody, the positions of the residues forming the loop structure and the residues close to the loop structure can specifically refer to the ranges from amino acid 7 (Kabat numbering) to amino acid 19 (Kabat numbering) of the monodomain antibody, from amino acid 24 (Kabat numbering) to amino acid 34 (Kabat numbering), from amino acid 39 (Kabat numbering) to amino acid 46 (Kabat numbering), from amino acid 49 (Kabat numbering) to amino acid 62 (Kabat numbering), from amino acid 50 (Kabat numbering) to amino acid 56 (Kabat numbering), from amino acid 89 (Kabat numbering) to amino acid 97 (Kabat numbering), and from amino acid 96 (Kabat numbering) to amino acid 107 (Kabat numbering).

[0162] In this embodiment, where the single-domain antibody is a single-domain VL antibody, the cleavage site / protease cleavage sequence is introduced at one or more positions within one or more sequences selected from the following sequences of the single-domain antibody: Sequences of single-domain antibodies from amino acid 7 (Kabat numbering) to amino acid 19 (Kabat numbering), sequences of single-domain antibodies from amino acid 24 (Kabat numbering) to amino acid 34 (Kabat numbering), sequences of single-domain antibodies from amino acid 39 (Kabat numbering) to amino acid 46 (Kabat numbering), sequences of single-domain antibodies from amino acid 49 (Kabat numbering) to amino acid 62 (Kabat numbering), sequences of single-domain antibodies from amino acid 50 (Kabat numbering) to amino acid 56 (Kabat numbering), sequences of single-domain antibodies from amino acid 89 (Kabat numbering) to amino acid 97 (Kabat numbering), and sequences of single-domain antibodies from amino acid 96 (Kabat numbering) to amino acid 107 (Kabat numbering).

[0163] Multiple cleavage sites / protease cleavage sequences can be provided within a ligand-binding molecule; for example, they can be provided at multiple locations within a single-domain antibody in the ligand-binding molecule.

[0164] In some embodiments of the present invention, the biological activity of a ligand is inhibited by binding to an uncleaved ligand-binding molecule. The embodiments in which the biological activity of a ligand is inhibited are not limited, but examples include embodiments in which the binding of an uncleaved ligand-binding molecule to a ligand substantially or significantly interferes with or competes with the binding of the ligand to its binding partner. When an antibody or fragment thereof having ligand-neutralizing activity is used as the ligand-binding molecule, the biological activity of the ligand can be inhibited by the ligand-binding molecule exerting its neutralizing activity upon binding to the ligand.

[0165] In one embodiment of the present invention, it is preferable that the uncleaved ligand-binding molecule can sufficiently neutralize the biological activity of the ligand by binding to it. That is, it is preferable that the biological activity of the ligand bound to the uncleaved ligand-binding molecule is lower than the biological activity of the ligand not bound to the uncleaved ligand-binding molecule. Although not limited thereto, for example, the biological activity of the ligand bound to the uncleaved ligand-binding molecule may be 90% or less, preferably 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, particularly preferably 20% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less compared to the biological activity of the ligand not bound to the uncleaved ligand-binding molecule. By sufficiently neutralizing the biological activity of the ligand, it is expected that when the ligand-binding molecule is administered, it will be possible to prevent the ligand from exerting its biological activity before reaching the target tissue.

[0166] In one embodiment of the present invention, the binding activity of the cleaved ligand-binding molecule to the ligand is preferably lower than the binding activity of the ligand's in vivo natural binding partner (e.g., a natural receptor for the ligand). However, the binding activity of the cleaved ligand-binding molecule to the ligand is preferably 90% or less, preferably 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, particularly preferably 20% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less compared to the amount of ligand binding between the in vivo natural binding partner (per unit binding partner). The indicator of binding activity may be any desired indicator as appropriate, for example, the dissociation constant (KD) may be used. When using the key-to-dissociation (KD) as an indicator for evaluating binding activity, a larger KD of the cleaved ligand-binding molecule relative to the ligand compared to the KD of the natural binding partner in vivo indicates that the binding activity of the cleaved ligand-binding molecule relative to the ligand is weaker than that of the natural binding partner in vivo. The KD of the cleaved ligand-binding molecule relative to the ligand is, for example, 1.1 times or more, preferably 1.5 times or more, 2 times or more, 5 times or more, 10 times or more, and particularly preferably 100 times or more, compared to the KD of the natural binding partner in vivo. By having low binding activity, or almost no binding activity, after cleavage, it is expected that the ligand-binding molecule will release the ligand and prevent it from rebinding to another ligand molecule.

[0167] It is desirable that the suppressed biological activity of the ligand be restored after the ligand-binding molecule is cleaved. It is also desirable that the ligand-binding molecule's inhibitory function on ligand activity be weakened by the reduced binding of the cleaved ligand-binding molecule to the ligand. Those skilled in the art can confirm the biological activity of a ligand by known methods, such as detecting the binding of the ligand to its binding partner.

[0168] In some embodiments of the present invention, the uncleaved ligand-binding molecule has a longer half-life than the monodomain antibody alone. Examples of embodiments that make the blood half-life of the ligand-binding molecule longer than that of the monodomain antibody alone include, but are not limited to, increasing the molecular weight of the ligand-binding molecule, fusing the monodomain antibody with a moiety that has FcRn binding ability, fusing the monodomain antibody with an albumin-binding ability, or fusing the monodomain antibody with a PEGylated moiety.

[0169] In this invention, it is preferable to compare the half-lives of a single-domain antibody and a ligand-binding molecule using the half-lives in human blood. If it is difficult to measure the blood half-life in humans, the blood half-life in humans can be predicted based on the blood half-life in mice (e.g., normal mice, human antigen-expressing transgenic mice, human FcRn-expressing transgenic mice, etc.) or monkeys (e.g., cynomolgus monkeys, etc.).

[0170] One embodiment of extending the blood half-life of a ligand-binding molecule beyond that of a single-domain antibody is to increase the molecular weight of the ligand-binding molecule. In a preferred embodiment, the molecular weight of the ligand-binding molecule is 60 kDa or greater. Molecules with a molecular weight of 60 kDa or greater are generally less likely to be cleared by the kidneys when present in the blood, and therefore likely to have a longer blood half-life (see J Biol Chem. 1988 Oct 15;263(29):15064-70).

[0171] One embodiment of extending the blood half-life of a ligand-binding molecule beyond that of a single-domain antibody involves fusing a single-domain antibody with a region capable of binding to FcRn. The region capable of binding to FcRn typically has an FcRn-binding domain. The FcRn-binding domain refers to the region capable of binding to FcRn, and any structure can be used as long as it is capable of binding to FcRn. The transport region, including the FcRn binding domain, can be taken up into cells via the FcRn salvage pathway and then returned to the plasma. For example, the relatively long retention time (slow disappearance) of IgG molecules in plasma is due to the function of FcRn, which is known as a salvage receptor for IgG molecules. IgG molecules taken up into endosomes by pinocytosis bind to FcRn expressed in endosomes under acidic conditions. IgG molecules that do not bind to FcRn proceed to lysosomes and are degraded there, but IgG molecules that do bind to FcRn migrate to the cell surface and dissociate from FcRn under neutral conditions in plasma, returning to the plasma. The FcRn binding region is preferably a region that directly binds to FcRn. A preferred example of an FcRn binding region is the Fc region of an antibody. However, regions that can bind to polypeptides that have the ability to bind to FcRn, such as albumin and IgG, can indirectly bind to FcRn via albumin or IgG, so the FcRn binding region in the present invention may be a region that binds to such polypeptides that have the ability to bind to FcRn.

[0172] The binding activity of the FcRn binding domain in the present invention to FcRn, particularly human FcRn, can be measured by methods known to those skilled in the art, as described in the section on binding activity, and the conditions can be appropriately determined by those skilled in the art. The binding activity to human FcRn can be evaluated as KD (Dissociation constant), apparent KD (Apparent dissociation constant), dissociation rate (kd), or apparent dissociation rate (kd). These can be measured by methods known to those skilled in the art. For example, Biacore (GE Healthcare), scatchard plots, flow cytometers, etc., can be used.

[0173] The conditions for measuring the binding activity of the FcRn binding region to FcRn can be appropriately selected by those skilled in the art and are not particularly limited. For example, it can be measured under conditions of MES buffer and 37°C, as described in WO2009 / 125825. Furthermore, the measurement of the binding activity of the FcRn binding region of the present invention to FcRn can be performed by methods known to those skilled in the art, for example, by using Biacore (GE Healthcare). The binding activity between the FcRn binding region and FcRn can be evaluated by flowing FcRn, the FcRn binding region, or the transport portion containing the FcRn binding region as an analyte to the FcRn binding region, the transport portion containing the FcRn binding region, or a tip with immobilized FcRn, respectively.

[0174] The binding affinity between the FcRn binding region and FcRn may be evaluated at any pH between pH 4.0 and pH 6.5 as the pH used for measurement conditions. Preferably, a pH between pH 5.8 and pH 6.0, which is close to the pH in early endosomes in vivo, is used to determine the binding affinity between the FcRn binding region and human FcRn. The binding affinity between the FcRn binding region and FcRn may be evaluated at any temperature between 10°C and 50°C as the temperature used for measurement conditions. Preferably, a temperature between 15°C and 40°C is used to determine the binding affinity between the FcRn binding region and human FcRn. More preferably, any temperature between 20°C and 35°C, such as any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C, is also used to determine the binding affinity between the FcRn binding region and FcRn. The temperature of 25°C is a non-limiting example of an embodiment of the present invention.

[0175] One example of an FcRn binding region, though not limited to this, is the Fc region of an IgG antibody. When using the Fc region of an IgG antibody, the type is not limited, and it is possible to use Fc regions of IgG1, IgG2, IgG3, IgG4, etc. For example, it is possible to use an Fc region containing one sequence selected from the amino acid sequences shown in SEQ ID NOs: 103, 104, 105, and 106.

[0176] Furthermore, not only the Fc region of natural IgG antibodies, but also modified Fc regions with one or more amino acid substitutions can be used, as long as they retain FcRn binding ability. For example, EU numbering in the IgG antibody Fc region: 237th, 238th, 239th, 248th, 250th, 252nd, 254th, 255th, 256th, 257th, 258th, 265th, 270th, 286th, 289th, 297th, 298th, 303rd, 305th, 307th, 308th, 309th, 311th, 312th, 314th It is possible to use a modified Fc region containing an amino acid sequence in which at least one amino acid selected from positions 315, 317, 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 is substituted with another amino acid.

[0177] More specifically, EU numbering in the Fc region of IgG antibodies Amino acid substitution where Gly at position 237 is replaced with Met. An amino acid substitution where the 238th Pro is replaced with Ala. An amino acid substitution where Ser at position 239 is replaced with Lys. An amino acid substitution where Lys at position 248 is replaced with Ile. Amino acid substitutions that replace the 250th Thr with Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr. Amino acid substitutions that replace Met at position 252 with Phe, Trp, or Tyr. An amino acid substitution where Ser at position 254 is replaced with Thr. Amino acid substitution where Arg at position 255 is replaced with Glu, Amino acid substitutions that replace the 256th Thr with Asp, Glu, or Gln. Amino acid substitutions that replace the 257th Pro with Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val. An amino acid substitution in which Glu at position 258 is replaced with His. An amino acid substitution in which Asp at position 265 is replaced with Ala. An amino acid substitution where Asp at position 270 is replaced with Phe. Amino acid substitutions that replace the 286th Asn with Ala or Glu. Amino acid substitution that replaces the 289th Thr with His, An amino acid substitution in which the 297th Asn is replaced with Ala. An amino acid substitution in which Ser at position 298 is replaced with Gly. An amino acid substitution in which the 303rd Val is replaced with Ala. An amino acid substitution in which the 305th Val is replaced with Ala. Amino acid substitutions that replace the 307th Thr with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr. Amino acid substitutions that replace the 308th Val with Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr. Amino acid substitutions that replace the 309th Leu or Val with Ala, Asp, Glu, Pro, or Arg. Amino acid substitutions that replace the 311th Gln with Ala, His, or Ile. Amino acid substitutions that replace Asp at position 312 with Ala or His. Amino acid substitutions that replace the 314th Leu with Lys or Arg. Amino acid substitutions that replace the 315th Asn with Ala or His, An amino acid substitution in which Lys at position 317 is replaced with Ala. Amino acid substitution where Asn at position 325 is replaced with Gly. Amino acid substitution where Ile at position 332 is replaced with Val. An amino acid substitution where Lys at position 334 is replaced with Leu. An amino acid substitution where the 360th Lys is replaced with His. An amino acid substitution in which Asp at position 376 is replaced with Ala. An amino acid substitution where Glu at position 380 is replaced with Ala. An amino acid substitution in which Glu at position 382 is replaced with Ala. Amino acid substitutions that replace the 384th Asn or Ser with Ala, Amino acid substitutions that replace the 385th Gly with Asp or His, Amino acid substitution where Gln at position 386 is replaced with Pro. An amino acid substitution where Pro at position 387 is replaced with Glu. Amino acid substitutions that replace the 389th Asn with Ala or Ser. An amino acid substitution in which Ser at position 424 is replaced with Ala. Amino acid substitutions that replace the 428th Met with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr. An amino acid substitution where His at position 433 is replaced with Lys. Amino acid substitutions that replace the 434th Asn with Ala, Phe, His, Ser, Trp, or Tyr, and Amino acid substitutions that replace Tyr or Phe at position 436 with His It is possible to use a modified Fc region that includes at least one amino acid substitution selected from the above.

[0178] From another perspective, EU numbering in the IgG antibody Fc region. Met at amino acid position 237, Ala at the 238th amino acid, Lys at the 239th amino acid, Ile at the 248th amino acid, Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr at the 250th amino acid, Phe, Trp, or Tyr at the 252nd amino acid, Thr at amino acid 254, Glu at the 255th amino acid, Asp, Glu, or Gln at the 256th amino acid, Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val at amino acid position 257 His at the 258th amino acid, Ala at the 265th amino acid, Phe at amino acid 270, Ala or Glu at the 286th amino acid, His at the 289th amino acid, Ala at the 297th amino acid, Gly at the 298th amino acid, Ala at amino acid position 303, Ala at amino acid position 305, Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr at amino acid position 307, Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr at amino acid position 308, Ala, Asp, Glu, Pro, or Arg at amino acid position 309, Ala, His, or Ile at the 311th amino acid, Ala or His at the 312th amino acid, Lys or Arg at the 314th amino acid, Ala or His at the 315th amino acid, Ala at amino acid position 317, Gly at amino acid position 325, Val at amino acid position 332, Leu at amino acid position 334, His at amino acid position 360, Ala at amino acid position 376, Ala at amino acid position 380, Ala at amino acid position 382, Ala at amino acid position 384, Asp or His at the 385th amino acid, Pro at amino acid 386, Glu at amino acid position 387, Ala or Ser at the 389th amino acid, Ala at amino acid position 424, Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr at amino acid position 428, Lys at amino acid position 433, Ala, Phe, His, Ser, Trp, or Tyr at amino acid position 434, and His at amino acid position 436 It is possible to use an Fc region containing at least one amino acid selected from the following.

[0179] Another embodiment of extending the blood half-life of a ligand-binding molecule beyond that of a single-domain antibody involves fusing a single-domain antibody with an albumin-binding moiety. Albumin is not excreted by the kidneys and has FcRn-binding properties, resulting in a long blood half-life of 17-19 days (J Clin Invest. 1953 Aug; 32(8): 746-768). Therefore, proteins bound to albumin become bulkier and can indirectly bind to FcRn, leading to an increased blood half-life, as reported (Antibodies 2015, 4(3), 141-156).

[0180] Furthermore, one embodiment of extending the blood half-life of a ligand-binding molecule beyond that of a single-domain antibody involves fusing a single-domain antibody with a PEGylated portion. It is believed that PEGylation increases the bulk of the protein and simultaneously suppresses its degradation by proteases in the blood, thereby extending the protein's blood half-life (J Pharm Sci. 2008 Oct;97(10):4167-83).

[0181] In some embodiments of the present invention, the ligand-binding molecule includes the antibody Fc region. In one specific embodiment, the ligand-binding molecule includes the CH2 and CH3 domains of a human IgG antibody. In another specific embodiment, the ligand-binding molecule includes a portion extending from Cys226 or Pro230 of the human IgG1 antibody heavy chain to the carboxyl terminus of the heavy chain. However, the lysine (Lys447) or glycine-lysine (Gly446-Lys447) at the C-terminus of the Fc region may or may not be present.

[0182] Ligand In this specification, the term “ligand” refers to a bioactive molecule. Bioactive molecules typically function by interacting with receptors on the cell surface, thereby biologically stimulating, inhibiting, or otherwise modulating them, and these are usually thought to be involved in intracellular signaling pathways that harbor such receptors.

[0183] In this specification, the term "ligand" encompasses any desired molecule that exerts biological activity by interacting with a biomolecule. For example, a ligand not only means a molecule that interacts with a receptor, but also a molecule that exerts biological activity by interacting with that molecule. For instance, receptors that interact with such molecules, or their binding fragments, are also included in the definition of ligand. For example, the ligand-binding site of a protein known as a receptor, or a protein containing a site on which such a receptor interacts with other molecules, are included in the definition of ligand in this invention. Specifically, soluble receptors, soluble fragments of receptors, extracellular domains of transmembrane receptors, and polypeptides containing these are included in the definition of ligand in this invention.

[0184] The ligands of the present invention can typically exert desired biological activity by binding to one or more binding partners. The binding partners of the ligand can be extracellular, intracellular, or transmembrane proteins. In one embodiment, the binding partner of the ligand is an extracellular protein, such as a soluble receptor. In another embodiment, the binding partner of the ligand is a membrane-bound receptor. The ligands of the present invention can specifically bind to their binding partners with dissociation constants (KD) of 10 μM, 1 μM, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 500 pM, 400 pM, 350 pM, 300 pM, 250 pM, 200 pM, 150 pM, 100 pM, 50 pM, 25 pM, 10 pM, 5 pM, 1 pM, 0.5 pM, or 0.1 pM or less.

[0185] Examples of biologically active molecules include, but are not limited to, cytokines, chemokines, polypeptide hormones, growth factors, apoptosis-inducing factors, PAMPs, DAMPs, nucleic acids, or fragments thereof. In detailed embodiments, interleukins, interferons, hematopoietic factors, the TNF superfamily, chemokines, cell growth factors, the TGF-β family, myokines, adipokines, or neurotrophic factors may be used as ligands. In more detailed embodiments, ligands may include CXCL10, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFN-α, IFN-β, IFN-g, MIG, I-TAC, RANTES, MIP-1a, MIP-1b, IL-1R1 (Interleukin-1 receptor, type I), IL-1R2 (Interleukin-1 receptor, type II), IL-1RAcP (Interleukin-1 receptor accessory protein), or IL-1Ra (protein accession No. NP_776214, mRNA accession No. NM_173842.2).

[0186] Chemokines are a family of homogeneous serum proteins between 7 and 16 kDa, originally characterized by their ability to induce leukocyte migration. Most chemokines possess four characteristic cysteine ​​(Cys) and are classified into the chemokine classes CXC, or alpha; CC, or beta; C, or gamma; and CX3C, or delta, based on the motif indicated by the first two cysteines. Two disulfide bonds are formed between the first and third cysteines, and between the second and fourth cysteines. Generally, disulfide crosslinking is considered necessary, and Clark-Lewis and collaborators reported that disulfide bonds are decisive to chemokine activity, at least for CXCL10 (Clark-Lewis et al., J. Biol. Chem. 269:16075-16081, 1994). The only exception to the rule of having four cysteine ​​residues is lymphotactin, which has only two. Therefore, lymphotactin manages to maintain its functional structure with only one disulfide bond. Furthermore, the CXC or alpha subfamilies are classified into two groups based on the presence of an ELR motif (Glu-Leu-Arg) preceding the first cysteine: ELR-CXC chemokines and non-ELR-CXC chemokines (see, for example, Clark-Lewis, above, and Belperio et al., "CXC Chemokines in Angiogenesis," J. Leukoc. Biol. 68:1-8, 2000).

[0187] Interferon-inducible protein 10 (IP-10 or CXCL10) is induced by interferon-γ and TNF-α and produced by keratinocytes, endothelial cells, fibroblasts, and monocytes. IP-10 is thought to play a role in the recruitment of activated T cells to sites of tissue inflammation (Dufour, et al., "IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking," J Immunol., 168:3195-204, 2002). Furthermore, IP-10 may play a role in hypersensitivity reactions. Furthermore, it may also play a role in the development of inflammatory demyelinating neuropathies (Kieseier, et al., "Chemokines and chemokine receptors in inflammatory demyelinating neuropathies: a central role for IP-10," Brain 125:823-34, 2002).

[0188] Studies have shown that IP-10 may be useful for stem cell engraftment following transplantation (Nagasawa, T., Int. J. Hematol. 72:408-11, 2000), stem cell recruitment (Gazitt, Y., J. Hematother Stem Cell Res 10:229-36, 2001; Hattori et al., Blood 97:3354-59, 2001), and enhancement of antitumor immunity (Nomura et al., Int. J. Cancer 91:597-606, 2001; Mach and Dranoff, Curr. Opin. Immunol. 12:571-75, 2000). For example, the biological activity of chemokines has been discussed in reports known to those skilled in the art (Bruce, L. et al., "Radiolabeled Chemokine binding assays," Methods in Molecular Biology (2000) vol. 138, pp129-134; Raphaele, B. et al., "Calcium Mobilization," Methods in Molecular Biology (2000) vol. 138, pp143-148; Paul D. Ponath et al., "Transwell Chemotaxis," Methods in Molecular Biology (2000) vol. 138, pp113-120 Humana Press. Totowa, New Jersey).

[0189] Examples of CXCL10's biological activities include binding to the CXCL10 receptor (CXCR3), CXCL10-induced calcium facilitation, CXCL10-induced cell chemotaxis, binding of CXCL10 to glycosaminoglycans, and CXCL10 oligomerization. Methods for measuring the biological activity of CXCL10 include measuring the cell migration activity of CXCL10, the Reporter assay using CXCR3 stable expression cell lines (see PLoS One. 2010 Sep 13;5(9):e12700.), and PathHunter, which utilizes B-Arrestin recruitment induced in the early stages of GPCR signaling. TM Examples include the β-Arrestin recruitment assay.

[0190] Interleukin-12 (IL-12) is a heterodimer cytokine consisting of disulfide-linked glycosyl polypeptide chains of 30 and 40 kD. Cytokines are synthesized and secreted by antigen-presenting cells, including dendritic cells, monocytes, macrophages, B cells, Langerhans cells, and keratinocytes, as well as natural killer (NK) cells. IL-12 mediates various biological processes and has been referred to as an NK cell stimulating factor (NKSF), a T cell stimulating factor, a cytotoxic T lymphocyte maturation factor, and an EBV-transformed B cell lineage factor.

[0191] Interleukin-12 can bind to IL-12 receptors expressed on the cytoplasmic membrane of cells (e.g., T cells, NK cells), thereby altering (e.g., initiating, inhibiting) biological processes. For example, the binding of IL-12 to the IL-12 receptor stimulates the proliferation of pre-activated T and NK cells, enhances the cytolytic activity of cytotoxic T cells (CTLs), NK cells, and LAK (lymphokine-activated killer) cells, induces the production of γ interferon (IFNγ) by T and NK cells, and induces the differentiation of naive Th0 cells into Th1 cells that produce IFNγ and IL-2. In particular, IL-12 is absolutely essential for the generation of cytolytic cells (e.g., NK, CTLs) and for establishing cellular immune responses (e.g., Th1 cell-mediated immune responses). Thus, IL-12 is absolutely crucial in the generation and regulation of both prophylactic immunity (e.g., eradication of infectious diseases) and pathological immune responses (e.g., autoimmunity).

[0192] Methods for measuring the biological activity of IL-12 include measuring the cell proliferation activity of IL-12, STAT4 reporter assays, IL-12-induced cell activation (cell surface marker expression, cytokine production, etc.), and IL-12-induced promotion of cell differentiation.

[0193] The protein Programmed Death 1 (PD-1) is an inhibitory member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells (Okazaki et al. (2002) Curr. Opin. Immunol. 14:391779-82, Bennett et al. (2003) J Immunol 170:711-8). CD28 and ICOS, the first members of this family, were discovered through their functional effects on increased T cell proliferation after the addition of monoclonal antibodies (Hutloff et al. (1999) Nature 397:263-266, Hansen et al. (1980) Immunogenics 10:247-260). PD-1 was discovered by screening for different expression levels in apoptotic cells (Ishida et al. (1992) EMBO J. 11:3887-95). Other members of the family, CTLA-4 and BTLA, were discovered by screening for different expression levels in cytotoxic T lymphocytes and TH1 cells, respectively. CD28, ICOS, and CTLA-4 all have unpaired cysteine ​​residues, which enable homodimerization. In contrast, PD-1 is thought to exist as a monomer and does not have the unpaired cysteine ​​characteristic of other CD28 family members.

[0194] The PD-1 gene is a 55 kDa type I transmembrane protein encoding a gene that is part of the Ig gene superfamily. PD-1 contains a membrane-proximal immunoreceptor tyrosine inhibitor motif (ITIM) and a membrane-distal tyrosine-based switch motif (ITSM). PD-1 is structurally similar to CTLA-4 but lacks the MYPPPY motif (SEQ ID NO: 102), which is important for B7-1 and B7-2 binding. Two ligands for PD-1, PD-L1 and PD-L2, have been identified and have been shown to negatively regulate T cell activation upon binding to PD-1 (Freeman et al. (2000) J Exp Med 192:1027-34, Latchman et al. (2001) Nat Immunol 2:261-8, Carter et al. (2002) Eur J Immunol 32:634-43). Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1 but not to other CD28 family members. PD-L1, one ligand for PD-1, is abundant in various human cancers (Dong et al. (2002) Nat. Med. 8:787-9). The interaction between PD-1 and PD-L1 results in a decrease in tumor-infiltrating lymphocytes, reduced T cell receptor-mediated proliferation, and immune evasion by cancer cells (Dong et al. (2003) J. Mol. Med. 81:281-7, Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314, Konishi et al. (2004) Clin. Cancer Res. 10:5094-100). Immunosuppression can be reversed by inhibiting the local interaction between PD-L1 and PD-1, and the effect is additive when the interaction between PD-L2 and PD-2 is similarly inhibited (Iwai et al. (2002) Proc. Nat' l. Acad. Sci. USA 99:12293-7, Brown et al. (2003) J. Immunol. 170:1257-66).

[0195] PD-1 is an inhibitory member of the CD28 family expressed on activated B cells, T cells, and myeloid cells. PD-1-deficient animals develop a variety of autoimmune phenotypes, including autoimmune cardiomyopathy and lupus-like syndrome with arthritis and nephritis (Nishimura et al. (1999) Immunity 11:141-51, Nishimura et al. (2001) Science 291:319-22). Furthermore, PD-1 has been shown to play an important role in autoimmune encephalomyelitis, systemic lupus erythematosus, graft-versus-host disease (GVHD), type 1 diabetes mellitus, and rheumatoid arthritis (Salama et al. (2003) J Exp Med 198:71-78, Prokunia and Alarcon-Riquelme (2004) Hum Mol Genet 13:R143, Nielsen et al. (2004) Lupus 13:510). In mouse B-cell tumor lines, PD-1 ITSM is BCR-mediated Ca 2+ It has been revealed that this process is essential for inhibiting tyrosine phosphorylation of downstream effector molecules (Okazaki et al. (2001) PNAS 98:13866-71).

[0196] In some embodiments of the present invention, the ligand is a cytokine. Cytokines are a family of secreted cell signaling proteins involved in immunomodulatory and inflammatory processes, secreted by glial cells in the nervous system and numerous cells in the immune system. Cytokines can be classified as proteins, peptides, or glycoproteins and encompass a large and diverse family of regulators. Cytokines bind to cell surface receptors and induce intracellular signaling, which can lead to the regulation of enzyme activity, upregulation or downregulation of several genes and their transcription factors, or feedback inhibition. In some embodiments, the cytokines of the present invention include immunomodulatory factors such as interleukins (ILs) and interferons (IFNs). Suitable cytokines may include proteins derived from one or more of the following types: the four α-helix bundle families (including the IL-2 subfamily, the IFN subfamily, and the IL-10 subfamily); the IL-1 family (including IL-1 and IL-8); and the IL-17 family. Cytokines may also include those classified as type 1 cytokines that enhance cellular immune responses (e.g., IFN-γ, TGF-β, etc.) or type 2 cytokines that favor antibody responses (e.g., IL-4, IL-10, IL-13, etc.).

[0197] In some embodiments of the present invention, the ligand is a chemokine. Chemokines generally act as chemotaxis, recruiting immune effector cells to chemokine expression sites. This is considered useful for expressing specific chemokine genes, for example, together with cytokine genes, for the purpose of recruiting other immune system components to the treatment site. Such chemokines include CXCL10, RANTES, MCAF, MIP1-α, and MIP1-β. Those skilled in the art will recognize that certain cytokines also have chemotaxis and can be classified under the term chemokines.

[0198] Furthermore, in some embodiments of the present invention, modified cytokines, chemokines, etc. (e.g., Annu Rev Immunol. 2015;33:139-67.) or fusion proteins containing them (e.g., Stem Cells Transl Med. 2015 Jan; 4(1): 66-73.) can be used as ligands.

[0199] In some embodiments of the present invention, the ligand is selected from CXCL10, PD-1, IL-12, IL-6R, IL-1R1, IL-1R2, IL-1RAcP, and IL-1Ra. The CXCL10, PD-1, IL-12, IL-6R, IL-1R1, IL-1R2, IL-1RAcP, and IL-1Ra may have the same sequence as the naturally occurring CXCL10, PD-1, IL-12, IL-6R, IL-1R1, IL-1R2, IL-1RAcP, and IL-1Ra, or they may be modified compounds that have a different sequence from the naturally occurring CXCL10, PD-1, IL-12, IL-6R, IL-1R1, IL-1R2, IL-1RAcP, and IL-1Ra but retain physiological activity while having the corresponding naturally occurring ligand. To obtain ligand variants, the ligand sequence may be artificially modified for various purposes, preferably by making modifications that prevent protease cleavage (protease-resistant).

[0200] Examples of ligand-binding molecules In some embodiments of the present invention, the ligand-binding molecule comprises only a single-domain antibody into which a protease cleavage sequence has been introduced. In some other embodiments of the present invention, the ligand-binding molecule comprises an Fc region. When using the Fc region of an IgG antibody, the type is not limited, and Fc regions such as IgG1, IgG2, IgG3, and IgG4 can be used. For example, an Fc region containing one sequence selected from the amino acid sequences shown in SEQ ID NOs: 103, 104, 105, and 106, or modified Fc region variants of these Fc regions can be used. Furthermore, in some embodiments of the present invention, the ligand-binding molecule comprises the antibody constant region.

[0201] In some embodiments of the present invention, the ligand-binding molecule is a fusion protein of a monodomain antibody into which a protease cleavage sequence has been introduced and an Fc region. In a more specific embodiment, the ligand-binding molecule comprises a series of peptide chains consisting of a monodomain antibody into which a protease cleavage sequence has been introduced and an antibody Fc region, extending from the N-terminus to the C-terminus. In another specific embodiment, the ligand-binding molecule is a dimer protein comprising two series of peptide chains consisting of a monodomain antibody into which a protease cleavage sequence has been introduced, an antibody hinge region, and an antibody Fc region. In yet another specific embodiment, the ligand-binding molecule is a dimer protein comprising two series of peptide chains consisting of a monodomain antibody into which a protease cleavage sequence has been introduced, an antibody hinge region, an antibody CH2, and an antibody CH3.

[0202] Ligand and ligand-binding molecule complex In some embodiments of the present invention, an uncleaved ligand-binding molecule forms a complex with a ligand by antigen-antibody binding. In more specific embodiments, the complex of the ligand-binding molecule and the ligand is formed by non-covalent bonding between the ligand-binding molecule and the ligand, for example, antigen-antibody binding. Accordingly, the present invention also provides a method for producing a complex, comprising the steps of contacting a ligand-binding molecule with a ligand and recovering a complex formed by the ligand binding to the ligand-binding molecule. In the present invention, the ligand-binding molecule and the ligand can be brought into contact under conditions that enable their binding. The resulting complex can be incorporated into a pharmaceutical composition, for example, by compounding it with a pharmaceutically acceptable carrier.

[0203] A fusion protein in which a ligand and a ligand-binding molecule are fused. In some embodiments of the present invention, an uncleaved ligand-binding molecule fuses with a ligand to form a fusion protein, and the ligand-binding molecule portion and the ligand portion within the fusion protein further interact via antigen-antibody binding. The ligand-binding molecule and the ligand may fuse via or without a linker. Even when the ligand-binding molecule and the ligand in the fusion protein are fused via or without a linker, a non-covalent bond between the ligand-binding molecule portion and the ligand portion still exists. In other words, even in embodiments where the ligand-binding molecule is fused with the ligand, the non-covalent bond between the ligand-binding molecule portion and the ligand portion is similar to that in embodiments where the ligand-binding molecule and the ligand are not fused. When the ligand-binding molecule is cleaved, its non-covalent bond is weakened. That is, the binding between the ligand-binding molecule and the ligand is weakened. In a preferred embodiment of the present invention, a ligand-binding molecule and a ligand are fused via a linker. Any peptide linker that can be introduced by genetic engineering, or a synthetic compound linker (see, for example, Protein Engineering, 9 (3), 299-305, 1996) can be used as the linker for fusing the ligand-binding molecule and the ligand; however, a peptide linker is preferred in this embodiment. The length of the peptide linker is not particularly limited and can be appropriately selected by those skilled in the art depending on the purpose. For example, but not limited to, in the case of a peptide linker: Ser Gly·Ser(GS) Ser·Gly(SG) Gly·Gly·Ser(GGS) Gly·Ser·Gly (GSG) Ser·Gly·Gly (SGG) Gly·Ser·Ser (GSS) Ser·Ser·Gly (SSG) Ser·Gly·Ser(SGS) Gly·Gly·Gly·Ser(GGGS, Sequence ID: 83) Gly·Gly·Ser·Gly (GGSG, Sequence ID: 84) Gly·Ser·Gly·Gly (GSGG, Sequence ID: 85) Ser·Gly·Gly·Gly (SGGG, Sequence ID: 86) Gly·Ser·Ser·Gly(GSSG, Sequence ID: 87) Gly·Gly·Gly·Gly·Ser(GGGGS, Sequence ID: 88) Gly·Gly·Gly·Ser·Gly (GGGSG, Sequence ID: 89) Gly·Gly·Ser·Gly·Gly(GGSGG, Sequence ID: 90) Gly·Ser·Gly·Gly·Gly(GSGGG, Sequence ID: 91) Gly·Ser·Gly·Gly·Ser(GSGGS, Sequence ID: 92) Ser·Gly·Gly·Gly·Gly (SGGGG, Sequence ID: 93) Gly·Ser·Ser·Gly·Gly (GSSGG, Sequence ID: 94) Gly·Ser·Gly·Ser·Gly(GSGSG, Sequence ID: 95) Ser·Gly·Gly·Ser·Gly (SGGSG, Sequence ID: 96) Gly·Ser·Ser·Ser·Gly(GSSSG, Sequence ID: 97) Gly·Gly·Gly·Gly·Gly·Ser(GGGGGS, Sequence ID: 98) Ser·Gly·Gly·Gly·Gly·Gly(SGGGGG, Sequence ID: 99) Gly·Gly·Gly·Gly·Gly·Gly·Ser(GGGGGGS, Sequence ID: 100) Ser·Gly·Gly·Gly·Gly·Gly·Gly(SGGGGGG, Sequence ID: 101) (Gly·Gly·Gly·Gly·Ser(GGGGS, Sequence ID: 88))n (Ser·Gly·Gly·Gly·Gly(SGGGG, Sequence ID: 93))n Examples include [n is an integer greater than or equal to 1]. However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0204] Synthetic compound linkers (chemical crosslinking agents) are crosslinking agents commonly used for crosslinking peptides, such as N-hydroxysuccinimide (NHS), disuccinimidylsverate (DSS), bis(sulfosuccinimidyl)sverate (BS3), dithiobis(succinimidylpropionate) (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), ethylene glycol bis(succinimidylsuccinate) (EGS), ethylene glycol bis(sulfosuccinimidylsuccinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimideoxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimideoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES), and these crosslinking agents are commercially available.

[0205] In some embodiments of the present invention, the ligand-binding molecule and ligand fusion protein is a fusion protein in which the N-terminus of the ligand-binding molecule and the C-terminus of the ligand are fused, either via a linker or without a linker. Several embodiments of the structure of the ligand-binding molecule and ligand fusion protein of the present invention are listed below. The embodiments below are described in order from the N-terminus to the C-terminus. Ligand - Single-domain antibody Ligand - Linker - Single-domain antibody Ligand - Single-domain antibody - Antibody constant region (or fragment thereof) Ligand - Linker - Single-domain antibody - Antibody constant region (or fragment thereof) Ligand - Single-domain antibody - Antibody hinge region - Antibody CH2 - Antibody CH3 Ligand - Linker - Single-domain antibody - Antibody hinge region - Antibody CH2 - Antibody CH3

[0206] treatment As used herein, “treatment” (and its grammatical derivatives, e.g., “to treat,” “to treat,” etc.) means a clinical intervention intended to modify the natural course of the individual being treated, and may be carried out for preventive purposes or during the course of a clinical condition. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, symptom reduction, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction of the rate of disease progression, recovery or mitigation of the disease state, and remission or improved prognosis. In some embodiments, the single-domain antibody-containing ligand-binding molecules of the present invention can control the biological activity of the ligand and are used to delay the onset of disease or slow the progression of disease.

[0207] Pharmaceutical composition The present invention also relates to a pharmaceutical composition (drug) comprising a single-domain antibody-containing ligand-binding molecule and a pharmaceutically acceptable carrier, a pharmaceutical composition (drug) comprising a single-domain antibody-containing ligand-binding molecule, a ligand, and a pharmaceutically acceptable carrier, and a pharmaceutical composition (drug) comprising a fusion protein in which a single-domain antibody-containing ligand-binding molecule and a ligand are fused, and a pharmaceutically acceptable carrier.

[0208] In this invention, a pharmaceutical composition generally refers to a drug used for the treatment or prevention of a disease, or for examination and diagnosis. Furthermore, in the present invention, the term "pharmaceutical composition containing a ligand-binding molecule" can be rephrased as "a method for treating a disease, comprising administering a ligand-binding molecule to a target for treatment," or as "the use of a ligand-binding molecule in the manufacture of a pharmaceutical for treating a disease." The term "pharmaceutical composition containing a ligand-binding molecule" can also be rephrased as "the use of a ligand-binding molecule for treating a disease." The term "pharmaceutical composition comprising a ligand-binding molecule and a ligand" can also be rephrased as "a method for treating a disease comprising administering a ligand-binding molecule and a ligand to a target for treatment," or as "the use of ligand-binding molecules and ligands in the manufacture of pharmaceuticals for treating diseases." The term "pharmaceutical composition comprising a ligand-binding molecule and a ligand" can also be rephrased as "the use of ligand-binding molecules and ligands for treating diseases." The term "pharmaceutical composition containing a fusion protein" can also be rephrased as "a method for treating a disease, comprising administering a fusion protein to a target," or as "the use of a fusion protein in the manufacture of a pharmaceutical for treating a disease." The term "pharmaceutical composition containing a fusion protein" can also be rephrased as "the use of a fusion protein for treating a disease."

[0209] The pharmaceutical compositions of the present invention can be formulated using methods known to those skilled in the art. For example, they can be administered parenterally in the form of sterile solutions with water or other pharmaceutically acceptable liquids, or as injectable suspensions. For example, they can be formulated by mixing them with pharmacokinetically acceptable carriers or media, specifically sterile water or saline solution, vegetable oil, emulsifiers, suspensions, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, etc., in a unit dose form generally required for pharmaceutical practice. The amount of active ingredient in these formulations is set to obtain an appropriate volume within the indicated range.

[0210] Sterile compositions for injection can be formulated in accordance with standard formulation procedures using a vehicle such as distilled water for injection. Examples of aqueous solutions for injection include physiological saline, glucose, and isotonic solutions containing other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). Appropriate solubilizers, such as alcohols (ethanol, etc.), polyalcohols (propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (polysorbate 80™, HCO-50, etc.), may be used in combination.

[0211] Examples of oily solutions include sesame oil and soybean oil, and benzyl benzoate and / or benzyl alcohol may also be used as solubilizers. Furthermore, buffers (e.g., phosphate buffer and sodium acetate buffer), analgesics (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants may be added. The prepared injection solution is usually filled into appropriate ampoules.

[0212] The pharmaceutical composition of the present invention is preferably administered by parenteral administration. For example, compositions in the form of injection, nasal administration, pulmonary administration, or transdermal administration may be administered. For example, it may be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.

[0213] The method of administration may be appropriately selected depending on the patient's age and symptoms. The dosage of the pharmaceutical composition containing the ligand-binding molecule may be set, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight per dose. Alternatively, for example, a dosage of 0.001 to 100,000 mg per patient may be set, but the present invention is not necessarily limited to these values. The dosage and method of administration will vary depending on the patient's weight, age, symptoms, etc., but a person skilled in the art can set an appropriate dosage and method of administration considering these conditions.

[0214] In some embodiments of the present invention, a composition containing a ligand-binding molecule can be administered to an individual. The ligand-binding molecule administered to the individual binds to a ligand already present in the individual, for example, in the blood or tissue, and is further transported within the body while bound to the ligand. The ligand-binding molecule transported to the target tissue is cleaved in the target tissue, weakening its binding to the ligand and releasing the bound ligand in the target tissue. The released ligand can exert biological activity in the target tissue and treat diseases originating from the target tissue. In embodiments in which the ligand-binding molecule suppresses the biological activity of the ligand while it is bound to the ligand, and the ligand-binding molecule is cleaved specifically in the target tissue, the biological activity of the ligand can be exerted only after cleavage in the target tissue without the ligand exhibiting biological activity during transport, thereby treating diseases and minimizing systemic side effects.

[0215] The present invention provides a method for suppressing or inhibiting the systemic effects of an endogenous ligand, comprising the step of administering the ligand-binding molecule of the present invention. Alternatively, the present invention provides a pharmaceutical composition for suppressing or inhibiting the systemic effects of an endogenous ligand, comprising the ligand-binding molecule as an active ingredient. Alternatively, the present invention relates to the use of the ligand-binding molecule in suppressing or inhibiting the systemic effects of an endogenous ligand. Furthermore, the present invention relates to the use of the ligand-binding molecule in the manufacture of a pharmaceutical composition for suppressing or inhibiting the systemic effects of an endogenous ligand. In these embodiments, the systemic effects of the endogenous ligand are suppressed or inhibited by the ligand-binding molecule of the present invention, and then expressed as tissue-specific effects in the target tissue.

[0216] In some embodiments of the present invention, a composition containing a ligand-binding molecule and a composition containing a ligand can be administered to an individual separately or simultaneously. Alternatively, a composition containing both a ligand-binding molecule and a ligand can be administered to an individual. When a composition containing both a ligand-binding molecule and a ligand is administered to an individual, the ligand-binding molecule and the ligand in the composition may form a complex. When both a ligand-binding molecule and a ligand are administered to an individual, the ligand-binding molecule binds to the administered ligand and is transported within the body while bound to the ligand. Upon delivery to a target tissue, the ligand-binding molecule is cleaved in the target tissue, reducing its binding to the ligand and releasing the bound ligand in the target tissue. The released ligand can exert biological activity in the target tissue, treating diseases originating from the target tissue. In embodiments where the ligand-binding molecule suppresses the biological activity of the ligand while bound, and the ligand-binding molecule is cleaved specifically in the target tissue, the ligand's biological activity can be exerted only after cleavage in the target tissue, without the ligand exhibiting biological activity during transport, thus treating diseases and minimizing systemic side effects. Ligand-binding molecules administered to an organism can bind not only to the administered ligand but also to ligands naturally present within the organism, allowing for the transport of both the naturally present ligand and the administered ligand within the organism while bound. In other words, the present invention also provides a method for producing a ligand-ligand-binding molecule complex, comprising the steps of contacting a ligand-binding monodomain antibody-containing molecule with a ligand and recovering a complex consisting of the monodomain antibody-containing molecule and the ligand. The complex of the present invention can be incorporated into a pharmaceutical composition, for example, by compounding it with a pharmaceutically acceptable carrier.

[0217] In some embodiments of the present invention, a fusion protein, which is a fusion of a ligand-binding molecule and a ligand, can be administered to an individual. In these embodiments, the ligand-binding molecule and ligand in the fusion protein form the fusion protein with or without the linker, but a non-covalent bond between the ligand-binding molecule portion and the ligand portion still exists. When a fusion protein, which is a fusion of a ligand-binding molecule and a ligand, is administered to an individual, the fusion protein is transported in the body, and the ligand-binding molecule portion in the fusion protein is cleaved in the target tissue, thereby weakening the non-covalent bond between the ligand-binding molecule portion and the ligand, and releasing the ligand and a portion of the ligand-binding molecule from the fusion protein. The released ligand and a portion of the ligand-binding molecule exert the biological activity of the ligand in the target tissue, and can treat diseases originating from the target tissue. In embodiments in which the biological activity of the ligand is suppressed when the ligand-binding molecule is bound to the ligand, and the ligand-binding molecule is cleaved specifically in the target tissue, the biological activity of the ligand can be exerted only after cleavage in the target tissue, without the biological activity of the ligand in the fusion protein being exerted during transport, thereby treating diseases and minimizing systemic side effects.

[0218] In the manner described above, a method for ligand delivery is provided. That is, the present invention provides a method for delivering a ligand to a target tissue, comprising the step of administering a ligand to which the ligand conjugate of the present invention is attached. Alternatively, the present invention provides a composition for delivering a ligand to a target tissue, comprising a ligand to which a ligand conjugate is attached. Furthermore, the present invention relates to the use of a ligand to which a ligand conjugate is attached in the delivery of a ligand to a target tissue. The present invention also relates to the use of a ligand to which a ligand conjugate is attached in the manufacture of a composition for delivering a ligand to a target tissue.

[0219] Method for producing ligand-binding molecules and fusion proteins The present invention also relates to a ligand-binding molecule containing a single-domain antibody whose binding to a ligand is weakened when cleaved, or to a method for producing a fusion protein obtained by fusing the ligand-binding molecule with a ligand. In one embodiment of the present invention, a method for producing a ligand-binding molecule or a fusion protein is provided, which includes introducing a protease cleavage sequence into a single-domain antibody contained in the ligand-binding molecule.

[0220] Examples of methods for introducing a protease cleavage sequence into a single-domain antibody contained in a ligand-binding molecule include inserting the protease cleavage sequence into the amino acid sequence of the single-domain antibody contained in the ligand-binding molecule, or replacing a portion of the amino acid sequence of the single-domain antibody contained in the ligand-binding molecule with the protease cleavage sequence.

[0221] Inserting amino acid sequence A into amino acid sequence B means dividing amino acid sequence B into two parts without deleting any, and connecting the two parts with amino acid sequence A (i.e., creating a new amino acid sequence such as "first half of amino acid sequence B - amino acid sequence A - second half of amino acid sequence B"). Introducing amino acid sequence A into amino acid sequence B means dividing amino acid sequence B into two parts and connecting the two parts with amino acid sequence A. In addition to inserting amino acid sequence A into amino acid sequence B, it is also possible to delete one or more amino acid residues, including amino acid residues in amino acid sequence B adjacent to amino acid sequence A, and then connect the two parts with amino acid sequence A (i.e., replacing a part of amino acid sequence B with amino acid sequence A).

[0222] An example of a method for obtaining a ligand-binding molecule containing a single-domain antibody is a method for obtaining a single-domain antibody that has the ability to bind to a ligand. Single-domain antibodies can be obtained, for example, by a method using a known single-domain antibody production method. The single-domain antibody obtained by this method may be used directly as a ligand-binding molecule, or it may be used after the single-domain antibody has been further linked to another amino acid sequence.

[0223] The method for producing single-domain antibodies is similar to that for producing ordinary antibodies and is well known to those skilled in the art. For example, in the case of monoclonal antibodies, they may be produced by hybridoma (Kohler and Milstein, Nature 256:495 (1975)) or recombinant methods (U.S. Patent No. 4,816,567). They may also be isolated from phage antibody libraries (Clackson et al., Nature 352:624-628 (1991); Marks et al., J.Mol.Biol. 222:581-597 (1991)). They may also be isolated from a single B cell clone (N. Biotechnol. 28(5): 253-457 (2011)).

[0224] The ligand-binding molecule of the present invention can be produced by introducing a protease cleavage sequence into the ligand-binding molecule. More specifically, the ligand-binding molecule of the present invention can be produced by introducing a protease cleavage sequence into a single-domain antibody within the ligand-binding molecule. Optionally, it is possible to confirm whether the ligand-binding molecule is cleaved by treating it with a protease corresponding to the protease cleavage sequence. For example, by contacting a molecule into which a protease cleavage sequence has been introduced with a protease and examining the molecular weight of the product after protease treatment using electrophoresis such as SDS-PAGE, it is possible to confirm whether or not the protease cleavage sequence has been cleaved.

[0225] The present invention also relates to a polynucleotide that encodes a single-domain antibody-containing ligand-binding molecule whose binding to a ligand is weakened when cleaved, or to a polynucleotide that encodes a fusion protein obtained by fusing the ligand-binding molecule with a ligand.

[0226] The polynucleotides in this invention are typically loaded (inserted) into a suitable vector and introduced into host cells. The vector is not particularly limited as long as it stably holds the inserted nucleic acid. For example, if E. coli is used as the host, the pBluescript vector (Stratagene) is preferred as a cloning vector, but various commercially available vectors can be used. When using a vector for the purpose of producing the ligand-binding molecule or fusion protein of this invention, an expression vector is particularly useful. The expression vector is not particularly limited as long as it is a vector that expresses the ligand-binding molecule in vitro, in E. coli, in cultured cells, or in living organisms. For example, the pBEST vector (Promega) is preferred for in vitro expression, the pET vector (Invitrogen) is preferred for E. coli, the pME18S-FL3 vector (GenBank Accession No. AB009864) is preferred for cultured cells, and the pME18S vector (Mol Cell Biol. 8:466-472 (1988)) is preferred for living organisms. The insertion of the DNA of the present invention into a vector can be carried out by conventional methods, for example, by a ligase reaction using restriction enzyme sites (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.4-11.11).

[0227] There are no particular restrictions on the host cells used, and various host cells can be used depending on the purpose. Examples of cells used to express ligand-binding molecules or fusion proteins include bacterial cells (e.g., Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, Bacillus subtilis), fungal cells (e.g., yeast, Aspergillus), insect cells (e.g., Drosophila S2, Spodoptera SF9), animal cells (e.g., CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells), and plant cells. Vector introduction into host cells can be performed by known methods such as calcium phosphate precipitation, electroporation (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), lipofectamine (GIBCO-BRL), and microinjection.

[0228] To secrete ligand-binding molecules or fusion proteins expressed in host cells into the lumen of the endoplasmic reticulum, the pericellular lumen, or the extracellular environment, appropriate secretion signals can be incorporated into the target ligand-binding molecule or fusion protein. These signals may be endogenous or heterologous to the target ligand-binding molecule or fusion protein.

[0229] A method for producing ligand-binding molecules or fusion proteins typically includes a step of recovering the ligand-binding molecules or fusion proteins. In the above production method, if the ligand-binding molecules or fusion proteins of the present invention are secreted into the culture medium, the culture medium is recovered. If the ligand-binding molecules or fusion proteins of the present invention are produced inside cells, the cells are first lysed, and then the ligand-binding molecules or fusion proteins are recovered.

[0230] To recover and purify the ligand-binding molecule or fusion protein of the present invention from recombinant cell cultures, known methods can be used, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyl apatite chromatography, and lectin chromatography.

[0231] It will be understood by those skilled in the art that any combination of one or more embodiments described herein is included in the present invention, provided that it does not contradict the common technical knowledge of those skilled in the art. Furthermore, any invention that excludes any combination of one or more embodiments described herein from the present invention should be considered as an invention intended and described herein, provided that it does not contradict the common technical knowledge of those skilled in the art. [Examples]

[0232] The following are examples of the methods and compositions of the present invention. In light of the general description above, it will be understood that various other embodiments may be implemented.

[0233] Example 1: Challenges of previously reported immunocytokines and protease-activating cytokines Immunocytokines that target antigens expressed in cancer tissue have generally been created by fusing the target cytokine to the terminal of targeting IgG or scFv (Expert Opin Investig Drugs. 2009 Jul;18(7):991-1000., Curr Opin Immunol. 2016 Jun;40:96-102.). Because cytokines such as IL-2, IL-12, and TNF are highly toxic, it is hoped that delivering these cytokines to the tumor site using antibodies will reduce side effects while enhancing efficacy (Non-patent Literature 4, 5, 6). However, all of these methods have challenges, such as not showing sufficient clinical efficacy with systemic administration, having a narrow therapeutic window, and being too toxic to be administered systemically. A major reason for this is that even immunocytokines, when administered systemically, are exposed to the entire body, which can cause systemic effects and toxicity, or they can only be administered at extremely low doses to avoid toxicity. Furthermore, immunocytokines that bind to cancer antigens are internalized and eliminated by cancer cells within the tumor, making it difficult to expose the tumor site to cytokines in some cases. There are also reports that the antitumor effect was the same whether immunocytokines were antibodies that bind to cancer antigens fused with IL-2 or antibodies that do not bind to cancer antigens fused with IL-2 (Non-Patent Literature 7).

[0234] To mitigate the systemic effects of immunocytokines, a major challenge, molecules have been reported in which cytokines and cytokine receptors are linked by a linker that is cleaved by a protease highly expressed in cancer. Cytokines are inhibited by cytokine receptors linked by the linker, but when the linker is cleaved by the protease, the cytokine receptor is released, and the cytokine becomes active. Examples include molecules in which TNFalpha and TNFR are linked by a linker cleaved by uPA (Cancer Immunol Immunother. 2006 Dec;55(12):1590-600.), and molecules in which IL-2 and IL-2R are linked by a linker cleaved by MMP-2 (Immunology. 2011 Jun;133(2):206-20). However, even before linker cleavage, the cytokines in these molecules possess biological activity, and linker cleavage only increases their activity by about 10 times. Two reasons for this are that the affinity between cytokines and cytokine receptors is not strong, so cytokines retain some activity even before protease cleavage, or that cytokine receptors can still bind to cytokines even after the linker has been cleaved by the protease, thereby inhibiting the biological activity of cytokines.

[0235] Example 2: Challenges in the application of chemokines to cancer immunotherapy Chemokines (Nature Immunology 9, 949 - 952 (2008)) are basic proteins that exert their effects via G protein-coupled receptors and belong to a group of cytokines. They act on specific leukocytes that express receptors and have the activity (chemotaxis) of causing leukocytes to migrate in the direction of the concentration gradient of the substance (Nat Cell Biol. 2016 Jan;18(1):43-53.). Chemokines are known to be produced in large quantities at the site of inflammation and to cause the migration of leukocytes from the blood vessels into the inflammatory tissue. Since controlling chemokines can control the migration of leukocytes, it is thought that this could be utilized in cancer immunotherapy. If T cells, antigen-presenting cells, and M1 macrophages can be migrated to the local site of a solid tumor, it is thought that an antitumor effect can be induced. Cytokines can exert their effects even when administered systemically, but chemokines cause cells to migrate to tissues with high concentrations through a concentration gradient, so the desired effect cannot be obtained by administering chemokines systemically. For this reason, cancer immunotherapy using systemic administration of chemokines (chemokine therapy) is not considered practical.

[0236] Example 3: Concept of a ligand-binding molecule that can release a target tissue-specific ligand by introducing a protease cleavage sequence. As shown in Examples 1 and 2, previously reported cytokine / chemokine therapies have the following problems. 1. In the case of immunocytokines, even if cytokines are targeted to solid tumors using antibodies, side effects occur because cytokines act systemically, or they can only be administered at low doses to avoid side effects, making it impossible to achieve high exposure within the tumor. 2. In cytokines activated by proteases, where cytokine receptors (or antibodies) and cytokines are linked by a linker that can be cleaved by proteases, the neutralization of cytokine activity is insufficient, and the cytokine retains some activity even before protease cleavage. 3. In the case of cytokines activated by proteases, cytokine receptors (or antibodies) can still bind to the cytokine even after the linker has been cleaved by the protease, thus inhibiting the biological activity of the cytokine.

[0237] To solve this problem, we believe it is important to meet the following conditions. 1. Systemically, ligands such as cytokines or chemokines are sufficiently inhibited (biological activity is minimized) by ligand-binding molecules. 2. The biological activity of the ligand is restored by cleavage by protease (it becomes an active ligand). 3. Cleavage by protease causes the ligand-binding molecule to lose its ligand-binding activity.

[0238] As a pharmaceutical composition that satisfies the above conditions, we have devised a molecule in which binding to a ligand is weakened by cleavage of a cleavage site. First, a single-domain antibody against the ligand is obtained, and then a cleavage site is introduced into the single-domain antibody to create a ligand-binding molecule containing the single-domain antibody with the cleavage site.

[0239] Example 4: Example of a ligand-binding molecule containing a single-domain antibody with a cleavage site introduced. Figures 1-4 show examples of ligand-binding molecules containing single-domain antibodies with cleavage sites introduced. Figure 1 shows an example of a ligand-binding molecule containing only a single-domain antibody into which a cleavage site has been introduced. When the cleavage site is not cleaved, the single-domain antibody can bind to the ligand, and if the affinity of the single-domain antibody for the ligand is sufficiently strong, the biological activity of the ligand is sufficiently inhibited. Even if this ligand-binding molecule and ligand are administered systemically, the ligand in the ligand-ligand complex is neutralized and therefore does not exert its biological activity, and the ligand-ligand complex has a longer half-life than the ligand alone. In the case where the cleavage site is a protease cleavage sequence that is cleaved by a tumor tissue-specific protease, when a ligand-binding molecule and ligand are administered systemically, the complex formed is cleaved by a protease highly expressed in the tumor tissue. When the protease cleavage sequence in the single-domain antibody is cleaved, the single-domain antibody can no longer bind to the ligand, the neutralization of the ligand is released, and it becomes possible for it to exert its biological effect in the tumor tissue.

[0240] Figure 2 shows an example of a fusion polypeptide formed by fusing a ligand with a ligand to a ligand, which contains only a single-domain antibody with a cleavage site introduced into it. When the cleavage site is not cleaved, the single-domain antibody in the fusion polypeptide can bind to the ligand, and if the affinity of the single-domain antibody to the ligand is sufficiently strong, the biological activity of the ligand is sufficiently inhibited. Even if this fusion polypeptide is administered systemically, the ligand in the fusion polypeptide is neutralized and therefore does not exert its biological activity, and the fusion polypeptide has a longer half-life than the ligand alone. In the case where the cleavage site is a protease cleavage sequence that is cleaved by a tumor tissue-specific protease, when the fusion polypeptide is administered systemically, the protease cleavage sequence in the single-domain antibody is cleaved by the protease highly expressed in the tumor tissue. As a result, the single-domain antibody in the fusion polypeptide can no longer bind to the ligand, the neutralization of the ligand is released, and a portion of the fusion polypeptide containing the ligand is released, allowing it to exert its biological effect in the tumor tissue.

[0241] Figures 3 and 4 show examples of a single-domain antibody with a cleavage site, a ligand-binding molecule containing an antibody hinge region and an antibody Fc region, and a fusion polypeptide of the ligand-binding molecule and the ligand. Similar to the embodiments in Figures 1 and 2, the ligand can be neutralized by the single-domain antibody in the uncleaved state, and the ligand can be released and exert its biological effect in the cleaved state. Furthermore, in the examples in Figures 3 and 4, since the antibody Fc region is included in the uncleaved complex or fusion polypeptide, the half-life in the uncleaved state is expected to be even longer than in the examples in Figures 1 and 2.

[0242] Example 5: Ligand-binding molecule containing an anti-human IL-6R monodomain antibody into which a protease cleavage sequence has been introduced. 5-1 Production of ligand-binding molecules by introducing protease cleavage sequences into polypeptides containing anti-human IL-6R monodomain antibodies A ligand-binding molecule containing a monodomain antibody with a protease-cleaved sequence was created by fusion of the antibody hinge region shown in SEQ ID NO: 121 and the N-terminus of the antibody Fc region sequence with the C-terminus of a monodomain antibody against human IL-6R (SEQ ID NO: 120). First, a sequence containing a peptide sequence (LSGRSDNH, SEQ ID NO: 3) that has been reported to be cleaved by a cancer-specific matryptase (MT-SP1) was inserted into the polypeptide IL6R90-G1T3dCHdC to create the ligand-binding molecule shown in Table 2. This molecule was expressed by transient expression using Expi293 (Life Technologies) and purified using a method known to the art using Protein A. IL6R90-G1T3dCHdC was also expressed and purified as a control molecule without the protease-cleaved sequence. The ligand-binding molecules and control molecules produced are dimeric proteins as shown in Figure 3.

[0243] [Table 2]

[0244] 5-2 Evaluation of the binding of a ligand-binding molecule containing an anti-human IL-6R monodomain antibody into which a protease cleavage sequence has been introduced to human IL-6R. 5-2-1 Protease treatment To the 0.1 mg / mL of ligand-binding molecule prepared in Example 5-1, Recombinant Human Matriptase / ST14 Catalytic Domain (hMT-SP1, R&D systemsm 3946-SE-010) was added to a final concentration of 25 nM, and the solution was incubated overnight at 37°C to obtain a solution containing protease-treated ligand-binding molecule. The conditions for preparing the solution containing untreated ligand-binding molecule were the same as those for preparing the solution containing the same volume of PBS instead of protease and storing it under the same conditions.

[0245] 5-2-2 Confirmation of protease cleavage of ligand-binding molecules (SDS-PAGE) The cleavage of ligand-binding molecules by the protease treatment performed in Example 5-2-1 was confirmed by SDS-PAGE. 9 μL of the solution containing the protease-treated ligand-binding molecules / untreated ligand-binding molecules prepared in Example 5-2-1 was mixed with 3 μL of sample buffer and incubated at 95°C for 1 minute. Next, electrophoresis was performed using Mini-PROTEAN TGX gel (4-20% 15well) (Bio-Rad #456-1096), and the proteins were stained with Sample Blue Safe Stain (novex, LC6065). The results are shown in Figure 5. As shown in Figure 5, control molecules without protease cleavage sequences (Lane 12, 13) had bands in the same position regardless of whether they were protease-treated or untreated, whereas ligand-binding molecules into which each protease cleavage sequence was introduced had new bands that appeared only after protease treatment. This indicates that the single-domain antibody-containing ligand-binding molecules into which each protease cleavage sequence was introduced were cleaved by protease treatment.

[0246] 5-2-3 Evaluation of the binding of protease-treated ligand-binding molecules and protease-untreated ligand-binding molecules to human IL-6R 20 μL of a solution containing protease-treated ligand-binding molecules / untreated ligand-binding molecules prepared in Example 5-2-1 was mixed with 60 μL of PBS to prepare the IL-6R binding evaluation sample. IL-6R binding of the sample was evaluated by biolayer interferometry (BLI). The IL-6R binding evaluation sample and IL-6R were dispensed into a tilted bottom (TW384) microplate (Forte bio, 18-5076). A Protein G sensor (ForteBio, 18-0022) was hydrated with PBS, and measurements were performed using an Octet RED 384 at 25°C. A baseline measurement was performed for 30 seconds in a well containing PBS, followed by 200 seconds of antibody binding to the Protein G sensor. Another baseline measurement was performed for 30 seconds in a well containing PBS, followed by 180 seconds of binding measurement in a well containing human 500 nM IL-6R, and then 180 seconds of dissociation measurement in a well containing PBS. Figure 6 shows a real-time binding graph illustrating the binding process. As shown in Figure 6, when using each ligand-binding molecule with a protease cleavage sequence introduced, the amount of human IL-6R bound decreased compared to when using the untreated ligand-binding molecule.

[0247] Example 6: Preparation and evaluation of a fusion protein (ligand-binding molecule-IL-6R fusion protein) between a ligand-binding molecule containing an anti-human IL-6R monodomain antibody into which a protease cleavage sequence has been introduced and human IL-6R. 6-1. Synthesis of a fusion protein between a ligand-binding molecule containing an anti-human IL-6R monodomain antibody into which a protease cleavage sequence has been introduced and human IL-6R. The ligand-binding molecule prepared in Example 5-1 was fused to the C-terminus of human IL-6R (SEQ ID NO: 128) via various linkers consisting of glycine and serine at its N-terminus to produce ligand-binding molecule-IL-6R fusion proteins (Table 3). These fusion proteins were expressed transiently using Expi293 (Life Technologies) by methods known to those skilled in the art, and purified using Protein A by methods known to those skilled in the art. Each of the produced fusion proteins is a dimeric protein having two peptide chains with the sequences shown in Table 3.

[0248] [Table 3]

[0249] As a control molecule that does not contain a protease cleavage sequence, a molecule (Table 4) was prepared by fusing the N-terminus of IL6R90-G1T3dCHdC (SEQ ID NO: 122) with the C-terminus of IL-6R via a linker, and this molecule was expressed and purified. The prepared control molecules are also dimeric proteins having two peptide chains with the sequences shown in Table 4.

[0250] [Table 4]

[0251] 6-2 Evaluation of ligand-binding molecule-IL-6R fusion protein cleavage by protease 6-2-1 Protease treatment To 0.1 mg / mL of the ligand-binding molecule-IL-6R fusion protein prepared in Example 6-1, Recombinant Human Matriptase / ST14 Catalytic Domain (hMT-SP1, R&D systemsm 3946-SE-010) was added to a final concentration of 25 nM, and the solution was incubated overnight at 37°C to obtain a solution containing the protease-treated fusion protein. The conditions for preparing the solution containing the untreated fusion protein were the same as those for preparing the solution containing the protease-treated fusion protein, with the same volume of PBS added instead of protease and stored under the same conditions.

[0252] 6-2-2 Confirmation of protease cleavage of anti-IL-6R monodomain antibody-IL-6R fusion protein (SDS-PAGE) The ligand-binding molecule-IL-6R fusion protein was confirmed to have been cleaved by the protease treatment performed in Example 6-2-1 using SDS-PAGE. 9 μL of a solution containing the protease-treated fusion protein / untreated fusion protein prepared in Example 6-2-1 was mixed with 3 μL of sample buffer and incubated at 95°C for 1 minute. Electrophoresis was then performed using Mini-PROTEAN TGX gel (4-20% 15well) (Bio-Rad #456-1096), and the proteins were stained with Sample Blue Safe Stain (novex, LC6065). The results are shown in Figure 7. As shown in Figure 7, while the control molecule without the protease cleavage sequence had the same band position regardless of whether it was protease-treated or untreated, each fusion protein containing a single-domain antibody-containing ligand-binding molecule into which the protease cleavage sequence was introduced had a new band that appeared only after protease treatment. This indicates that each fusion protein containing a single-domain antibody-containing ligand-binding molecule into which the protease cleavage sequence was introduced was cleaved by protease treatment.

[0253] 6-2-3 Preparation of a molecule containing a biotinylated anti-IL-6R monodomain antibody As a method for detecting the release of IL-6R fused to a ligand-binding molecule, a method was used in which biotin was added to a molecule containing a monodomain antibody that recognizes IL-6R, and the binding of free IL-6R to the biotinylated anti-IL-6R monodomain antibody molecule was detected. The biotinylated anti-IL-6R monodomain antibody molecule was prepared as follows: An anti-IL-6R monodomain antibody molecule having the same monodomain antibody partial sequence as IL6R90-G1T3dCH1dC (SEQ ID NO: 122) was prepared, and a biotin addition sequence (AviTag sequence, SEQ ID NO: 159) was added to its C-terminus to create IL6R90-FcBAPdC (SEQ ID NO: 160). A gene fragment encoding IL6R90-FcBAPdC was prepared and introduced into an animal cell expression vector by a method known to those skilled in the art. At this time, a gene expressing EBNA1 and a gene expressing biotin ligase were introduced simultaneously, and biotin was added for the purpose of biotin labeling. Cells into which the gene was introduced were cultured at 37°C and 8% CO2, and the target biotinylated anti-IL-6R monodomain antibody-containing molecule (IL6R90-bio) was secreted into the culture supernatant. IL6R90-bio was purified from the culture supernatant by methods known to the art.

[0254] 6-2-4 Evaluation of IL-6R release from fusion proteins by protease treatment The release of IL-6R by protease treatment of the fusion protein was evaluated using biolayer interferometry (BLI) with a biotinylated anti-IL-6R monodomain antibody-containing molecule (IL6R90-bio) prepared in Example 6-2-3.

[0255] The protease-treated fusion protein, untreated fusion protein, and IL6R90-bio prepared in Example 6-2-1 were dispensed into different wells of Tilted bottom (TW384) Microplates (ForteBio, 18-5076). The Streptavidin biosensor (ForteBio, 18-5021) was hydrated with PBS, and measurements were performed using Octet RED 384 at 27°C. A baseline measurement was performed for 30 seconds in the well containing PBS, and then IL6R90-bio was allowed to bind to the Streptavidin sensor for 200 seconds. Another baseline measurement was performed for 30 seconds in the well containing PBS, and then binding was measured for 180 seconds in the well containing either the protease-treated or untreated fusion protein, and dissociation was measured for 180 seconds in the well containing PBS. A real-time binding graph showing the binding process is shown in Figure 8. As shown in Figure 8, in the case of a ligand-binding molecule containing a single-domain antibody into which a protease cleavage sequence was introduced, the amount of human IL-6R binding to IL6R90-bio increased after protease treatment compared to the case without protease treatment. In other words, protease treatment weakened the binding activity of the single-domain antibody in the fusion protein to IL-6R, and IL-6R was released from the fusion protein.

[0256] Example 7: Evaluation of the introduction of diverse protease cleavage sequences 7-1 Production of IgG antibodies into which diverse protease cleavage sequences have been introduced An expression vector for MRA (heavy chain: MRAH-G1T4 (SEQ ID NO: 119), light chain: MRAL-k0 (SEQ ID NO: 118)), a neutralizing antibody (IgG antibody) against human IL-6R, was prepared by methods known to those skilled in the art. Table 5 shows peptide sequences known to be cleaved at MMP-2, MMP-7, and MMP-9, as well as peptide sequences containing a movable linker made of a glycine-serine polymer near these sequences.

[0257] [Table 5]

[0258] These insertion sequences were inserted near the boundary between the variable and constant regions of the heavy chain of the MRA antibody, resulting in the following modified heavy chains: MEIVHG4SMP2MP9G4S-MEIVHG4SMP2MP9G4SG1T4 (SEQ ID NO: 111), MEIVHG4SMP2.2G4S-MEIVHG4SMP2.2G4SG1T4 (SEQ ID NO: 112), MEIVHG4SMP2.4G4S-MEIVHG4SMP2.4G4SG1T4 (SEQ ID NO: 113), MEIVHG4SMP9G4S-MEIVHG4SMP9G4SG1T4 (SEQ ID NO: 114), MEIVHMP2.1-MEIVHMP2.1G1T4 (SEQ ID NO: 115), MEIVHMP2.3-MEIVHMP2.3G1T4 (SEQ ID NO: 116), and MEIVHMP7.2-MEIVHMP7.2G1T4. We designed (heavy chain sequence number: 117) and prepared expression vectors encoding these modified heavy chains using methods known to the art. These modified heavy chains and MRA light chains were combined to express the MRA variants shown in Table 6 by transient expression using FreeStyle293 cells (Invitrogen) or Expi293 cells (Life Technologies) using methods known to those skilled in the art, and then purified using methods known to those skilled in the art with Protein A.

[0259] [Table 6]

[0260] 7-2. Evaluation of protease cleavage of IgG antibodies into which diverse protease cleavage sequences have been introduced. We verified whether the MRA variant prepared in 7-1 could be cleaved by proteases. Recombinant human MMP-2 (R&D Systems, 902-MP-010), recombinant human MMP-7 (R&D Systems, 907-MP-010), and recombinant human MMP-9 (R&D Systems, 911-MP-010) were used as proteases. The proteases were mixed with 1 mM p-aminophenylmercuric acetate (APMA; abcam, ab112146) and activated at 37°C for 1 and 24 hours, respectively, before use. Figures 11, 12, and 13 show the results of evaluating protease cleavage by reducing SDS-PAGE after reacting with 50 nM, 100 nM, or 500 nM protease, 50 μg / mL antibody, and either an assay buffer (MMP Activity Assay Kit (Fluorometric - Green) (ab112146), Component C: Assay Buffer) or 20 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, pH 7.2 (hereinafter referred to as Tris) at 37°C for 20 hours. The MRA-modified antibodies were reacted with the proteases shown in Table 2. In MMP-2, the cuttings are MEIVHG4SMP2MP9G4S-MEIVHG4SMP2MP9G4SG1T4 / MRAL-k0, MEIVHG4SMP2.2G4S-MEIVHG4SMP2.2G4SG1T4 / MRAL-k0, MEIVHG4SMP2.4G4S-MEIVHG4SMP2.4G4SG1T4 / MRAL-k0, MEIVHMP2.1-MEIVHMP2.1G1T4 / MRAL-k0, MEIVHMP2.3-MEIVHMP2.3G1T4 / MRAL-k0; in MMP-7, the cutting is MEIVHMP7.2-MEIVHMP7.2G1T4 / MRAL-k0; in MMP-9, MEIVHG4SMP2MP9G4S-MEIVHG4SMP2MP9G4SG1T4 / MRAL-k0, Cutting of MEIVHG4SMP9G4S-MEIVHG4SMP9G4SG1T4 / MRAL-k0 was observed.

[0261] Example 8: Preparation and evaluation of a fusion protein (ligand-binding molecule-PD-1 fusion protein) of a ligand-binding molecule containing an anti-human PD-1 monodomain antibody into which a protease cleavage sequence has been introduced, and human PD-1. 8-1. Synthesis of a fusion protein of a ligand-binding molecule containing an anti-human PD-1 monodomain antibody into which a protease cleavage sequence has been introduced, and human PD-1. PD102C3-G1T3noCyshinge (SEQ ID NO: 161) and PD102C12-G1T3noCyshinge (SEQ ID NO: 162) were prepared by fusing a human antibody constant region to a single-domain antibody that binds to human PD-1, using methods known to the art. Next, protease cleavage sequences were introduced into PD102C3-G1T3noCyshinge and PD102C12-G1T3noCyshinge, and then their respective N-terminuses were fused to the C-terminus of the extracellular domain of human PD-1 (SEQ ID NO: 163) via various linkers consisting of glycine and serine to produce ligand-binding molecule-PD-1 fusion proteins (Table 7). These fusion proteins were expressed transiently using Expi293 (Life Technologies) using methods known to the art, and purified using Protein A using methods known to the art. The prepared fusion proteins are dimeric proteins having two peptide chains with the sequences shown in Table 7.

[0262] [Table 7]

[0263] 8-2. Evaluation of ligand-binding molecule-PD-1 fusion protein cleavage by protease 8-2-1. Protease treatment To 0.1 mg / mL of the ligand-binding molecule-PD-1 fusion protein prepared in Example 8-1, Recombinant Human Matriptase / ST14 Catalytic Domain (hMT-SP1, R&D systemsm 3946-SE-010) was added to a final concentration of 25 nM, and the solution was incubated overnight at 37°C to obtain a solution containing the protease-treated fusion protein. The conditions for preparing the solution containing the untreated fusion protein were the same as those for preparing the solution containing the protease-treated fusion protein, with the same volume of PBS added instead of protease and stored under the same conditions.

[0264] 8-2-2. Confirmation of protease cleavage of anti-PD-1 monodomain antibody-PD-1 fusion protein (SDS-PAGE) The ligand-binding molecule-PD-1 fusion protein was confirmed by SDS-PAGE to be cleaved by the protease treatment performed in Example 8-2-1. 9 μL of a solution containing the protease-treated fusion protein / untreated fusion protein prepared in Example 8-2-1 was mixed with 3 μL of sample buffer and incubated at 95°C for 1 minute. Electrophoresis was then performed using Mini-PROTEAN TGX gel (4-20% 15well) (Bio-Rad #456-1096), and the proteins were stained with Sample Blue Safe Stain (novex, LC6065). The results are shown in Figure 9. As shown in Figure 9, each fusion protein containing a single-domain antibody-containing ligand-binding molecule into which each protease cleavage sequence was introduced showed a new band that appeared only after protease treatment, indicating that each fusion protein containing a single-domain antibody-containing ligand-binding molecule into which each protease cleavage sequence was introduced was cleaved by protease treatment.

[0265] 8-2-3. Preparation of molecules containing biotinylated anti-PD-1 monodomain antibody As a method for detecting the release of PD-1 fused to a ligand-binding molecule, we used a method in which biotin was added to a single-domain antibody-containing molecule that recognizes PD-1, and the binding of free PD-1 to the biotinylated anti-PD-1 single-domain antibody-containing molecule was detected. The biotinylated anti-PD-1 single-domain antibody-containing molecule was prepared as follows: A single-domain antibody-containing molecule having the same single-domain antibody partial sequence as PD102C3-G1T3noCyshinge was prepared, and a biotin addition sequence (AviTag sequence, SEQ ID NO: 159) was added to its C-terminus to create PD102C3-FcBAPdC (SEQ ID NO: 168). Furthermore, a molecule containing an anti-PD-1 monodomain antibody having the same monodomain antibody partial sequence as PD102C12-G1T3noCyshinge was prepared, and a biotin addition sequence (AviTag sequence, SEQ ID NO: 159) was added to its C-terminus to create PD102C12-FcBAPdC (SEQ ID NO: 169). Gene fragments encoding PD102C3-FcBAPdC and PD102C12-FcBAPdC, respectively, were prepared and introduced into animal cell expression vectors by methods known to the art. At this time, a gene expressing EBNA1 and a gene expressing biotin ligase were introduced simultaneously, and biotin was added for the purpose of biotin labeling. Cells into which the genes were introduced were cultured at 37°C and 8% CO2, and the target biotinylated anti-PD-1 monodomain antibody-containing molecule (PD1-bio) was secreted into the culture supernatant. PD1-bio was purified from the culture supernatant by methods known to the art.

[0266] 8-2-4 Evaluation of PD-1 release from fusion proteins by protease treatment The release of PD-1 by protease treatment of the fusion protein was evaluated using biolayer interferometry (BLI) with a biotinylated anti-PD-1 monodomain antibody-containing molecule (PD1-bio) prepared in Example 8-2-3.

[0267] The protease-treated fusion protein, untreated fusion protein, and PD1-bio prepared in Example 8-2-1 were dispensed into different wells of Tilted bottom (TW384) Microplates (ForteBio, 18-5076). PD1-Bio was selected as appropriate, either PD102C3-FcBAPdC or PD102C12-FcBAPdC, to bind to the same epitope as the single-domain antibody in the measurement sample. The Streptavidin biosensor (ForteBio, 18-5021) was hydrated with PBS, and measurements were performed using Octet RED 384 at 27°C. A baseline measurement was performed for 30 seconds in the well containing PBS, followed by 200 seconds of binding of PD1-bio to the Streptavidin sensor. Baseline measurements were performed again for 30 seconds in wells containing PBS, followed by 180 seconds of binding measurement in wells containing either protease-treated or untreated fusion proteins, and then 180 seconds of dissociation measurement in wells containing PBS. A real-time binding graph showing the binding process is shown in Figure 10. As shown in Figure 10, in the case of a single-domain antibody-containing ligand-binding molecule-PD-1 fusion protein into which a protease cleavage sequence was introduced, the amount of human PD-1 binding to PD1-bio increased in the protease-treated case compared to the untreated case. That is, protease treatment weakened the binding activity of the single-domain antibody in the fusion protein to PD-1, and PD-1 was released from the fusion protein.

[0268] The invention described herein has been described in detail with examples and illustrations for the purpose of aiding clear understanding, but the descriptions and illustrations herein should not be construed as limiting the scope of the invention. All disclosures of patent and scientific documents cited herein are expressly incorporated herein by reference throughout. [Industrial applicability]

[0269] The ligand-binding molecule of the present invention is transported in vivo while bound to the ligand, and is cleaved in diseased tissue, weakening its binding to the ligand and allowing the ligand to be released specifically in the diseased tissue. Therefore, diseased tissue can be specifically exposed to the ligand. Furthermore, since the ligand-binding molecule suppresses the biological activity of the ligand during transport, the risk of the ligand acting systemically is reduced, making it extremely useful in the treatment of diseases.

Claims

1. A ligand-binding molecule comprising a monodomain antibody, wherein the monodomain antibody is capable of binding to a ligand and has at least one cleavage site introduced into the monodomain antibody, the binding of the ligand-binding molecule to the ligand when the cleavage site is cleaved is attenuated compared to the binding of the ligand-binding molecule to the ligand when the cleavage site is not cleaved, the monodomain antibody is a VHH or monodomain VH antibody, the cleavage site comprises a protease cleavage sequence, the cleavage site is introduced at one or more positions selected from the sequence of amino acids 12 to 17 (Kabat numbering) of the monodomain antibody, and the protease cleavage sequence is 4 to 18 amino acids long.

2. The ligand-binding molecule according to claim 1, wherein the cleavage site containing the protease cleavage sequence is introduced at one or more positions selected from (a) to (e) below: (a) The sequence of the 12th amino acid (Kabat numbering) to the 13th amino acid (Kabat numbering) of the single-domain antibody; (b) The sequence of the 13th amino acid (Kabat numbering) to the 14th amino acid (Kabat numbering) of the single-domain antibody; (c) The sequence of the 14th amino acid (Kabat numbering) to the 15th amino acid (Kabat numbering) of the single-domain antibody; (d) The sequence of the 15th amino acid (Kabat numbering) to the 16th amino acid (Kabat numbering) of the single-domain antibody; (e) The sequence of the 16th amino acid (Kabat numbering) to the 17th amino acid (Kabat numbering) of the single-domain antibody.

3. The ligand-binding molecule according to claim 1 or 2, wherein the protease is a target tissue-specific protease.

4. The ligand-binding molecule according to any one of claims 1 to 3, wherein the protease cleavage sequence introduced into the single-domain antibody is introduced at the position of a residue forming a loop structure in the single-domain antibody, or at the position of a residue close to the loop structure.

5. The ligand-binding molecule according to any one of claims 1 to 4, wherein the protease is a cancer tissue-specific protease or an inflammatory tissue-specific protease.

6. The ligand-binding molecule according to any one of claims 1 to 5, wherein the protease is at least one protease selected from matryptase, urokinase (uPA), and metalloproteinase.

7. The ligand-binding molecule according to any one of claims 1 to 6, wherein the protease cleavage sequence is a sequence comprising one or more sequences selected from the sequences shown in SEQ ID NOs: 2 to 82 and 170 to 925.

8. The ligand-binding molecule according to any one of claims 1 to 7, wherein the ligand is released from the ligand-binding molecule when the cleavage site or the protease cleavage sequence is cleaved.

9. The ligand-binding molecule according to any one of claims 1 to 8, wherein the ligand is a biologically active molecule, and the single-domain antibody has neutralizing activity against the ligand.

10. A complex formed of the ligand and the ligand-binding molecule described in any one of claims 1 to 9.

11. A fusion protein comprising the ligand described above and a ligand-binding molecule according to any one of claims 1 to 9.

12. A pharmaceutical composition comprising a ligand-binding molecule according to any one of claims 1 to 9, or a complex according to claim 10, or a fusion protein according to claim 11.

13. A polynucleotide encoding a ligand-binding molecule according to any one of claims 1 to 9 or a fusion protein according to claim 11.

14. A vector comprising a polynucleotide as described in claim 13.

15. A host cell comprising the polynucleotide described in claim 13 or the vector described in claim 14.

16. A method for producing a ligand-binding molecule according to any one of claims 1 to 9, a complex according to claim 10, or a fusion protein according to claim 11, comprising the step of culturing the host cells according to claim 15.

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