Protease substrates and polypeptides containing protease cleavage sequences

Abstract

The present invention pertains to: a protease substrate; a peptide sequence that can be cleaved by a protease; a polypeptide including a protease cleavage sequence; methods for producing those; a pharmaceutical composition that contains a polypeptide including a protease cleavage sequence; and a method for releasing a ligand or an antigen binding domain by cleaving a protease cleavage sequence included in a polypeptide.

Description

Technical Field

[0001] The present disclosure provides protease substrates, peptide sequences cleavable by proteases, polypeptides comprising protease cleavage sequences and methods for producing them, pharmaceutical compositions comprising polypeptides comprising protease cleavage sequences, and methods for releasing antigen-binding domains or ligands by cleavage of protease cleavage sequences comprised in polypeptides.

Background Art

[0002] Proteases are enzymes that cleave peptide bonds between amino acid residues. Some proteases are known to break specific peptide bonds based on the presence of specific amino acid sequences within proteins. Proteases occur naturally in all organisms and are involved in a variety of physiological reactions ranging from simple degradation to highly regulated pathways. However, many pathological conditions are associated with deregulated protease expression and / or activity. Thus, inappropriate proteolysis may play a major role in the development and progression of cancer, as well as cardiovascular, inflammatory, neurodegenerative, eukaryotic, bacterial, viral, and parasitic diseases.

[0003] Accordingly, there is a need to identify new substrates for proteases and to use these substrates in a variety of therapeutic, diagnostic, and prophylactic applications.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present disclosure has been made in view of such circumstances, and one of its objects is to provide protease substrates, peptide sequences cleavable by proteases, polypeptides containing protease cleavage sequences, methods for producing them, pharmaceutical compositions containing polypeptides containing protease cleavage sequences, and methods for releasing antigen-binding domains or ligands by cleavage of the protease cleavage sequences contained in the polypeptides. **Means for Solving the Problems**

[0006] The inventors of the present disclosure conducted intensive research to achieve the above object, and as a result, found compounds that can be used as protease substrates, particularly peptide sequences that can be used as protease substrates and are cleavable by proteases, and found that it is useful for the treatment of diseases including administering polypeptides containing peptide sequences cleavable by proteases (abbreviated as protease cleavage sequences), and that polypeptides containing such protease cleavage sequences are useful in the production of medicaments for the treatment of diseases. Further, the inventors of the present disclosure created polypeptides containing such protease cleavage sequences and methods for producing them, and completed the present disclosure.

[0007] The present disclosure is based on such findings, and specifically includes the embodiments exemplified below. [1] A protease substrate having a higher cleavage rate by protease than a protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3. [2] The protease substrate according to [1], having a higher cleavage rate by human uPA than a protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3. [3] The protease substrate according to any one of [1] to [2], having a higher cleavage rate by human MT-SP1 than a protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3. [4] A protease substrate according to any one of [1] to [3], which has a higher cleavage rate by mouse uPA than a protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3. [5] A protease substrate according to any one of [1] to [4], which has a higher cleavage rate by mouse MT-SP1 than a protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3. [6] A protease substrate according to any one of [1] to [5], which has a higher ratio of the cleavage rate by human uPA to the cleavage rate by human serum than a protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3. [7] A protease substrate according to any one of [1] to [6], which has a higher ratio of the cleavage rate by human MT-SP1 to the cleavage rate by human serum than a protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3. [8] A protease substrate according to any one of [1] to [7], which has a higher ratio of the cleavage rate by mouse uPA to the cleavage rate by human serum than a protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3. [9] A protease substrate according to any one of [1] to [8], which has a higher ratio of the cleavage rate by mouse MT-SP1 to the cleavage rate by human serum than a protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3.

[10] A protease substrate according to any one of [1] to [9], which is not cleaved by human serum.

[11] A protease substrate according to any one of [1] to

[10] , which contains at least one sequence selected from the following: The sequence from the 4th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 4th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 6th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 1st amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 14th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, the sequence from the 5th amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, the sequence from the 5th amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, a sequence represented by any of SEQ ID NOs: 5 to 18003.

[12] A protease substrate comprising at least one sequence selected from the following: The sequence from the 4th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 4th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 6th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 1st amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 14th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, the sequence from the 5th amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, the sequence from the 5th amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, a sequence represented by any of SEQ ID NOs: 5 to 18003.

[13] The protease substrate according to

[12] , wherein the protease is matriptase and / or urokinase.

[14] The protease substrate according to any one of

[12] to

[13] , wherein the protease is at least one protease selected from human MT-SP1, mouse MT-SP1, human uPA, and mouse uPA.

[15] The protease substrate according to any one of

[12] to

[14] , having a higher cleavage rate by the protease as compared with a protease substrate containing any one of the sequences of SEQ ID NOs: 1, 2, and 3.

[16] The protease substrate according to any one of

[12] to

[15] , having a higher cleavage rate by human uPA as compared with a protease substrate containing any one of the sequences of SEQ ID NOs: 1, 2, and 3.

[17] A protease substrate described in any one of

[12] to

[16] , which has a higher cleavage rate by human MT-SP1 compared to a protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3.

[18] A protease substrate described in any one of

[12] to

[17] , which has a higher cleavage rate by mouse uPA compared to a protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3.

[19] A protease substrate described in any one of

[12] to

[18] , which has a higher cleavage rate by mouse MT-SP1 compared to a protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3.

[20] A protease substrate described in any one of

[12] to

[19] , which has a higher ratio of the cleavage rate by human uPA to the cleavage rate by human serum compared to a protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3.

[21] A protease substrate described in any one of

[12] to

[20] , which has a higher ratio of the cleavage rate by human MT-SP1 to the cleavage rate by human serum compared to a protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3.

[22] A protease substrate described in any one of

[12] to

[21] , which has a higher ratio of the cleavage rate by mouse uPA to the cleavage rate by human serum compared to a protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3.

[23] A protease substrate described in any one of

[12] to

[22] , which has a higher ratio of the cleavage rate by mouse MT-SP1 to the cleavage rate by human serum compared to a protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3.

[24] A protease substrate described in any one of

[12] to

[23] , which is not cleaved by human serum.

[25] Use of at least one sequence selected from the following as a protease cleavage sequence: The sequence from the 4th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 4th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 6th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 1st amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 14th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, the sequence from the 5th amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, the sequence from the 5th amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, a sequence represented by any of SEQ ID NOs: 5 to 18003.

[26] A polypeptide comprising at least one sequence selected from the following: The sequence from the 4th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 4th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 6th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 1st amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 14th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, the sequence from the 5th amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, the sequence from the 5th amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, the sequence represented by any of SEQ ID NOs: 5 to 18003.

[27] The sequence from the 4th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 4th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 6th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 1st amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 14th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, the sequence from the 5th amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, the sequence from the 5th amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, the polypeptide according to

[26] , wherein the sequence selected from any of the sequences shown by SEQ ID NOs: 5 to 18003 is a protease cleavage sequence cleavable by a protease.

[28] A method for producing the polypeptide according to

[26] or

[27] .

[29] A polynucleotide encoding the polypeptide according to

[26] or

[27] .

[30] A vector containing the polynucleotide according to

[29] .

[31] A host cell containing the polynucleotide according to

[29] or the vector according to

[30] .

[32] A method for producing the polypeptide according to

[26] or

[27] , comprising the step of culturing the host cell according to

[31] .

[33] A method for producing the polypeptide according to

[32] , comprising the step of isolating the polypeptide from the culture supernatant.

[0008] The polypeptide according to [A-1]

[27] , wherein the polypeptide comprises an antigen-binding domain and a transport moiety, and the transport moiety has a suppression domain that suppresses the antigen-binding activity of the antigen-binding domain. [A-2] The suppression of the antigen-binding activity of the antigen-binding domain by the suppression domain in the state where the protease cleavage sequence is cleaved by a protease is weaker than the suppression of the antigen-binding activity of the antigen-binding domain by the suppression domain in the state where the protease cleavage sequence is uncleaved. The polypeptide according to [A-1]. [A-3] The antigen-binding domain has a shorter half-life in blood than the uncleaved polypeptide. The polypeptide according to [A-1] or [A-2]. [A-4] The antigen-binding domain has a shorter half-life in blood than the transport moiety. The polypeptide according to any one of [A-1] to [A-3]. [A-5] The molecular weight of the antigen-binding domain is smaller than the molecular weight of the transport moiety. The polypeptide according to any one of [A-1] to [A-4]. [A-6] The molecular weight of the antigen-binding domain is 60 kDa or less. The polypeptide according to any one of [A-1] to [A-5]. [A-7] The transport moiety has FcRn-binding activity, and the antigen-binding domain has no FcRn-binding activity or has a weaker FcRn-binding activity than the transport moiety. The polypeptide according to any one of [A-1] to [A-6]. [A-8] The antigen-binding domain is detachable from the polypeptide, and the antigen-binding activity of the antigen-binding domain in the state of being detached from the polypeptide is higher than the antigen-binding activity in the state of not being detached from the polypeptide. The polypeptide according to any one of [A-1] to [A-7]. [A-9] The antigen-binding activity of the antigen-binding domain is suppressed by the association of the antigen-binding domain and the suppression domain of the transport moiety. The polypeptide according to any one of [A-1] to [A-8]. [A-10] The polypeptide according to [A-8], wherein the protease cleavage sequence is cleaved by a protease so that the antigen-binding domain can be released from the polypeptide. [A-11] The polypeptide according to [A-9], wherein the protease cleavage sequence is cleaved by a protease so that the association between the antigen-binding domain and the inhibitory domain of the transport moiety is eliminated. [A-12] The polypeptide according to any one of [A-1] to [A-11], wherein the protease is matriptase and / or urokinase. [A-13] The polypeptide according to any one of [A-1] to [A-11], wherein the protease is at least one protease selected from human MT-SP1, mouse MT-SP1, human uPA, and mouse uPA. [A-14] The polypeptide according to any one of [A-1] to [A-13], wherein a first flexible linker is further added to one end of the protease cleavage sequence. [A-15] The polypeptide according to [A-14], wherein the first flexible linker is a flexible linker composed of a glycine-serine polymer. [A-16] The polypeptide according to [A-14] or [A-15], wherein a second flexible linker is further added to the other end of the protease cleavage sequence. [A-17] The polypeptide according to [A-16], wherein the second flexible linker is a flexible linker composed of a glycine-serine polymer. [A-18] The polypeptide according to any one of [A-1] to [A-17], wherein the antigen-binding domain includes a single-domain antibody or is a single-domain antibody, and the inhibitory domain of the transport moiety inhibits the antigen-binding activity of the single-domain antibody. [A-19] The polypeptide according to [A-18], wherein the single-domain antibody is VHH, or VH having antigen-binding activity in a single domain, or VL having antigen-binding activity in a single domain. [A-20] The antigen-binding domain includes a single-domain antibody, the inhibitory domain of the transport moiety is a VHH, or an antibody VH, or an antibody VL, and the antigen-binding activity of the single-domain antibody is inhibited by the VHH, or the antibody VH, or the antibody VL, the polypeptide according to any one of [A-1] to [A-19]. [A-21] The antigen-binding domain includes a single-domain antibody, the inhibitory domain of the transport moiety is a VHH, or an antibody VH, or an antibody VL, and the antigen-binding activity of the single-domain antibody is inhibited by associating with the VHH, or the antibody VH, or the antibody VL, the polypeptide according to any one of [A-1] to [A-20]. [A-22] The single-domain antibody is a VHH, or a VH having antigen-binding activity with a single domain, the inhibitory domain of the transport moiety is an antibody VL, and the VHH or VH having antigen-binding activity with a single domain is inhibited from having antigen-binding activity by associating with the antibody VL, the polypeptide according to any one of [A-18] to [A-21]. [A-23] The single-domain antibody is a VHH, and the VHH has an amino acid substitution at at least one position selected from the amino acids at positions 37, 44, 45, or 47 (all Kabat numbering), the polypeptide according to any one of [A-18] to [A-22]. [A-24] The single-domain antibody is a VHH, and the VHH contains at least one amino acid selected from the amino acids 37V, 44G, 45L, or 47W (all Kabat numbering), the polypeptide according to any one of [A-18] to [A-22]. [A-25] The single-domain antibody is a VHH, and the VHH contains at least one amino acid substitution selected from the amino acid substitutions F37V, Y37V, E44G, Q44G, R45L, H45L, G47W, F47W, L47W, T47W, or S47W (all Kabat numbering), the polypeptide according to any one of [A-18] to [A-22]. [A-26] The polypeptide according to any one of [A-18] to [A-22], wherein the single-domain antibody is a VHH, and the VHH has amino acid substitutions at at least one pair of positions selected from 37 / 44, 37 / 45, 37 / 47, 44 / 45, 44 / 47, 45 / 47, 37 / 44 / 45, 37 / 44 / 47, 37 / 45 / 47, 44 / 45 / 47, and 37 / 44 / 45 / 47 (all Kabat numbering). [A-27] The polypeptide described in any one of [A-18] to [A-22], wherein the single domain antibody is a VHH, and the VHH contains at least one pair of amino acids selected from 37V / 44G, 37V / 45L, 37V / 47W, 44G / 45L, 44G / 47W, 45L / 47W, 37V / 44G / 45L, 37V / 44G / 47W, 37V / 45L / 47W, 44G / 45L / 47W, and 37V / 44G / 45L / 47W (all Kabat numbering). [A-28] A polypeptide described in any one of [A-18] to [A-22], wherein the single domain antibody is a VHH, and the VHH contains at least one set of amino acid substitutions selected from F37V / R45L, F37V / G47W, R45L / G47W, and F37V / R45L / G47W (all Kabat numbering). [A-29] The polypeptide according to any one of [A-18] to [A-21], wherein the single-domain antibody is a VL having antigen-binding activity as a single domain, the inhibitory domain of the transporter is an antibody VH, and the antigen-binding activity of the VL having antigen-binding activity as a single domain is inhibited by associating with the antibody VH. [A-30] The polypeptide according to any one of [A-1] to [A-29], wherein the transport moiety has an FcRn binding region. [A-31] The polypeptide according to any one of [A-1] to [A-30], wherein the transport moiety comprises an antibody constant region. [A-32] The polypeptide according to [A-31], wherein the antibody constant region of the transporter and the antigen-binding domain are fused with or without a linker. [A-33] The transport portion contains a constant region of an antibody heavy chain, and the constant region of the antibody heavy chain and the antigen-binding domain are fused via a linker or without a linker, the polypeptide according to [A-31]. [A-34] The transport portion contains a constant region of an antibody light chain, and the constant region of the antibody light chain and the antigen-binding domain are fused via a linker or without a linker, the polypeptide according to [A-31]. [A-35] In the polypeptide, the N-terminus of the constant region of the antibody heavy chain of the transport portion and the C-terminus of the antigen-binding domain are fused via a linker or without a linker, and the protease cleavage sequence is located in the sequence of the antigen-binding domain or on the antigen-binding domain side from the amino acid at position 122 (EU numbering) of the constant region of the heavy chain antibody, the polypeptide according to [A-33]. [A-36] In the polypeptide, the N-terminus of the constant region of the antibody light chain of the transport portion and the C-terminus of the antigen-binding domain are fused via a linker or without a linker, and the protease cleavage sequence is located in the sequence of the antigen-binding domain or on the antigen-binding domain side from the amino acid at position 113 (Kabat numbering) of the constant region of the light chain antibody, the polypeptide according to [A-34]. [A-37] In the polypeptide, the N-terminus of the constant region of the antibody of the transport portion and the C-terminus of the antigen-binding domain are fused via a linker or without a linker, the antigen-binding domain is a single-domain antibody or VHH prepared from VH, and the protease cleavage sequence is located in the sequence of the constant region of the antibody or on the constant region side from the amino acid at position 109 (Kabat numbering) of the single-domain antibody of the antigen-binding domain, the polypeptide according to any one of [A-32] to [A-35]. [A-38] In the polypeptide, the N-terminus of the constant region of the antibody of the transport portion and the C-terminus of the antigen-binding domain are fused via a linker or without a linker, and the protease cleavage sequence is located near the boundary between the antigen-binding domain and the constant region of the antibody, the polypeptide according to [A-32]. [A-39] The polypeptide has the N-terminus of the constant region of the antibody heavy chain of the transport portion and the C-terminus of the antigen-binding domain fused either via a linker or without a linker, and the protease cleavage sequence is located near the boundary between the antigen-binding domain and the constant region of the antibody heavy chain, the polypeptide according to [A-33]. [A-40] The polypeptide has the N-terminus of the constant region of the antibody light chain of the transport portion and the C-terminus of the antigen-binding domain fused either via a linker or without a linker, and the protease cleavage sequence is located near the boundary between the antigen-binding domain and the constant region of the antibody light chain, the polypeptide according to [A-34]. [A-41] The antigen-binding domain is a single-domain antibody or VHH prepared from VH, and the protease cleavage sequence is located between the amino acid at position 109 (Kabat numbering) of the single-domain antibody of the antigen-binding domain and the amino acid at position 122 (EU numbering) of the constant region of the antibody heavy chain, the polypeptide according to [A-39]. [A-42] The antigen-binding domain is a single-domain antibody or VHH prepared from VH, and the protease cleavage sequence is located between the amino acid at position 109 (Kabat numbering) of the single-domain antibody of the antigen-binding domain and the amino acid at position 113 (Kabat numbering) of the constant region of the antibody light chain, the polypeptide according to [A-40]. [A-43] The antigen-binding domain is a single-domain antibody prepared from VL, and the protease cleavage sequence is located between the amino acid at position 104 (Kabat numbering) of the single-domain antibody of the antigen-binding domain and the amino acid at position 122 (EU numbering) of the constant region of the antibody heavy chain, the polypeptide according to [A-39]. [A-44] The antigen-binding domain is a single-domain antibody prepared from VL, and the protease cleavage sequence is located between the amino acid at position 109 (Kabat numbering) of the single-domain antibody of the antigen-binding domain and the amino acid at position 113 (Kabat numbering) of the constant region of the antibody light chain, the polypeptide according to [A-40]. [A-45] The antibody constant region of the polypeptide is an IgG antibody constant region, and the polypeptide described in any one of [A-31] to [A-44]. [A-46] The polypeptide is an IgG antibody-like molecule, and the polypeptide described in any one of [A-1] to [A-45]. [A-47] When the antigen-binding domain is in an un-released state and the measurement is performed using the BLI (Bio-Layer Interferometry) method (Octet), no binding between the antigen-binding domain and the antigen is observed, and the polypeptide described in any one of [A-1] to [A-46]. [A-48] A second antigen-binding domain is further linked to the antigen-binding domain, and the polypeptide described in any one of [A-1] to [A-47]. [A-49] The second antigen-binding domain has an antigen-binding specificity different from that of the antigen-binding domain, and the polypeptide described in [A-48]. [A-50] The second antigen-binding domain includes a second single-domain antibody, and the polypeptide described in [A-48] or [A-49]. [A-51] The antigen-binding domain is a single-domain antibody, the second antigen-binding domain is a second single-domain antibody, the antigen-binding domain and the second antigen-binding domain are releasable from the polypeptide, and in the released state of the antigen-binding domain and the second antigen-binding domain, the single-domain antibody and the second single-domain antibody form a bispecific antigen-binding molecule, and the polypeptide described in [A-50]. [A-52] The polypeptide further has another antigen-binding domain different from the antigen-binding domain, and the antigen-binding activity of the polypeptide is suppressed by linking the another antigen-binding domain to the transport portion of the polypeptide, and the polypeptide described in any one of [A-1] to [A-51]. [A-53] The another antigen-binding domain has an antigen-binding specificity different from that of the antigen-binding domain, and the polypeptide described in [A-52]. [A-54] A pharmaceutical composition comprising the polypeptide according to any one of [A-1] to [A-53]. [A-55] A method for producing the polypeptide according to any one of [A-1] to [A-53]. [A-56] A polynucleotide encoding the polypeptide according to any one of [A-1] to [A-53]. [A-57] A vector comprising the polynucleotide described in [A-56]. [A-58] A host cell comprising the polynucleotide according to [A-56] or the vector according to [A-57]. [A-59] A method for producing a polypeptide according to any one of [A-1] to [A-53], comprising a step of culturing the host cell according to [A-58]. [A-60] A method for producing the polypeptide according to [A-59], comprising a step of isolating the polypeptide from a culture supernatant. A method for releasing the antigen-binding domain from the polypeptide, comprising cleavage of at least one site having a sequence selected from the sequences shown by any of SEQ ID NOs: 5 to 18003 by a protease, wherein the sequence is from the 4th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 4th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 6th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 1st amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 14th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, the sequence from the 5th amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, the sequence from the 5th amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003. [A-62] The method according to [A-61], wherein the polypeptide further comprises a transport portion, and the transport portion has a suppression domain that suppresses the antigen-binding activity of the antigen-binding domain. [A-63] The method according to [A-62], wherein the suppression of the antigen-binding activity of the antigen-binding domain by the suppression domain in the state where the protease cleavage sequence is cleaved by a protease is weaker than the suppression of the antigen-binding activity of the antigen-binding domain by the suppression domain in the state where the protease cleavage sequence is uncleaved. [A-64] The method according to [A-62] or [A-63], wherein the antigen-binding domain has a shorter blood half-life than the uncleaved polypeptide. The method according to any one of [A-62] to [A-64], wherein the antigen-binding domain has a shorter half-life in blood than the transport moiety. The method according to any one of [A-62] to [A-65], wherein the molecular weight of the antigen-binding domain is smaller than the molecular weight of the transport moiety. The method according to any one of [A-62] to [A-66], wherein the molecular weight of the antigen-binding domain is 60 kDa or less. The method according to any one of [A-62] to [A-67], wherein the transport moiety has FcRn-binding activity, and the antigen-binding domain has no FcRn-binding activity or has weaker FcRn-binding activity than the transport moiety. The method according to any one of [A-62] to [A-68], wherein the antigen-binding activity of the antigen-binding domain in a state of being free from the polypeptide is higher than the antigen-binding activity in a state of not being free from the polypeptide. The method according to any one of [A-62] to [A-69], wherein the antigen-binding activity of the antigen-binding domain is suppressed by the association of the antigen-binding domain and the inhibitory domain of the transport moiety. The method according to [A-70], wherein the association between the antigen-binding domain and the inhibitory domain of the transport moiety is eliminated by cleavage of the protease cleavage sequence by a protease. The method according to any one of [A-62] to [A-71], wherein the protease is matriptase and / or urokinase. The method according to any one of [A-62] to [A-71], wherein the protease is at least one protease selected from human MT-SP1, mouse MT-SP1, human uPA, and mouse uPA. The method according to any one of [A-62] to [A-73], wherein a first flexible linker is further added to one end of the protease cleavage sequence. [A-75] The method according to [A-74], wherein the first movable linker is a movable linker composed of a glycine-serine polymer. [A-76] The method according to [A-74] or [A-75], wherein a second movable linker is further added to the other end of the protease cleavage sequence. [A-77] The method according to [A-76], wherein the second movable linker is a movable linker composed of a glycine-serine polymer. [A-78] The method according to any one of [A-62] to [A-77], wherein the antigen-binding domain comprises a single-domain antibody or is a single-domain antibody, and the inhibitory domain of the transport moiety inhibits the antigen-binding activity of the single-domain antibody. [A-79] The method according to [A-78], wherein the single-domain antibody is a VHH, or a VH having antigen-binding activity in a single domain, or a VL having antigen-binding activity in a single domain. [A-80] The method according to any one of [A-62] to [A-79], wherein the antigen-binding domain comprises a single-domain antibody, the inhibitory domain of the transport moiety is a VHH, or an antibody VH, or an antibody VL, and the antigen-binding activity of the single-domain antibody is inhibited by the VHH, or the antibody VH, or the antibody VL. [A-81] The method according to any one of [A-62] to [A-80], wherein the antigen-binding domain comprises a single-domain antibody, the inhibitory domain of the transport moiety is a VHH, or an antibody VH, or an antibody VL, and the antigen-binding activity of the single-domain antibody is inhibited by associating with the VHH, or the antibody VH, or the antibody VL. [A-82] The method according to any one of [A-78] to [A-81], wherein the single-domain antibody is a VHH, or a VH having antigen-binding activity in a single domain, the inhibitory domain of the transport moiety is an antibody VL, and the antigen-binding activity of the VHH or the VH having antigen-binding activity in a single domain is inhibited by associating with the antibody VL. [A-83] A method described in any one of [A-78] to [A-82], wherein the single-domain antibody is a VHH, and the VHH has an amino acid substitution at at least one position selected from amino acids 37, 44, 45, or 47 (all Kabat numbering). [A-84] A method described in any one of [A-78] to [A-82], wherein the single domain antibody is a VHH, and the VHH contains at least one amino acid selected from amino acids 37V, 44G, 45L, or 47W (all Kabat numbering). [A-85] A method described in any one of [A-78] to [A-82], wherein the single domain antibody is a VHH, and the VHH contains at least one amino acid substitution selected from the amino acid substitutions F37V, Y37V, E44G, Q44G, R45L, H45L, G47W, F47W, L47W, T47W, or S47W (all Kabat numbering). [A-86] The method described in any one of [A-78] to [A-82], wherein the single-domain antibody is a VHH, and the VHH has amino acid substitutions at at least one pair of positions selected from 37 / 44, 37 / 45, 37 / 47, 44 / 45, 44 / 47, 45 / 47, 37 / 44 / 45, 37 / 44 / 47, 37 / 45 / 47, 44 / 45 / 47, and 37 / 44 / 45 / 47 (all Kabat numbering). [A-87] The method described in any one of [A-78] to [A-82], wherein the single domain antibody is a VHH, and the VHH contains at least one pair of amino acids selected from 37V / 44G, 37V / 45L, 37V / 47W, 44G / 45L, 44G / 47W, 45L / 47W, 37V / 44G / 45L, 37V / 44G / 47W, 37V / 45L / 47W, 44G / 45L / 47W, and 37V / 44G / 45L / 47W (all Kabat numbering). [A-88] The single-domain antibody is a VHH, and the VHH contains at least one set of amino acid substitutions selected from F37V / R45L, F37V / G47W, R45L / G47W, F37V / R45L / G47W (all Kabat numbering), and is the method according to any one of [A-78] to [A-82]. [A-89] The single-domain antibody is a VL having antigen-binding activity in a single domain, the inhibitory domain of the transport portion is an antibody VH, and the VL having antigen-binding activity in a single domain is suppressed in antigen-binding activity by associating with the antibody VH, and is the method according to any one of [A-78] to [A-81]. [A-90] The transport portion has an FcRn binding region, and is the method according to any one of [A-62] to [A-89]. [A-91] The transport portion includes an antibody constant region, and is the method according to any one of [A-62] to [A-90]. [A-92] The antibody constant region of the transport portion and the antigen-binding domain are fused via a linker or without a linker, and is the method according to [A-91]. [A-93] The transport portion includes an antibody heavy chain constant region, and the antibody heavy chain constant region and the antigen-binding domain are fused via a linker or without a linker, and is the method according to [A-91]. [A-94] The transport portion includes an antibody light chain constant region, and the antibody light chain constant region and the antigen-binding domain are fused via a linker or without a linker, and is the method according to [A-91]. [A-95] The polypeptide has the N-terminus of the antibody heavy chain constant region of the transport portion and the C-terminus of the antigen-binding domain fused via a linker or without a linker, and the protease cleavage sequence is located on the antigen-binding domain side from the amino acid at position 122 (EU numbering) of the heavy chain antibody constant region in the sequence of the antigen-binding domain, and is the method according to [A-93]. [A-96] The polypeptide has the N-terminus of the antibody light chain constant region of the transport portion and the C-terminus of the antigen-binding domain fused via a linker or without a linker, and the protease cleavage sequence is located in the sequence of the antigen-binding domain or on the antigen-binding domain side from the amino acid at position 113 (Kabat numbering) of the light chain antibody constant region, according to the method described in [A-94]. [A-97] The polypeptide has the N-terminus of the antibody constant region of the transport portion and the C-terminus of the antigen-binding domain fused via a linker or without a linker, the antigen-binding domain is a single-domain antibody or VHH prepared from VH, and the protease cleavage sequence is located in the sequence of the antibody constant region or on the antibody constant region side from the amino acid at position 109 (Kabat numbering) of the single-domain antibody of the antigen-binding domain, according to any one of the methods described in [A-92] to [A-95]. [A-98] The polypeptide has the N-terminus of the antibody constant region of the transport portion and the C-terminus of the antigen-binding domain fused via a linker or without a linker, and the protease cleavage sequence is located near the boundary between the antigen-binding domain and the antibody constant region, according to the method described in [A-92]. [A-99] The polypeptide has the N-terminus of the antibody heavy chain constant region of the transport portion and the C-terminus of the antigen-binding domain fused via a linker or without a linker, and the protease cleavage sequence is located near the boundary between the antigen-binding domain and the antibody heavy chain constant region, according to the polypeptide described in [A-93]. [A-100] The polypeptide has the N-terminus of the antibody light chain constant region of the transport portion and the C-terminus of the antigen-binding domain fused via a linker or without a linker, and the protease cleavage sequence is located near the boundary between the antigen-binding domain and the antibody light chain constant region, according to the method described in [A-34]. [A-101] The antigen-binding domain is a single-domain antibody or VHH prepared from VH, and the protease cleavage sequence is located between the amino acid at position 109 (Kabat numbering) of the single-domain antibody of the antigen-binding domain and the amino acid at position 122 (EU numbering) of the constant region of the antibody heavy chain, according to the method described in [A-99]. [A-102] The antigen-binding domain is a single-domain antibody or VHH prepared from VH, and the protease cleavage sequence is located between the amino acid at position 109 (Kabat numbering) of the single-domain antibody of the antigen-binding domain and the amino acid at position 113 (Kabat numbering) of the constant region of the antibody light chain, according to the method described in [A-100]. [A-103] The antigen-binding domain is a single-domain antibody prepared from VL, and the protease cleavage sequence is located between the amino acid at position 104 (Kabat numbering) of the single-domain antibody of the antigen-binding domain and the amino acid at position 122 (EU numbering) of the constant region of the antibody heavy chain, according to the method described in [A-99]. [A-104] The antigen-binding domain is a single-domain antibody prepared from VL, and the protease cleavage sequence is located between the amino acid at position 109 (Kabat numbering) of the single-domain antibody of the antigen-binding domain and the amino acid at position 113 (Kabat numbering) of the constant region of the antibody light chain, according to the method described in [A-100]. [A-105] The antibody constant region of the polypeptide is an IgG antibody constant region, according to the method described in any one of [A-91] to [A-104]. [A-106] The polypeptide is an IgG antibody-like molecule, according to the method described in any one of [A-62] to [A-105]. [A-107] When the antigen-binding domain is in an un-released state and measured using the BLI (Bio-Layer Interferometry) method (Octet), no binding between the antigen-binding domain and the antigen is observed, according to the method described in any one of [A-62] to [A-106]. [A-108] The method according to any one of [A-62] to [A-107], wherein a second antigen-binding domain is further linked to the antigen-binding domain. [A-109] The method according to [A-108], wherein the second antigen-binding domain has an antigen-binding specificity different from that of the antigen-binding domain. [A-110] The method according to [A-108] or [A-109], wherein the second antigen-binding domain comprises a second single-domain antibody. [A-111] The antigen-binding domain is a single-domain antibody, the second antigen-binding domain is a second single-domain antibody, the antigen-binding domain and the second antigen-binding domain are detachable from the polypeptide, and in the free state of the antigen-binding domain and the second antigen-binding domain, the single-domain antibody and the second single-domain antibody form a bispecific antigen-binding molecule. The method according to [A-110]. [A-112] The polypeptide further has another antigen-binding domain different from the antigen-binding domain, and the antigen-binding activity of the another antigen-binding domain is also suppressed by linking it to the transport portion of the polypeptide. The method according to any one of [A-62] to [A-111]. [A-113] The method according to [A-112], wherein the another antigen-binding domain has an antigen-binding specificity different from that of the antigen-binding domain.

[0009] [B-1] The polypeptide according to

[27] , wherein the polypeptide is a ligand-binding molecule capable of binding to a ligand, and the binding of the ligand-binding molecule to the ligand in the state where the protease cleavage sequence is cleaved is weaker than the binding of the ligand-binding molecule to the ligand in the state where the protease cleavage sequence is uncleaved. Ligand-binding molecule. [B-2] The ligand-binding molecule according to [B-1], wherein in the state where the protease cleavage sequence is cleaved, the ligand is released from the ligand-binding molecule. [B-3] The protease is matriptase and / or urokinase, and the ligand-binding molecule according to any one of [B-1] to [B-2]. [B-4] The protease is at least one protease selected from human MT-SP1, mouse MT-SP1, human uPA, and mouse uPA, and the ligand-binding molecule according to any one of [B-1] to [B-3]. [B-5] A first flexible linker is further added to one end of the protease cleavage sequence, and the ligand-binding molecule according to any one of [B-1] to [B-4]. [B-6] The first flexible linker is a flexible linker composed of a glycine-serine polymer, and the ligand-binding molecule according to [B-5]. [B-7] A second flexible linker is further added to the other end of the protease cleavage sequence, and the ligand-binding molecule according to [B-5] or [B-6]. [B-8] The second flexible linker is a flexible linker composed of a glycine-serine polymer, and the ligand-binding molecule according to [B-7]. [B-9] The ligand-binding molecule includes an antibody VH, an antibody VL, and an antibody constant region, and the ligand-binding molecule according to any one of [B-1] to [B-8]. [B-10] The protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence and the first flexible linker and the second flexible linker are located within the antibody constant region, and the ligand-binding molecule according to [B-9]. [B-11] The protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence and the first flexible linker and the second flexible linker are introduced at any position in the sequence from amino acid 118 (EU numbering) of the antibody heavy chain constant region to amino acid 140 (EU numbering) of the antibody heavy chain constant region, and the ligand-binding molecule according to [B-10]. [B-12] The protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are introduced at any position in the sequence from amino acid 108 (Kabat numbering) to amino acid 131 (Kabat numbering) of the antibody light chain constant region, of the ligand-binding molecule according to [B-10]. [B-13] The protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are located within the antibody VH or within the antibody VL, of the ligand-binding molecule according to [B-9]. [B-14] The protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are introduced at any position in the sequence selected from the group consisting of from amino acid 7 (Kabat numbering) to amino acid 16 (Kabat numbering), from amino acid 40 (Kabat numbering) to amino acid 47 (Kabat numbering), from amino acid 55 (Kabat numbering) to amino acid 69 (Kabat numbering), from amino acid 73 (Kabat numbering) to amino acid 79 (Kabat numbering), from amino acid 83 (Kabat numbering) to amino acid 89 (Kabat numbering), from amino acid 95 (Kabat numbering) to amino acid 99 (Kabat numbering), and from amino acid 101 (Kabat numbering) to amino acid 113 (Kabat numbering) of the antibody VH, of the ligand-binding molecule according to [B-13]. [B-15] The protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are introduced at any position in a sequence selected from the group consisting of from the 7th to 19th amino acids (Kabat numbering) of the antibody VL, from the 39th to 46th amino acids (Kabat numbering) of the antibody VL, from the 49th to 62nd amino acids (Kabat numbering) of the antibody VL, and from the 96th to 107th amino acids (Kabat numbering) of the antibody VL, the ligand-binding molecule according to [B-13]. [B-16] The protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are located near the boundary between the antibody constant region and the antibody VH, and / or near the boundary between the antibody constant region and the antibody VL, the ligand-binding molecule according to [B-9]. [B-17] The protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are introduced at any position in the sequence from the 109th amino acid (Kabat numbering) of the antibody VH to the 122nd amino acid (EU numbering) of the antibody heavy chain constant region, the ligand-binding molecule according to [B-17]. [B-18] The protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are introduced at any position in the sequence from the 104th amino acid (Kabat numbering) of the antibody VL to the 113th amino acid (Kabat numbering) of the antibody light chain constant region, the ligand-binding molecule according to [B-16]. [B-19] The antibody VL and the antibody VH in the ligand-binding molecule are associated, and the association is eliminated by cleavage of the protease cleavage sequence with a protease, the ligand-binding molecule according to any one of [B-9] to [B-18]. [B-20] The ligand is a molecule having biological activity, and the ligand-binding molecule is the ligand-binding molecule according to any one of [B-1] to [B-19], which inhibits the biological activity of the ligand upon binding to the ligand. [B-21] The ligand is a cytokine or chemokine, and the ligand-binding molecule is the ligand-binding molecule according to any one of [B-1] to [B-20]. [B-22] The ligand is a ligand selected from interleukin, interferon, hematopoietic factor, TNF superfamily, chemokine, cell growth factor, and TGF-β family, and the ligand-binding molecule is the ligand-binding molecule according to any one of [B-1] to [B-20]. [B-23] The ligand-binding molecule is an IgG antibody, and the ligand-binding molecule is the ligand-binding molecule according to any one of [B-1] to [B-22]. [B-24] The ligand-binding molecule according to any one of [B-1] to [B-23], which is bound to the ligand. [B-25] The ligand-binding molecule according to any one of [B-1] to [B-23], which is fused with the ligand. [B-26] The ligand-binding molecule according to [B-25], wherein the ligand-binding molecule does not bind to another ligand in the state of being fused with the ligand. [B-27] The ligand-binding molecule according to [B-25] or [B-26], wherein the ligand-binding molecule is fused with the ligand via a linker. [B-28] The ligand-binding molecule according to [B-27], wherein the linker does not contain a protease cleavage sequence. [B-29] A complex formed by the ligand and the ligand-binding molecule according to any one of [B-1] to [B-23], which is bound to the ligand. [B-30] A fusion protein in which the ligand is fused with the ligand-binding molecule according to any one of [B-1] to [B-23]. [B-31] The fusion protein according to [B-30], wherein the ligand-binding molecule does not bind to another ligand in the state of being fused with the ligand. [B-32] The fusion protein according to [B-30] or [B-31], wherein the ligand-binding molecule is fused to the ligand via a linker. [B-33] The fusion protein according to [B-32], wherein the linker does not contain a protease cleavage sequence. [B-34] The fusion protein according to [B-32] or [B-33], wherein the linker is a linker consisting of a glycine-serine polymer. [B-35] A pharmaceutical composition comprising the ligand-binding molecule according to any one of [B-1] to [B-28]. [B-36] A pharmaceutical composition comprising the ligand-binding molecule according to any one of [B-1] to [B-24] and a ligand. [B-37] A pharmaceutical composition comprising the complex according to [B-29]. [B-38] A pharmaceutical composition comprising the fusion protein according to any one of [B-30] to [B-34]. [B-39] A method for producing the ligand-binding molecule according to any one of [B-1] to [B-28]. [B-40] The production method according to [B-39], comprising introducing a protease cleavage sequence into a molecule capable of binding to a ligand. [B-41] A method for producing the fusion protein according to any one of [B-30] to [B-34], comprising fusing a ligand-binding molecule having a protease cleavage sequence with its ligand. [B-42] A polynucleotide encoding the ligand-binding molecule according to any one of [B-1] to [B-28]. [B-43] A vector comprising the polynucleotide according to [B-42]. [B-44] A host cell comprising the polynucleotide according to [B-42] or the vector according to [B-43]. [B-45] A method for producing the ligand-binding molecule according to any one of [B-1] to [B-28], comprising culturing the host cell according to [B-44]. A method for producing the ligand-binding molecule according to [B-45], which comprises the step of isolating the polypeptide from the culture supernatant. [B-47] A polynucleotide encoding the fusion protein according to any one of [B-30] to [B-34]. [B-48] A vector containing the polynucleotide according to [B-46]. [B-49] A host cell containing the polynucleotide according to [B-46] or the vector according to [B-48]. [B-50] A method for producing the fusion protein according to any one of [B-30] to [B-34], which comprises the step of culturing the host cell according to [B-49]. [B-51] A method for producing the fusion protein according to [B-50], which comprises the step of isolating the polypeptide from the culture supernatant. A method for releasing a ligand bound to the ligand-binding molecule, which comprises cleaving a site having at least one sequence selected from the sequences represented by any of SEQ ID NOs: 5 to 18003 by a protease, the site being a sequence from the 4th amino acid to the 15th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201, a sequence from the 4th amino acid to the 13th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201, a sequence from the 6th amino acid to the 13th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201, a sequence from the 1st amino acid to the 12th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993, a sequence from the 3rd amino acid to the 12th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993, a sequence from the 3rd amino acid to the 11th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993, a sequence from the 3rd amino acid to the 10th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993, a sequence from the 3rd amino acid to the 14th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003, a sequence from the 5th amino acid to the 12th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003, a sequence from the 5th amino acid to the 10th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003. [B-53] A method for releasing the ligand from the fusion protein, which comprises cleavage of at least one site having a sequence selected from the sequences shown by any of SEQ ID NOs: 5 to 18003 by a protease. The site is the sequence from the 4th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 4th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 6th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 1st amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 14th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, the sequence from the 5th amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, the sequence from the 5th amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003. [B-54] The method according to [B-53], wherein the ligand-binding molecule is fused to the ligand via a linker. [B-55] The method according to [B-54], wherein the linker does not contain a protease cleavage sequence. [B-56] The method according to [B-54] or [B-55], wherein the linker is a linker consisting of a glycine-serine polymer. [B-57] The method according to any one of [B-53] to [B-56], wherein the ligand-binding molecule does not bind to another ligand in the state of being fused to the ligand. [B-58] The method according to any one of [B-52] to [B-57], wherein the binding of the ligand-binding molecule to the ligand in the state where the protease cleavage sequence is cleaved is weaker than the binding of the ligand-binding molecule to the ligand in the state where the protease cleavage sequence is uncleaved. [B-59] The method according to any one of [B-52] to [B-58], wherein the protease is matriptase and / or urokinase. [B-60] The method according to any one of [B-52] to [B-59], wherein the protease is at least one protease selected from human MT-SP1, mouse MT-SP1, human uPA, and mouse uPA. [B-61] The method according to any one of [B-52] to [B-60], wherein a first flexible linker is further added to one end of the protease cleavage sequence. [B-62] The method according to [B-61], wherein the first flexible linker is a flexible linker composed of a glycine-serine polymer. [B-63] The method according to [B-61] or [B-62], wherein a second flexible linker is further added to the other end of the protease cleavage sequence. [B-64] The method according to [B-63], wherein the second flexible linker is a flexible linker composed of a glycine-serine polymer. [B-65] The method according to any one of [B-52] to [B-64], wherein the ligand-binding molecule comprises an antibody VH, an antibody VL, and an antibody constant region. [B-66] The method according to [B-65], wherein the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are located within the antibody constant region. [B-67] The method according to [B-66], wherein the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence and the first and second flexible linkers are introduced at any position in the sequence from amino acid 118 (EU numbering) to amino acid 140 (EU numbering) of the antibody heavy chain constant region. [B-68] The method according to [B-66], wherein the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence and the first and second flexible linkers are introduced at any position in the sequence from amino acid 108 (Kabat numbering) to amino acid 131 (Kabat numbering) of the antibody light chain constant region. [B-69] The method according to [B-65], wherein the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence and the first and second flexible linkers are located within the antibody VH or the antibody VL. [B-70] The method according to [B-69], wherein the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence and the first and second flexible linkers are introduced at any position in a sequence selected from the group consisting of from amino acid 7 (Kabat numbering) to amino acid 16 (Kabat numbering), from amino acid 40 (Kabat numbering) to amino acid 47 (Kabat numbering), from amino acid 55 (Kabat numbering) to amino acid 69 (Kabat numbering), from amino acid 73 (Kabat numbering) to amino acid 79 (Kabat numbering), from amino acid 83 (Kabat numbering) to amino acid 89 (Kabat numbering), from amino acid 95 (Kabat numbering) to amino acid 99 (Kabat numbering), and from amino acid 101 (Kabat numbering) to amino acid 113 (Kabat numbering) of the antibody VH. [B-71] The method according to [B-69], wherein the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are introduced at any position in a sequence selected from the group consisting of from the 7th to the 19th amino acid (Kabat numbering) of the antibody VL, from the 39th to the 46th amino acid (Kabat numbering), from the 49th to the 62nd amino acid (Kabat numbering), and from the 96th to the 107th amino acid (Kabat numbering). [B-72] The method according to [B-65], wherein the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are located near the boundary between the antibody constant region and the antibody VH, and / or near the boundary between the antibody constant region and the antibody VL. [B-73] The method according to [B-73], wherein the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are introduced at any position in the sequence from the 109th amino acid (Kabat numbering) of the antibody VH to the 122nd amino acid (EU numbering) of the antibody heavy chain constant region. [B-74] The method according to [B-72], wherein the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are introduced at any position in the sequence from the 104th amino acid (Kabat numbering) of the antibody VL to the 113th amino acid (Kabat numbering) of the antibody light chain constant region. [B-75] The method according to any one of [B-65] to [B-74], wherein the antibody VL and the antibody VH in the ligand-binding molecule are associated, and the association is abolished by cleavage of the protease cleavage sequence with a protease. [B-76] The method according to any one of [B-52] to [B-75], wherein the ligand is a molecule having biological activity, and the ligand-binding molecule inhibits the biological activity of the ligand upon binding to the ligand. [B-77] The method according to any one of [B-52] to [B-76], wherein the ligand is a cytokine or a chemokine. [B-78] The method according to any one of [B-52] to [B-76], wherein the ligand is a ligand selected from the group consisting of interleukin, interferon, hematopoietic factor, TNF superfamily, chemokine, cell growth factor, and TGF-β family. [B-79] The method according to any one of [B-52] to [B-78], wherein the ligand-binding molecule is an IgG antibody.

[0010] [C-1] The polypeptide according to

[27] , wherein the polypeptide is a ligand-binding molecule capable of binding to a ligand, the ligand-binding molecule includes a single-domain antibody, the single-domain antibody is capable of binding to the ligand, and at least one protease cleavage sequence is introduced, and the binding of the ligand-binding molecule to the ligand in a state where the protease cleavage sequence is cleaved is attenuated compared to the binding of the ligand-binding molecule to the ligand in a state where the protease cleavage sequence is not cleaved. [C-2] The ligand-binding molecule according to [C-1], wherein the ligand is released from the ligand-binding molecule in a state where the protease cleavage sequence is cleaved. [C-3] The ligand-binding molecule according to [C-1] or [C-2], wherein the protease is matriptase and / or urokinase. [C-4] The ligand-binding molecule according to any one of [C-1] to [C-3], wherein the protease is at least one protease selected from the group consisting of human MT-SP1, mouse MT-SP1, human uPA, and mouse uPA. [C-5] The ligand-binding molecule according to any one of [C-1] to [C-4], wherein a first flexible linker is further added to one end of the protease cleavage sequence. [C-6] The ligand-binding molecule according to [C-5], wherein the first flexible linker is a flexible linker composed of a glycine-serine polymer. [C-7] The ligand-binding molecule according to [C-5] or [C-6], wherein a second flexible linker is further added to the other end of the protease cleavage sequence. [C-8] The ligand-binding molecule according to [C-7], wherein the second flexible linker is a flexible linker composed of a glycine-serine polymer. [C-9] The ligand-binding molecule according to any one of [C-1] to [C-8], wherein the single-domain antibody is a VHH, or a single-domain VH antibody, or a single-domain VL antibody. [C-10] The single-domain antibody is a VHH or a single-domain VH antibody, and the cleavage site, or the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are introduced at one or more positions included in one or more sequences selected from the following sequences of the single-domain antibody: the sequence from amino acid 7 (Kabat numbering) to amino acid 17 (Kabat numbering) of the single-domain antibody, the sequence from amino acid 12 (Kabat numbering) to amino acid 17 (Kabat numbering) of the single-domain antibody, the sequence from amino acid 31 (Kabat numbering) to amino acid 35b (Kabat numbering) of the single-domain antibody, the sequence from amino acid 40 (Kabat numbering) to amino acid 47 (Kabat numbering) of the single-domain antibody, the sequence from amino acid 50 (Kabat numbering) to amino acid 65 (Kabat numbering) of the single-domain antibody, the sequence from amino acid 55 (Kabat numbering) to amino acid 69 (Kabat numbering) of the single-domain antibody, the sequence from amino acid 73 (Kabat numbering) to amino acid 79 (Kabat numbering) of the single-domain antibody, the sequence from amino acid 83 (Kabat numbering) to amino acid 89 (Kabat numbering) of the single-domain antibody, the sequence from amino acid 95 (Kabat numbering) to amino acid 99 (Kabat numbering) of the single-domain antibody, the sequence from amino acid 95 (Kabat numbering) to amino acid 102 (Kabat numbering) of the single-domain antibody, the sequence from amino acid 101 (Kabat numbering) to amino acid 113 (Kabat numbering) of the single-domain antibody. [C-11] The single-domain antibody is a single-domain VL antibody, and the cleavage site, or the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are introduced at one or more positions included in one or more sequences selected from the following sequences of the single-domain antibody: the sequence from the 7th amino acid (Kabat numbering) to the 19th amino acid (Kabat numbering) of the single-domain antibody, the sequence from the 24th amino acid (Kabat numbering) to the 34th amino acid (Kabat numbering) of the single-domain antibody, the sequence from the 39th amino acid (Kabat numbering) to the 46th amino acid (Kabat numbering) of the single-domain antibody, the sequence from the 49th amino acid (Kabat numbering) to the 62nd amino acid (Kabat numbering) of the single-domain antibody, the sequence from the 50th amino acid (Kabat numbering) to the 56th amino acid (Kabat numbering) of the single-domain antibody, the sequence from the 89th amino acid (Kabat numbering) to the 97th amino acid (Kabat numbering) of the single-domain antibody, the sequence from the 96th amino acid (Kabat numbering) to the 107th amino acid (Kabat numbering) of the single-domain antibody. [C-12] The ligand is a molecule having biological activity, and the single-domain antibody inhibits the biological activity of the ligand by binding to the ligand, the ligand-binding molecule according to any one of [C-1] to [C-11]. [C-13] The ligand is a molecule having biological activity, and the single-domain antibody has neutralizing activity against the ligand, the ligand-binding molecule according to any one of [C-1] to [C-12]. [C-14] The ligand-binding molecule comprises only a single-domain antibody containing the cleavage site or the protease cleavage sequence, the ligand-binding molecule according to any one of [C-1] to [C-13]. [C-15] The ligand-binding molecule further comprises an antibody Fc region, the ligand-binding molecule according to any one of [C-1] to [C-14]. [C-16] The ligand-binding molecule is a ligand-binding molecule according to any one of [C-1] to [C-15], which contains a series of peptide chains consisting of a single-domain antibody - antibody Fc region from the N-terminus to the C-terminus. [C-17] The ligand-binding molecule is a dimer containing two series of peptide chains consisting of a single-domain antibody - antibody hinge region - antibody Fc region, which is a ligand-binding molecule according to [C-1] to [C-15]. [C-18] The antibody Fc region is an Fc region containing one sequence selected from the amino acid sequences represented by SEQ ID NOs: 18004 to 18007, or an Fc region variant obtained by modifying these Fc regions, which is a ligand-binding molecule according to [C-15] to [C-17]. [C-19] The ligand is a cytokine or chemokine, which is a ligand-binding molecule according to any one of [C-1] to [C-18]. [C-20] The ligand is a ligand selected from interleukin, interferon, hematopoietic factor, TNF superfamily, chemokine, cell growth factor, and TGF-β family, which is a ligand-binding molecule according to any one of [C-1] to [C-19]. [C-21] The ligand-binding molecule that binds to the ligand according to any one of [C-1] to [C-20]. [C-22] The ligand-binding molecule that is fused with the ligand according to any one of [C-1] to [C-20]. [C-23] When the ligand-binding molecule is fused with the ligand, the single-domain antibody contained in the ligand-binding molecule does not bind to another ligand, which is the ligand-binding molecule according to [C-22]. [C-24] The ligand-binding molecule is fused with the ligand via a linker, which is the ligand-binding molecule according to [C-22] or [C-23]. [C-25] The linker does not contain a protease cleavage sequence, which is the ligand-binding molecule according to [C-24]. [C-26] A complex formed by the ligand and a ligand-binding molecule described in any one of [C-1] to [C-20]. [C-27] A fusion protein in which the ligand and a ligand-binding molecule described in any one of [C-1] to [C-20] are fused. [C-28] The fusion protein according to [C-27], wherein when the ligand-binding molecule is fused with the ligand, the single-domain antibody does not further bind to another ligand. [C-29] The fusion protein according to [C-27] or [C-28], wherein the ligand-binding molecule is fused with the ligand via a linker. [C-30] The fusion protein according to [C-29], wherein the linker does not contain a protease cleavage sequence. [C-31] The fusion protein according to [C-29] or [C-30], which is fused in the order of ligand-linker-ligand-binding molecule from the N-terminus to the C-terminus. [C-32] A pharmaceutical composition comprising a ligand-binding molecule described in any one of [C-1] to [C-22]. [C-33] A pharmaceutical composition comprising a ligand-binding molecule described in any one of [C-1] to [C-21] and a ligand. [C-34] A pharmaceutical composition comprising the complex described in [C-26]. [C-35] A pharmaceutical composition comprising a fusion protein described in any one of [C-27] to [C-31]. [C-36] A method for producing a ligand-binding molecule described in any one of [C-1] to [C-20]. [C-37] The production method according to [C-36], which includes introducing a protease cleavage sequence into the single-domain antibody in the ligand-binding molecule containing the single-domain antibody. [C-38] A method for producing a fusion protein described in any one of [C-27] to [C-31], which includes fusing a ligand-binding molecule containing a single-domain antibody into which a protease cleavage sequence has been introduced with a ligand capable of binding to the single-domain antibody. A polynucleotide encoding a ligand-binding molecule according to any one of [C-1] to [C-20]. [C-40] A vector containing the polynucleotide according to [C-39]. [C-41] A host cell containing the polynucleotide according to [C-39] or the vector according to [C-40]. [C-42] A method for producing a ligand-binding molecule according to any one of [C-1] to [C-20], comprising the step of culturing the host cell according to [C-41]. [C-43] A method for producing the ligand-binding molecule according to [C-42], comprising the step of isolating the polypeptide from the culture supernatant. [C-44] A polynucleotide encoding a fusion protein according to any one of [C-27] to [C-31]. [C-45] A vector containing the polynucleotide according to [C-43]. [C-46] A host cell containing the polynucleotide according to [C-43] or the vector according to [C-45]. [C-47] A method for producing a fusion protein according to any one of [C-27] to [C-31], comprising the step of culturing the host cell according to [C-46]. [C-48] A method for producing the ligand-binding molecule according to [C-47], comprising the step of isolating the polypeptide from the culture supernatant. [C-49] A single domain antibody in a ligand-binding molecule capable of binding to a ligand, comprising a sequence from the 4th amino acid at the N-terminus to the 15th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201, a sequence from the 4th amino acid at the N-terminus to the 13th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201, a sequence from the 6th amino acid at the N-terminus to the 13th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201, a sequence from the 1st amino acid at the N-terminus to the 12th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993, a sequence from the 3rd amino acid at the N-terminus to the 12th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993, a sequence from the 3rd amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; a sequence from the 5th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; a sequence from the 5th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; a sequence from the 5th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; a sequence from the 5th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; or a sequence represented by any of SEQ ID NOs: 5 to 18003. [C-50] A method for releasing a ligand from a fusion protein of the ligand and the ligand-binding molecule, comprising cleavage of at least one site having a sequence selected from the sequences shown by any of SEQ ID NOs: 5 to 18003 by a protease, the sequence being from the 4th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence being from the 4th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence being from the 6th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence being from the 1st amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence being from the 3rd amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence being from the 3rd amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence being from the 3rd amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence being from the 3rd amino acid to the 14th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, the sequence being from the 5th amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, the sequence being from the 5th amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003. [C-51] The method according to [C-50], wherein the ligand-binding molecule is fused to the ligand via a linker. [C-52] The method according to [C-51], wherein the linker does not contain a protease cleavage sequence. [C-53] The method according to [C-51] or [C-52], wherein the linker is a linker consisting of a glycine-serine polymer. [C-54] The method according to any one of [C-50] to [C-53], wherein the ligand-binding molecule does not bind to another ligand in the state of being fused to the ligand. [C-55] The method according to any one of [C-49] to [C-54], wherein 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 protease cleavage sequence is uncleaved. [C-56] The method according to [C-55], wherein in the state where the protease cleavage sequence is cleaved, the ligand is released from the ligand-binding molecule. [C-57] The method according to [C-55] or [C-56], wherein the protease is matriptase and / or urokinase. [C-58] The method according to any one of [C-55] to [C-57], wherein the protease is at least one protease selected from human MT-SP1, mouse MT-SP1, human uPA, and mouse uPA. [C-59] The method according to any one of [C-55] to [C-58], wherein a first flexible linker is further added to one end of the protease cleavage sequence. [C-60] The method according to [C-59], wherein the first flexible linker is a flexible linker composed of a glycine-serine polymer. [C-61] The method according to [C-59] or [C-60], wherein a second flexible linker is further added to the other end of the protease cleavage sequence. [C-62] The method according to [C-61], wherein the second flexible linker is a flexible linker composed of a glycine-serine polymer. [C-63] The method according to any one of [C-55] to [C-62], wherein the single-domain antibody is a VHH, or a single-domain VH antibody, or a single-domain VL antibody. [C-64] The single-domain antibody is a VHH or a single-domain VH antibody, and the cleavage site, or the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence and the first flexible linker and the second flexible linker are introduced at one or more positions included in one or more sequences selected from the following sequences of the single-domain antibody: the sequence from the 7th amino acid (Kabat numbering) to the 17th amino acid (Kabat numbering) of the single-domain antibody, the sequence from the 12th amino acid (Kabat numbering) to the 17th amino acid (Kabat numbering) of the single-domain antibody, the sequence from the 31st amino acid (Kabat numbering) to the 35bth amino acid (Kabat numbering) of the single-domain antibody, the sequence from the 40th amino acid (Kabat numbering) to the 47th amino acid (Kabat numbering) of the single-domain antibody, the sequence from the 50th amino acid (Kabat numbering) to the 65th amino acid (Kabat numbering) of the single-domain antibody, the sequence from the 55th amino acid (Kabat numbering) to the 69th amino acid (Kabat numbering) of the single-domain antibody, the sequence from the 73rd amino acid (Kabat numbering) to the 79th amino acid (Kabat numbering) of the single-domain antibody, the sequence from the 83rd amino acid (Kabat numbering) to the 89th amino acid (Kabat numbering) of the single-domain antibody, the sequence from the 95th amino acid (Kabat numbering) to the 99th amino acid (Kabat numbering) of the single-domain antibody, the sequence from the 95th amino acid (Kabat numbering) to the 102nd amino acid (Kabat numbering) of the single-domain antibody, the sequence from the 101st amino acid (Kabat numbering) to the 113th amino acid (Kabat numbering) of the single-domain antibody. [C-65] The single-domain antibody is a single-domain VL antibody, and the cleavage site, or the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are introduced at one or more positions included in one or more sequences selected from the following sequences of the single-domain antibody: the sequence from the 7th amino acid (Kabat numbering) to the 19th amino acid (Kabat numbering) of the single-domain antibody, the sequence from the 24th amino acid (Kabat numbering) to the 34th amino acid (Kabat numbering) of the single-domain antibody, the sequence from the 39th amino acid (Kabat numbering) to the 46th amino acid (Kabat numbering) of the single-domain antibody, the sequence from the 49th amino acid (Kabat numbering) to the 62nd amino acid (Kabat numbering) of the single-domain antibody, the sequence from the 50th amino acid (Kabat numbering) to the 56th amino acid (Kabat numbering) of the single-domain antibody, the sequence from the 89th amino acid (Kabat numbering) to the 97th amino acid (Kabat numbering) of the single-domain antibody, the sequence from the 96th amino acid (Kabat numbering) to the 107th amino acid (Kabat numbering) of the single-domain antibody. [C-66] The ligand is a molecule having biological activity, and the single-domain antibody inhibits the biological activity of the ligand by binding to the ligand, according to the method described in any one of [C-55] to [C-65]. [C-67] The ligand is a molecule having biological activity, and the single-domain antibody has neutralizing activity against the ligand, according to the method described in any one of [C-55] to [C-66]. [C-68] The ligand-binding molecule comprises only a single-domain antibody containing the cleavage site or protease cleavage sequence, according to the method described in any one of [C-55] to [C-67]. [C-69] The ligand-binding molecule further comprises an antibody Fc region, according to the method described in any one of [C-55] to [C-68]. [C-70] The ligand-binding molecule contains a series of peptide chains consisting of a single-domain antibody-antibody Fc region from the N-terminus to the C-terminus, and is the method described in any one of [C-55] to [C-69]. [C-71] The ligand-binding molecule is a dimer containing two series of peptide chains consisting of a single-domain antibody-antibody hinge region-antibody Fc region, and is the method described in [C-55] to [C-69]. [C-72] The antibody Fc region is an Fc region containing one sequence selected from the amino acid sequences represented by SEQ ID NOs: 18004 to 18007, or an Fc region variant obtained by modifying these Fc regions, and is the method described in [C-69] to [C-71]. [C-73] The ligand is a cytokine or a chemokine, and is the method described in any one of [C-55] to [C-72]. [C-74] The ligand is a ligand selected from interleukin, interferon, hematopoietic factor, TNF superfamily, chemokine, cell growth factor, and TGF-β family, and is the method described in any one of [C-55] to [C-73].

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

[0023] Figure 1 shows an example of a polypeptide comprising a protease cleavage sequence of the present disclosure. In this example, the polypeptide is a single domain antibody comprising a protease cleavage sequence. When the protease cleavage sequence contained in the single domain antibody is uncleaved, the single domain antibody can bind to a ligand. When the protease cleavage sequence is cleaved, the single domain antibody is cleaved and cannot bind to the ligand, resulting in the release of the ligand.

Figure 8

[0023] Figure 1 shows an example of a polypeptide comprising a protease cleavage sequence according to the present disclosure. In this example, the polypeptide is a fusion protein of a ligand and a single domain antibody comprising the protease cleavage sequence. When the protease cleavage sequence contained 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 protease cleavage sequence 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 9

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Figure 12

Figure 13

Figure 14

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Figure 16

Figure 17

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Figure 20

Mode for Carrying Out the Invention

[0012] Amino acid In this specification, for example, amino acids are represented by one-letter codes or three-letter codes, or both, as represented by Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, Val / V. Natural amino acid The "natural amino acids" in this specification refer to the 20 types of amino acids contained in proteins. Specifically, they refer to Gly, Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, His, Glu, Asp, Gln, Asn, Cys, Met, Lys, Arg, Pro.

[0013] Peptide The "peptide" in this specification refers to a compound in which two or more amino acid molecules are bonded by the removal of one molecule of water from one amino group and the other carboxyl group. The number of amino acids contained in the peptide is not limited. Therefore, both oligopeptides and polypeptides are included in the peptide.

[0014] Polypeptide The polypeptide in the present disclosure generally refers to a peptide having a length of about 10 amino acids or more and a protein. When a chain of amino acids connected by peptide bonds from the N-terminus to the C-terminus is regarded as a series of peptide chains, the polypeptide of the present disclosure may be a complex protein formed by a plurality of series of peptide chains through interactions such as S-S bonds, hydrophobic interactions, and ionic bonds. Further, the polypeptide in the present disclosure is usually a polypeptide consisting of an artificially designed sequence, but is not particularly limited, and may be, for example, any of a synthetic polypeptide, a recombinant polypeptide, and the like. Furthermore, fragments of the above polypeptides are also included in the polypeptides of the present disclosure.

[0015] Isolated polypeptide The polypeptide of the present disclosure can refer to an isolated polypeptide. An "isolated" polypeptide is separated from the components of its original environment. In some embodiments, the polypeptide is purified to a purity of more than 95% or 99% as measured by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). When the polypeptide is an antibody, for a review of methods for evaluating the purity of the antibody, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0016] Protease As used herein, the term "protease" refers to an enzyme, usually an endopeptidase, such as an endopeptidase or exopeptidase that hydrolyzes peptide bonds.

[0017] Although not to be construed restrictively, specific types of proteases include cysteine proteases (including cathepsin families B, L, S, etc.), aspartyl proteases (cathepsin D, E, K, O, etc.), serine proteases (matriptase (including MT-SP1), cathepsin A and G, thrombin, plasmin, urokinase (uPA), tissue plasminogen activator (tPA), elastase, proteinase 3, thrombin, kallikrein, tryptase, chymase), metalloproteases (metalloproteases (MMP1-28) including both membrane-bound (MMP14-17 and MMP24-25) and secreted (MMP1-13 and MMP18-23 and MMP26-28)), protease A disintegrin and metalloprotease (ADAM), metalloprotease with an A disintegrin or thrombospondin motif (ADAMTS), meprin (meprin alpha, meprin beta), CD10 (CALLA), and prostate-specific antigen (PSA), legumain, TMPRSS3, TMPRSS4, neutrophil elastase (HNE), beta-secretase (BACE), fibroblast activation protein alpha (FAP), granzyme B, guanidino benzoatase (GB), hepsin, neprilysin, NS3 / 4A, HCV-NS3 / 4, calpain, ADAMDEC1, renin, cathepsin C, cathepsin V / L2, cathepsin X / Z / P, curpain, osteopontin 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), furin, and the like.

[0018] The protease of the present disclosure can be a protease closely related to diseased tissue. For example, (1) A protease that is expressed at a higher level in diseased tissue than in normal tissue, (2) A protease that has higher activity in diseased tissue than in normal tissue, (3) A protease that is expressed at a higher level in cells in diseased tissue than in normal cells, (4) A protease that has higher activity in target cells in diseased tissue than in normal cells. It can refer to any of these. Examples of diseased tissue include cancer tissue and inflammatory tissue.

[0019] The term "cancer tissue" means a tissue containing at least one cancer cell. Thus, it refers to all cell types contributing to the formation of a tumor mass containing cancer cells and endothelial cells, for example, as cancer tissue contains cancer cells and blood vessels. In this specification, a tumor refers to a foci of tumor tissue. The term "tumor" is generally used to mean a benign or malignant neoplasm.

[0020] In this specification, "inflammatory tissue" includes, for example, the following by way of illustration. · 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, kidney, and lung · Tissue in which rejection occurs in organ transplantation · Blood vessels and heart (myocardium) in arteriosclerosis and heart failure · Visceral fat in metabolic syndrome · Skin tissue in atopic dermatitis and other dermatitis · Spinal nerves in intervertebral disc herniation and chronic low back pain

[0021] Urokinase (uPA) Urokinase (uPA), also known as urokinase-type plasminogen activator, is a type of extracellular serine protease. The terms urokinase, urokinase-type plasminogen activator, and uPA are used interchangeably herein and include any suitable uPA that can be derived from any organism, is naturally occurring, is endogenously produced, and / or is in recombinant form as long as it has serine protease activity. For example, in one aspect of the present disclosure, uPA is in a two-chain active form (tc-uPA). Urokinase further includes uPA from another species such as human uPA or uPA from mammals such as primates (e.g., chimpanzee, cynomolgus monkey, or rhesus monkey); rodents (such as mouse or rat), lagomorphs (such as rabbit), or artiodactyls (such as cow, sheep, pig, or camel). The terms urokinase-type plasminogen activator and uPA further include uPA produced recombinantly. This includes any uPA "produced by recombinant gene technology" including, but not limited to, proteins expressed in mammalian cell lines or bacteria or yeast. Urokinase (uPA) is said to be highly associated with cancer tissue, and peptide sequences cleavable by urokinase (uPA) are cleaved more in cancer tissue than in normal tissue.

[0022] Matriptase Matriptase is a type of serine protease. Matriptase of the present disclosure encompasses any naturally occurring, endogenously produced, and / or recombinant form of matriptase that may be derived from any suitable organism, so long as it possesses serine protease activity. Matriptase includes human matriptase or matriptase from another species, such as a mammalian-derived matriptase, such as matriptase from a primate (e.g., chimpanzee, cynomolgus monkey, or rhesus monkey); a rodent (e.g., mouse or rat), a lagomorph (e.g., rabbit), or an artiodactyl (e.g., cow, sheep, pig, or camel). Matriptase also encompasses recombinantly produced matriptase. This includes any matriptase "produced by recombinant genetic technology," including, but not limited to, proteins expressed in mammalian cell lines, bacteria, or yeast. Matriptase includes MT-SP1, which encompasses any naturally occurring, endogenously produced, and / or recombinant form of MT-SP1 that may be derived from any suitable organism, so long as it possesses serine protease activity. MT-SP1 encompasses human MT-SP1 or MT-SP1 from another species, such as mammalian-derived MT-SP1, such as MT-SP1 from a primate (e.g., chimpanzee, cynomolgus monkey, or rhesus monkey); a rodent (e.g., mouse or rat), a lagomorph (e.g., rabbit), or an artiodactyl (e.g., cow, sheep, pig, or camel). MT-SP1 also encompasses recombinantly produced MT-SP1. This includes any MT-SP1 "produced by recombinant genetic technology," including, but not limited to, proteins expressed in mammalian cell lines, bacteria, or yeast. Matriptase (including MT-SP1) is said to be highly associated with cancer tissue, and peptide sequences that can be cleaved by matriptase (including MT-SP1) are cleaved more frequently in cancer tissue than in normal tissue. Since the sequences of human MT-SP1 and mouse MT-SP1 are quite similar, their enzymatic activities toward the same substrates are likely to be similar.

[0023] Method for confirming cleavage by protease As a method for evaluating the cleavage of the protease substrates or protease cleavage sequences described in this specification by proteases, the method described in Mol Cell Proteomics. 2014 Jun;13(6):1585-97. doi: 10.1074 / mcp.M113.033308. Epub 2014 Apr 4. is exemplified. Fix the protease substrate or protease cleavage sequence to be evaluated on a peptide array, treat the peptide array with a solution containing the protease to be evaluated, and calculate the cleavage rate using the fluorescence value measured from the chip as follows: TIFF0007716979000001.tif18170 The type and concentration of protease, treatment temperature, and treatment time used for the evaluation can be appropriately selected. Exemplarily, PBS containing 1000 nM human uPA, PBS containing 1000 nM mouse uPA, PBS containing 500 nM human MT-SP1, or PBS containing 500 nM mouse MT-SP1 can be used, and the treatment can be carried out at 37°C for 1 hour.

[0024] Alternatively, instead of using a protease-containing solution, the peptide array can be treated with serum (including human serum and mouse serum), and the cleavage rate can be calculated using the fluorescence value measured from the chip as follows: TIFF0007716979000002.tif18170 The type and concentration of serum, treatment temperature, and treatment time used for the evaluation can be appropriately selected. Exemplarily, human serum diluted to 80% concentration can be used as the treatment solution, and the treatment can be carried out at 37°C overnight. "Not cleaved by serum" in this specification means that the cleavage rate by serum measured and calculated by the above method is 1.5 or less, or the cleavage rate by serum measured and calculated by the above method is lower than that of the peptide shown in SEQ ID NO: 4.

[0025] As a method for qualitatively confirming whether the protease cleavage sequence contained in a polypeptide has been cleaved by a protease, it can be confirmed by subjecting a solution containing the polypeptide containing the protease cleavage sequence to SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and measuring the molecular weight of the fragment. It can also be confirmed by comparing the molecular weights of the polypeptide before protease treatment and the polypeptide after protease treatment.

[0026] As used herein, the term "cleaved" refers to a state in which a polypeptide is fragmented after modification of the protease cleavage sequence by a protease and / or reduction of the cysteine-cysteine disulfide bond of the protease cleavage sequence. As used herein, the term "not cleaved" refers to a state in which the portions on both sides of the protease cleavage sequence in a polypeptide are linked in the absence of cleavage of the protease cleavage sequence by a protease and / or in the absence of reduction of the cysteine-cysteine disulfide bond of the protease cleavage sequence.

[0027] In addition, by quantifying the amount of cleavage fragments after protease treatment separated by electrophoresis methods such as SDS-PAGE, it is possible to evaluate the protease cleavage sequence and the cleavage rate of a molecule into which the protease cleavage sequence has been introduced. The following method can be mentioned as a non-limiting aspect of the method for evaluating the cleavage rate of a molecule into which a protease cleavage sequence has been introduced. For example, when evaluating the cleavage rate of an antibody variant into which a protease cleavage sequence has been introduced using recombinant human u-Plasminogen Activator / Urokinase (human uPA, huPA) (R&D Systems; 1310-SE-010) or recombinant human Matriptase / ST14 Catalytic Domain (human MT-SP1, hMT-SP1) (R&D Systems; 3946-SE-010), after reacting for 1 hour under the conditions of 40 nM huPA or 3 nM hMT-SP1, 100 μg / mL antibody variant, PBS, and 37°C, it is subjected to capillary electrophoresis immunoassay. Wes (Protein Simple) can be used for the capillary electrophoresis immunoassay, but it is not limited to this, and as a method alternative to the capillary electrophoresis immunoassay, it may be detected by Western Blotting method after separation by SDS-PAGE or the like, and it is not limited to these methods. An anti-human lambda chain HRP-labeled antibody (abcam; ab9007) can be used for the detection of the light chain before and after cleavage, but any antibody that can detect the cleavage fragment can be used. By outputting the area of each peak obtained after protease treatment using software dedicated to Wes (Compass for SW; Protein Simple), the cleavage rate (%) of the antibody variant can be calculated by the formula (cleaved light chain peak area) * 100 / (cleaved light chain peak area + uncleaved light chain peak area). The calculation of the cleavage rate is possible as long as protein fragments before and after protease treatment can be detected, and the cleavage rate can be calculated for various proteins not limited to antibody variants into which a protease cleavage sequence has been introduced.

[0028] After administering a molecule introduced with a protease cleavage sequence to an animal, it is possible to calculate the cleavage rate in vivo by detecting the administered molecule in a blood sample. For example, after administering an antibody variant introduced with a protease cleavage sequence to a mouse, plasma is recovered from the blood sample, and the antibody is purified by a method known to those skilled in the art using Dynabeads Protein A (Thermo; 10001D), and the protease cleavage rate of the antibody variant can be evaluated by subjecting it to a capillary electrophoresis immunoassay. Wes (Protein Simple) can be used for the capillary electrophoresis immunoassay, but it is not limited thereto. As a method alternative to the capillary electrophoresis immunoassay, it may be detected by Western Blotting after separation by SDS-PAGE or the like, and it is not limited to these methods. An anti-human lambda chain HRP-labeled antibody (abcam; ab9007) can be used to detect the light chain of the antibody variant recovered from the mouse, but any antibody capable of detecting the cleavage fragment can be used. The area of each peak obtained by the capillary electrophoresis immunoassay is output using software dedicated to Wes (Compass for SW; Protein Simple), and by calculating (light chain peak area) / (heavy chain peak area) as the light chain residual ratio, it is possible to calculate the ratio of the full-length light chain remaining without being cleaved in the mouse body. Calculation of the cleavage efficiency in vivo is possible as long as protein fragments recovered from the living body can be detected, and molecules introduced with a protease cleavage sequence can calculate the cleavage rate not only for antibody variants but also for various proteins. By calculating the cleavage rate using the method described above, it is possible to compare, for example, the cleavage rates in vivo of antibody variants introduced with different cleavage sequences, and it is also possible to compare the cleavage rates of the same antibody variant between different animal models such as a normal mouse model and a tumor xenograft mouse model.

[0029] Protease substrate One aspect of the present disclosure relates to protease substrates. In one embodiment, a protease substrate refers to a peptide or polypeptide having an amino acid sequence that is hydrolyzed by the action of a protease. In another embodiment, the protease substrate is a peptide having a length of about 5 to 15 amino acids. The protease substrate of the present disclosure can be specifically modified (cleaved) by a protease at a rate of about 0.001 to 1500×10 4 M -1 S -1 or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500×10 4 M -1 S -1 per second.

[0030] In certain embodiments, the protease substrate of the present disclosure has a higher cleavage rate by a protease compared to a protease substrate comprising any one of the sequences of SEQ ID NO: 1, 2, or 3. In certain embodiments, the protease substrate of the present disclosure has a higher cleavage rate by human uPA compared to a protease substrate comprising any one of the sequences of SEQ ID NO: 1, 2, or 3. In certain embodiments, the cleavage rate of the protease substrate of the present disclosure by human uPA is 1.5 or more, 1.6 or more, 1.8 or more, 2 or more, 2.2 or more, 2.4 or more, 2.6 or more, 2.8 or more, 3 or more, 3.2 or more, 3.4 or more, 3.6 or more, 3.8 or more, 3.9 or more, or 4 or more. In certain embodiments, the protease substrate of the present disclosure has a higher cleavage rate by human MT-SP1 compared to a protease substrate comprising any one of the sequences of SEQ ID NO: 1, 2, or 3. In certain embodiments, the cleavage rate of the protease substrate of the present disclosure by human MT-SP1 is 1.5 or more, 1.6 or more, 1.8 or more, 2 or more, 2.2 or more, 2.4 or more, 2.6 or more, 2.8 or more, 3 or more, 3.2 or more, 3.4 or more, 3.6 or more, 3.8 or more, 3.9 or more, or 4 or more. In certain embodiments, the protease substrate of the present disclosure has a higher cleavage rate by mouse uPA compared to the protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3. In certain embodiments, the cleavage rate of the protease substrate of the present disclosure by mouse uPA is 1 or more, 1.1 or more, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 1.6 or more, 1.7 or more, 1.8 or more, 1.9 or more, 2 or more, 2.5 or more, 3 or more, 3.5 or more, or 4 or more. In certain embodiments, the protease substrate of the present disclosure has a higher cleavage rate by mouse MT-SP1 compared to the protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3. In certain embodiments, the cleavage rate of the protease substrate of the present disclosure by mouse MT-SP1 is 1.5 or more, 1.6 or more, 1.8 or more, 2 or more, 2.2 or more, 2.4 or more, 2.6 or more, 2.8 or more, 3 or more, 3.2 or more, 3.4 or more, 3.6 or more, 3.8 or more, 3.9 or more, or 4 or more. In certain embodiments, the protease substrate of the present disclosure has a higher ratio of the cleavage rate by human uPA to the cleavage rate by human serum compared to the protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3. In certain embodiments, the ratio of the cleavage rate of the protease substrate of the present disclosure by human uPA to the cleavage rate by human serum is 0.9 or more, 0.95 or more, 1 or more, 1.2 or more, 1.4 or more, 1.6 or more, 1.8 or more, 2 or more, 2.2 or more, 2.4 or more, 2.6 or more, 2.8 or more, 3 or more, 3.2 or more, 3.4 or more, 3.6 or more, 3.8 or more, or 4 or more. Here, when the cleavage rate by human serum is 1 or less, it can be used for the calculation of the ratio of the cleavage rate by human uPA to the cleavage rate by human serum after converting it to 1. In one embodiment, the protease substrate of the present disclosure has a higher ratio of the cleavage rate by human MT-SP1 to the cleavage rate by human serum than the protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3. In one embodiment, the ratio of the cleavage rate by human MT-SP1 to the cleavage rate by human serum of the protease substrate of the present disclosure is 0.7 or more, 0.8 or more, 0.9 or more, 0.95 or more, 1 or more, 1.2 or more, 1.4 or more, 1.6 or more, 1.8 or more, 2 or more, 2.2 or more, 2.4 or more, 2.5 or more, or 2.6 or more. Here, when the cleavage rate by human serum is 1 or less, it can be used for the calculation of the ratio of the cleavage rate by human MT-SP1 to the cleavage rate by human serum after converting it to 1. In one embodiment, the protease substrate of the present disclosure has a higher ratio of the cleavage rate by mouse uPA to the cleavage rate by human serum than the protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3. In one embodiment, the ratio of the cleavage rate by mouse uPA to the cleavage rate by human serum of the protease substrate of the present disclosure is 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, 0.95 or more, 1 or more, 1.1 or more, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 1.6 or more, 1.7 or more, 1.8 or more, 1.9 or more, or 2 or more. Here, when the cleavage rate by human serum is 1 or less, it can be used for the calculation of the ratio of the cleavage rate by mouse uPA to the cleavage rate by human serum after converting it to 1. In one embodiment, the protease substrate of the present disclosure has a higher ratio of the cleavage rate by mouse MT-SP1 to the cleavage rate by human serum than the protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3. In one embodiment, the ratio of the cleavage rate by mouse MT-SP1 to the cleavage rate by human serum of the protease substrate of the present disclosure is 0.7 or more, 0.8 or more, 0.9 or more, 0.95 or more, 1 or more, 1.2 or more, 1.4 or more, 1.6 or more, 1.8 or more, 2 or more, 2.2 or more, 2.4 or more, 2.5 or more, or 2.6 or more. Here, when the cleavage rate by human serum is 1 or less, it can be used for the calculation of the ratio of the cleavage rate by mouse MT-SP1 to the cleavage rate by human serum after converting it to 1. In one embodiment, the protease substrate of the present disclosure is not cleaved by human serum. As a specific example, the cleavage rate of the protease substrate of the present disclosure by human serum is 1.5 or less. As another specific example, the cleavage rate of the protease substrate of the present disclosure by human serum is lower than the cleavage rate of the peptide represented by SEQ ID NO: 4 by human serum.

[0031] As a more specific example, for example, the sequence from the 4th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 4th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 6th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 1st amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 14th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, the sequence from the 5th amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, the sequence from the 5th amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, and the peptide represented by any of the sequences shown in SEQ ID NOs: 5 to 18003 are useful as protease substrates that are hydrolyzed by the action of proteases. As another specific example, for example, a peptide represented by a sequence composed in the order of "sequence selected from the following Group A - sequence selected from the following Group B" from the N-terminus is also useful as a protease substrate that is hydrolyzed by the action of proteases: (Group A) The sequence from the 1st amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the second amino acid to the ninth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the third amino acid to the ninth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the fourth amino acid to the ninth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the fifth amino acid to the ninth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the sixth amino acid to the ninth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the seventh amino acid to the ninth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the eighth amino acid to the ninth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the first amino acid to the sixth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the second amino acid to the sixth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the third amino acid to the sixth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the fourth amino acid to the sixth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the fifth amino acid to the sixth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the first amino acid to the eighth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, The sequence from the second amino acid to the eighth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, The sequence from the third amino acid to the eighth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, The sequence from the 4th amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, The sequence from the 5th amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, The sequence from the 6th amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, The sequence from the 7th amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003; (Group B) The sequence from the 10th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 10th amino acid to the 14th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 10th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 10th amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 10th amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 7th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 14th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 9th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, The sequence from the 9th amino acid to the 14th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, The sequence from the 9th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, The sequence from the 9th amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, The sequence from the 9th amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, The sequence from the 9th amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003.

[0032] The protease substrate of the present disclosure can be utilized as a library for selecting, for example, a polypeptide having properties suitable for the intended purpose when incorporated into a polypeptide. Specifically, in order to selectively cleave a polypeptide by a protease localized in a lesion, its protease sensitivity can be evaluated. After being administered to a living body, the polypeptide may reach the lesion through contact with various proteases. Therefore, it is desirable to have sensitivity to the protease localized in the lesion while having as high resistance as possible to other proteases. In order to select a desirable protease substrate according to the intended purpose, if the sensitivity of each protease substrate to various proteases is comprehensively analyzed in advance, protease resistance can be known. Based on the obtained protease resistance spectrum, a protease substrate having the required sensitivity and resistance can be found. Alternatively, the polypeptide incorporated with the protease substrate reaches the lesion through various environmental stresses such as changes in pH, temperature, and redox stress, as well as only through the enzymatic action of the protease. Based on the information comparing the resistance of each protease substrate to such external factors, a protease substrate with desirable properties according to the purpose can also be selected.

[0033] Protease cleavage sequence One aspect of the present disclosure relates to a protease cleavage sequence. The protease cleavage sequence is a specific amino acid sequence that is specifically recognized by the protease when the polypeptide is hydrolyzed by the protease in an aqueous solution. In the present disclosure, the protease cleavage sequence may sometimes be referred to as a peptide sequence cleavable by the protease.

[0034] The protease cleavage sequence of the present disclosure can be specifically modified (cleaved) by the protease at a rate of about 0.001 to 1500×10 4 M -1 S -1 or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500×10 4 M -1 S -1

[0035] ​For example, any of the sequences from the 4th amino acid to the 15th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 5 to 17201, the sequence from the 4th amino acid to the 13th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 5 to 17201, the sequence from the 6th amino acid to the 13th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 5 to 17201, the sequence from the 1st amino acid to the 12th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 12th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 11th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 10th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 14th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 17994 to 18003, the sequence from the 5th amino acid to the 12th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 17994 to 18003, the sequence from the 5th amino acid to the 10th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 17994 to 18003, and any of the sequences represented by SEQ ID NOs: 5 to 18003 are useful as protease cleavage sequences. The present disclosure also relates to the use of these sequences as protease cleavage sequences. For example, any of the sequences composed in the order of "sequence selected from the following Group A - sequence selected from the following Group B" from the N-terminus are useful as protease cleavage sequences. The present disclosure also relates to the use of these sequences as protease cleavage sequences: (Group A) The sequence from the 1st amino acid to the 9th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 5 to 17201, The sequence from the 2nd amino acid to the 9th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 5 to 17201, The sequence from the 3rd amino acid to the 9th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 5 to 17201, The sequence from the 4th amino acid to the 9th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 5 to 17201, The sequence from the 5th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201 The sequence from the 6th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201 The sequence from the 7th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201 The sequence from the 8th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201 The sequence from the 1st amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993 The sequence from the 2nd amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993 The sequence from the 3rd amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993 The sequence from the 4th amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993 The sequence from the 5th amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993 The sequence from the 1st amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 2nd amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 3rd amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 4th amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 5th amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 6th amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 7th amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003; (Group B) The sequence from the 10th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 10th amino acid to the 14th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 10th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 10th amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 10th amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 7th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 14th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 9th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, The sequence from the 9th amino acid to the 14th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 9th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 9th amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 9th amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 9th amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003

[0036] In certain embodiments, the protease cleavage sequence is linked or otherwise attached to an antibody. For example, the protease cleavage sequence is used to link one or more agents to an antibody that binds to a predetermined target such that when the protease cleavage sequence is exposed to a protease, i.e., matriptase and / or uPA, the protease cleavage sequence is cleaved and the agent is released from the antibody.

[0037] In certain embodiments, the protease cleavage sequence of the present disclosure has a higher cleavage rate by protease compared to a protease substrate comprising any one of the sequences of SEQ ID NOs: 1, 2, 3. In certain embodiments, the protease cleavage sequence of the present disclosure has a higher cleavage rate by human uPA compared to a protease substrate comprising any one of the sequences of SEQ ID NOs: 1, 2, 3. In certain embodiments, the cleavage rate of the protease cleavage sequence of the present disclosure by human uPA is 1.5 or more, 1.6 or more, 1.8 or more, 2 or more, 2.2 or more, 2.4 or more, 2.6 or more, 2.8 or more, 3 or more, 3.2 or more, 3.4 or more, 3.6 or more, 3.8 or more, 3.9 or more, or 4 or more. In certain embodiments, the protease cleavage sequence of the present disclosure has a higher cleavage rate by human MT-SP1 compared to protease substrates containing any one of the sequences of SEQ ID NO: 1, 2, or 3. In certain embodiments, the cleavage rate of the protease cleavage sequence of the present disclosure by human MT-SP1 is 1.5 or more, 1.6 or more, 1.8 or more, 2 or more, 2.2 or more, 2.4 or more, 2.6 or more, 2.8 or more, 3 or more, 3.2 or more, 3.4 or more, 3.6 or more, 3.8 or more, 3.9 or more, or 4 or more. In certain embodiments, the protease cleavage sequence of the present disclosure has a higher cleavage rate by mouse uPA compared to protease substrates containing any one of the sequences of SEQ ID NO: 1, 2, or 3. In certain embodiments, the cleavage rate of the protease cleavage sequence of the present disclosure by mouse uPA is, in certain embodiments, the cleavage rate of the protease substrate of the present disclosure by mouse uPA is 1 or more, 1.1 or more, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 1.6 or more, 1.7 or more, 1.8 or more, 1.9 or more, 2 or more, 2.5 or more, 3 or more, 3.5 or more, or 4 or more. In certain embodiments, the protease cleavage sequence of the present disclosure has a higher cleavage rate by mouse MT-SP1 compared to protease substrates containing any one of the sequences of SEQ ID NO: 1, 2, or 3. In certain embodiments, the cleavage rate of the protease cleavage sequence of the present disclosure by mouse MT-SP1 is 1.5 or more, 1.6 or more, 1.8 or more, 2 or more, 2.2 or more, 2.4 or more, 2.6 or more, 2.8 or more, 3 or more, 3.2 or more, 3.4 or more, 3.6 or more, 3.8 or more, 3.9 or more, or 4 or more. In certain embodiments, the protease cleavage sequence of the present disclosure has a higher ratio of the cleavage rate by human uPA to the cleavage rate by human serum compared to protease substrates containing any one of the sequences of SEQ ID NO: 1, 2, or 3. In certain embodiments, the ratio of the cleavage rate of the protease cleavage sequence of the present disclosure by human uPA to the cleavage rate by human serum is 0.9 or more, 0.95 or more, 1 or more, 1.2 or more, 1.4 or more, 1.6 or more, 1.8 or more, 2 or more, 2.2 or more, 2.4 or more, 2.6 or more, 2.8 or more, 3 or more, 3.2 or more, 3.4 or more, 3.6 or more, 3.8 or more, or 4 or more. Here, when the cleavage rate by human serum is 1 or less, it can be used for the calculation of the ratio of the cleavage rate by human uPA to the cleavage rate by human serum after converting it to 1. In certain embodiments, the protease cleavage sequence of the present disclosure has a higher ratio of the cleavage rate by human MT-SP1 to the cleavage rate by human serum compared to the protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3. In certain embodiments, the ratio of the cleavage rate by human MT-SP1 to the cleavage rate by human serum of the protease cleavage sequence of the present disclosure is 0.7 or more, 0.8 or more, 0.9 or more, 0.95 or more, 1 or more, 1.2 or more, 1.4 or more, 1.6 or more, 1.8 or more, 2 or more, 2.2 or more, 2.4 or more, 2.5 or more, or 2.6 or more. Here, when the cleavage rate by human serum is 1 or less, it can be used for calculating the ratio of the cleavage rate by human MT-SP1 to the cleavage rate by human serum after converting it to 1. In certain embodiments, the protease cleavage sequence of the present disclosure has a higher ratio of the cleavage rate by mouse uPA to the cleavage rate by human serum compared to the protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3. In certain embodiments, the ratio of the cleavage rate by mouse uPA to the cleavage rate by human serum of the protease cleavage sequence of the present disclosure is 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, 0.95 or more, 1 or more, 1.1 or more, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 1.6 or more, 1.7 or more, 1.8 or more, 1.9 or more, or 2 or more. Here, when the cleavage rate by human serum is 1 or less, it can be used for calculating the ratio of the cleavage rate by mouse uPA to the cleavage rate by human serum after converting it to 1. In certain embodiments, the protease cleavage sequence of the present disclosure has a higher ratio of the cleavage rate by mouse MT-SP1 to the cleavage rate by human serum compared to the protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3. In certain embodiments, the ratio of the cleavage rate by mouse MT-SP1 to the cleavage rate by human serum of the protease cleavage sequence of the present disclosure is 0.7 or more, 0.8 or more, 0.9 or more, 0.95 or more, 1 or more, 1.2 or more, 1.4 or more, 1.6 or more, 1.8 or more, 2 or more, 2.2 or more, 2.4 or more, 2.5 or more, or 2.6 or more. Here, when the cleavage rate by human serum is 1 or less, it can be used for calculating the ratio of the cleavage rate by mouse MT-SP1 to the cleavage rate by human serum after converting it to 1. In one embodiment, the protease cleavage sequence of the present disclosure is not cleaved by human serum. As a specific example, the cleavage rate of the protease cleavage sequence of the present disclosure by human serum is 1.5 or less. As another specific example, the cleavage rate of the protease substrate of the present disclosure by human serum is lower than the cleavage rate of the peptide represented by SEQ ID NO: 4 by human serum.

[0038] The protease cleavage sequence of the present disclosure can be utilized as a library for selecting a sequence having properties suitable for a purpose, for example, when incorporated into a polypeptide. Specifically, in order to selectively cleave a polypeptide by a protease localized in a lesion, its protease sensitivity can be evaluated. After being administered to a living body, the polypeptide may reach the lesion through contact with various proteases. Therefore, it is desirable to have sensitivity to the protease localized in the lesion while having as high resistance as possible to other proteases. Depending on the purpose, in order to select a desirable protease cleavage sequence, the protease resistance can be known by comprehensively analyzing the sensitivity of each protease cleavage sequence to various proteases in advance. Based on the obtained protease resistance spectrum, a protease cleavage sequence having the required sensitivity and resistance can be found. Alternatively, the polypeptide incorporated with the protease cleavage sequence reaches the lesion through various environmental stresses such as changes in pH, temperature, and redox stress, as well as only the enzymatic action of the protease. Based on the information comparing the resistance of each protease cleavage sequence to such external factors, it is also possible to select a protease cleavage sequence having desirable properties according to the purpose.

[0039] In some embodiments, the protease cleavage sequence is cleavable by at least matriptase. In some embodiments, the protease cleavage sequence is cleavable by at least MT-SP1. In some embodiments, the protease cleavage sequence is cleavable by at least uPA. In some embodiments, the protease cleavage sequence is cleavable by at least matriptase and uPA. In some embodiments, the protease cleavage sequence is cleavable by at least MT-SP1 and uPA.

[0040] In some embodiments, the protease cleavage sequence is a substrate for matriptase and / or uPA and is a substrate resistant to cleavage by at least one other protease. In some embodiments, the protease cleavage sequence is a substrate for matriptase and / or uPA and is a substrate resistant to cleavage by at least plasmin. In some embodiments, the protease cleavage sequence is a substrate for matriptase and / or uPA and is a substrate resistant to cleavage by at least tissue plasminogen activator (tPA). In some embodiments, the protease cleavage sequence is a substrate for matriptase and / or uPA and is a substrate resistant to cleavage by at least human serum.

[0041] Amino acid modification For the modification of amino acids in the amino acid sequence of a polypeptide, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and Overlap extension PCR can be appropriately employed. Also, as methods for modifying amino acids to substitute amino acids other than natural amino acids, a plurality of known methods can also be employed (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. U.S.A. (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct(Protein Express)) containing a tRNA in which a non-natural amino acid is bound to a complementary amber suppressor tRNA of the UAG codon (amber codon), which is one of the stop codons, is also preferably used.

[0042] In this specification, the meaning of the term "and / or" used when representing the amino acid modification site includes all combinations in which "and" and "or" are appropriately combined. Specifically, for example, "the amino acids at positions 37, 45, and / or 47 are substituted" includes the following variations of amino acid modifications; (a) position 37, (b) position 45, (c) position 47, (d) positions 37 and 45, (e) positions 37 and 47, (f) positions 45 and 47, (g) positions 37, 45, and 47.

[0043] In this specification, as an expression representing an amino acid modification, an expression in which the one-letter code or three-letter code of the amino acid before and after the modification is written before and after the number representing the specific position may be appropriately used. For example, the modification F37V or Phe37Val used when adding an amino acid substitution in the antibody variable region represents the substitution of Phe at position 37 represented by Kabat numbering with Val. That is, the number represents the position of the amino acid represented by Kabat numbering, the one-letter code or three-letter code of the amino acid described before it represents the amino acid before substitution, and the one-letter code or three-letter code of the amino acid described after it represents the amino acid after substitution. Similarly, the modification P238A or Pro238Ala used when adding an amino acid substitution to the Fc region included in the antibody constant region represents the substitution of Pro at position 238 represented by EU numbering with Ala. That is, the number represents the position of the amino acid represented by EU numbering, the one-letter code or three-letter code of the amino acid described before it represents the amino acid before substitution, and the one-letter code or three-letter code of the amino acid described after it represents the amino acid after substitution.

[0044] "Inserting" amino acid sequence A into amino acid sequence B refers to dividing amino acid sequence B into two parts without deleting it and connecting the two parts with amino acid sequence A in between (that is, creating a new amino acid sequence such as "the first half of amino acid sequence B - amino acid sequence A - the second half of amino acid sequence B"). "Introducing" amino acid sequence A into amino acid sequence B refers to dividing amino acid sequence B into two parts and connecting the two parts with amino acid sequence A in between. 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 starting from the amino acid residues of amino acid sequence B adjacent to amino acid sequence A and then connect the two parts with amino acid sequence A (that is, replacing a part of amino acid sequence B with amino acid sequence A). In this specification, the term "near the boundary between part A and part B" refers to the part before and after the site where part A and part B in the polypeptide are connected and that does not significantly affect the secondary structure of part A and / or part B.

[0045] Antibody and antibody fragment As used herein, the term "antibody" is used in the broadest sense and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, encompassing various antibody structures as long as they exhibit the desired antigen-binding activity.

[0046] "Antibody fragment" refers to a molecule other than the complete antibody that includes a portion of the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0047] The terms "full-length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to the native antibody structure or having a heavy chain that includes the Fc region as defined herein.

[0048] In some embodiments herein, a protease cleavage sequence is included in the antibody, but regardless of whether a protease cleavage sequence is included, it can be expressed as an "antibody."

[0049] Variable region The term "variable region" or "variable domain" refers to the domain of 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, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) One VH or VL domain would be sufficient to confer antigen-binding specificity.

[0050] CDR As used herein, the term "complementary determining region" or "CDR" refers to regions of an array that are hypervariable and / or form structurally defined loops ("hypervariable loops") and / or antigen contact residues ("antigen contacts") in each region of the variable domain of an antibody or single domain antibody. Typically, an antibody contains six CDRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary antibody CDRs 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 contacts occurring at amino acid residues 27c - 36 (L1), 46 - 55 (L2), 89 - 96 (L3), 30 - 35b (H1), 47 - 58 (H2), and 93 - 101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732 - 745 (1996)); and, (d) Combinations of (a), (b), and / or (c) that include 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).

[0051] Generally, single-domain antibodies contain three CDRs: CDR1, CDR2, and CDR3. When the single-domain antibody is a VHH or a single-domain VH antibody, the CDRs of the single-domain antibody include, by way of example, 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 contacts 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) Combinations of (a), (b), and / or (c) that include CDR amino acid residues 26 - 35 (CDR1), 26 - 35b (CDR1), 49 - 65 (CDR2), 93 - 102 (CDR3), or 94 - 102 (CDR3).

[0052] When the single-domain antibody is a single-domain VL antibody, the CDRs of the single-domain antibody include, by way of 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 contacts 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) Combinations of (a), (b), and / or (c) that include CDR amino acid residues 46 - 56 (CDR2), 47 - 56 (CDR2), 48 - 56 (CDR2), or 49 - 56 (CDR2).

[0053] Unless otherwise indicated, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al. supra.

[0054] 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 CDRs and FRs typically appear in VH (or VL) in the following order: FR1 - H1(L1) - FR2 - H2(L2) - FR3 - H3(L3) - FR4. In a single - domain antibody, the sequences of CDRs and FRs typically appear in the following order: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4.

[0055] The "human consensus framework" is a framework that indicates the amino acid residues that most commonly occur in a selected group of human immunoglobulin VL or VH framework sequences. Usually, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Usually, the subgroup of sequences is the subgroup in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one aspect, for VL, the subgroup is subgroup κI by Kabat et al. as described above. In one aspect, for VH, the subgroup is subgroup III by Kabat et al. as described above.

[0056] Constant region As used herein, the term "constant region" or "constant domain" refers to the portion of an antibody other than the variable region. For example, an IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy chain domain or heavy chain variable domain, followed by a heavy chain constant region (CH) including the CH1 domain, the hinge region, the CH2 domain, and the CH3 domain. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called the 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 "comprising a constant region" may include all of the constant region or a part of the constant region.

[0057] Unless otherwise specified herein, the numbering of amino acid residues in the antibody variable region and antibody light and heavy chain constant regions follows the Kabat numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0058] The "class" of an antibody refers to the type of constant domain or constant region borne by the antibody's heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM. And some of these may be further divided into subclasses (isotypes). For example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively.

[0059] Fc region As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes the Fc region of the native sequence and variant Fc regions. In one aspect, for human IgG1, the heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or antibody heavy chain constant region follows the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0060] Hinge region The term "hinge region" or "antibody hinge region" can refer to the region composed of amino acids 216 to 230 in the EU numbering in the antibody heavy chain, or a part thereof.

[0061] Single-domain antibody As used herein, the term "single-domain antibody" is an antibody that can exhibit antigen-binding activity by its domain alone. The structure of the single-domain antibody is not limited as long as it can exhibit antigen-binding activity by its domain alone. Normal antibodies exemplified by IgG antibodies exhibit antigen-binding activity in a state where a variable region is formed by the pairing of VH and VL, whereas single-domain antibodies are known to be able to exhibit antigen-binding activity by the domain structure of the single-domain antibody itself without pairing with other domains. Single-domain antibodies usually have a relatively low molecular weight and exist in a monomeric form. In some embodiments, the form of the single-domain antibody is similar to the variable region of the antibody heavy chain or the variable region of the antibody light chain. Examples of single-domain antibodies include, but are not limited to, for example, the variable region (VHH) of the heavy-chain antibody of animals of the camelidae family, the V NAR such as antigen-binding molecules that are congenitally lacking in light chains, or antibody fragments containing all or part of the VH domain or all or part of the VL domain of an antibody. Examples of single-domain antibodies that are antibody fragments containing all or part of the VH / VL domain of an antibody include, but are not limited to, for example, single-domain antibodies artificially produced starting from human antibody VH or human antibody VL as described in US Patent No. 6,248,516 B1, etc. (hereinafter, single-domain VH antibodies, single-domain VL antibodies).

[0062] Single-domain antibodies can be obtained from animals capable of producing single-domain antibodies or by immunizing animals capable of producing single-domain antibodies. Examples of animals capable of producing single-domain antibodies include, but are not limited to, for example, camelids, transgenic animals into which a gene capable of producing a single-domain antibody has been introduced. Camelids include camels, llamas, alpacas, vicuñas, and guanacos, etc. Examples of transgenic animals into which a gene capable of producing a single-domain antibody has been introduced include, but are not limited to, the transgenic animals described in International Publication WO2015 / 143414 and US Patent Publication US2011 / 0123527 A1. Humanized single-domain antibodies can also be obtained by making the framework sequence of a single-domain antibody obtained from an animal the same as or similar to a human germline sequence. Humanized single-domain antibodies (for example, humanized VHH) are also an aspect of the single-domain antibodies described herein.

[0063] In addition, single-domain antibodies can be obtained from a polypeptide library containing single-domain antibodies by ELISA, panning, etc. Examples of polypeptide libraries containing single-domain antibodies include, but are not limited to, for example, naive antibody libraries obtained from various animals or humans (for example, 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 (for example, Journal of Applied Microbiology 2014 117:2 (528 - 536)), or synthetic antibody libraries created from antibody genes of various animals or humans (for example, Journal of Biomolecular Screening 2016 21:1 (35 - 43), Journal of Biological Chemistry 2016 291:24 (12641 - 12657), AIDS 2016 30:11 (1691 - 1701)).

[0064] In the present disclosure, there is an embodiment in which a protease cleavage sequence is included in a single-domain antibody, but regardless of whether a protease cleavage sequence is included or not, it can be expressed as a "single-domain antibody".

[0065] The single-domain antibodies herein, in some forms, generally a) an amino acid sequence consisting of four framework regions / sequences with three complementarity-determining regions / sequences inserted therebetween (the amino acid residue at position 11 according to Kabat numbering is selected from the group consisting of L, M, S, V, W, preferably L), and / or b) an amino acid sequence consisting of four framework regions / sequences with three complementarity-determining regions / sequences inserted therebetween (the amino acid residue at position 37 according to Kabat numbering is selected from the group consisting of F, Y, H, I, L, 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 therebetween (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 therebetween (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 therebetween (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 / arrays with three complementarity-determining regions / arrays inserted therein (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 / arrays with three complementarity-determining regions / arrays inserted therein (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 / arrays with three complementarity-determining regions / arrays inserted therein (the amino acid residue at position 103 according to Kabat numbering is selected from the group consisting of W, P, R, S, preferably W), and / or i) An amino acid sequence consisting of four framework regions / arrays with three complementarity-determining regions / arrays inserted therein (the amino acid residue at position 104 according to Kabat numbering is G or D, preferably G), and / or j) It can be defined as a polypeptide comprising an amino acid sequence consisting of four framework regions / arrays with three complementarity-determining regions / arrays inserted therein (the amino acid residue at position 108 according to Kabat numbering is selected from the group consisting of Q, L, R, preferably Q or L).

[0066] More specifically, but not exclusively, a single-domain antibody can be defined as a polypeptide comprising any one of the amino acid sequences consisting of four framework regions / arrays with three complementarity-determining regions / arrays inserted therein as follows: k) An amino acid sequence in which the amino acid residues at positions 43 to 46 according to Kabat numbering are KERE or KQRE; l) An amino acid sequence in which the amino acid residues at positions 44 to 47 according to Kabat numbering are GLEW; m) An amino acid sequence in which the amino acid residues at positions 83 to 84 according to Kabat numbering are KP or EP.

[0067] Chimeric antibody The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a particular source or species, while the remaining portion of the heavy chain and / or light chain is derived from a different source or species. A "chimeric single-domain antibody" refers to a single-domain antibody in which a portion of the single-domain antibody is derived from a particular source or species, while the remaining portion of the single-domain antibody is derived from a different source or species.

[0068] Humanized antibody A "humanized" antibody refers to a chimeric antibody that contains amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, typically two, variable domains, wherein in the variable region, all or substantially all of the CDRs correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A "humanized single-domain antibody" refers to a chimeric single-domain antibody that contains amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, a humanized single-domain antibody has all or substantially all of its CDRs corresponding to those of a non-human antibody and all or substantially all of its FRs corresponding to those of a human antibody. Even if some of the residues in the FR do not correspond to those of a human antibody in a humanized antibody, this may 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 single-domain antibody, it is necessary to make some of the residues in the FR different from those of a human antibody (C Vincke et al., The Journal of Biological Chemistry 284, 3273-3284). A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. The "humanized form" of an antibody (e.g., a non-human antibody) refers to the antibody that has undergone humanization.

[0069] Association As used herein, "association" can be paraphrased as, for example, a state in which two or more polypeptide regions interact. Generally, hydrophobic bonds, hydrogen bonds, ionic bonds, etc. are formed between the target polypeptide regions to form an aggregate. As a common example of association, in antibodies typified by natural antibodies, it is known that the heavy chain variable region (VH) and the light chain variable region (VL) maintain a pairing structure through non-covalent bonds between them. The dissociation of association can be paraphrased as, for example, the complete or partial elimination of the interaction state of two or more polypeptide regions. The dissociation of the association between VH and VL may mean that the interaction between VH and VL is completely eliminated or only a part of the interaction between VH and VL is eliminated.

[0070] FcRn binding region As used herein, the "FcRn binding region" refers to a region having binding affinity for FcRn, and any structure can be used as long as it has binding affinity for FcRn. A molecule containing an FcRn binding region can return to the plasma again after being taken into the cell by the salvage pathway of FcRn. For example, the relatively long retention time (slow disappearance) of IgG molecules in the plasma is because FcRn, known as the salvage receptor of IgG molecules, functions. IgG molecules taken into the endosome by pinocytosis bind to FcRn expressed in the endosome under acidic conditions in the endosome. IgG molecules that cannot bind to FcRn proceed to the lysosome and are degraded there, while IgG molecules that bind to FcRn migrate to the cell surface and dissociate from FcRn under neutral conditions in the plasma to return to the plasma again. The FcRn binding region is preferably a region that directly binds to FcRn. Preferred examples of the FcRn binding region include the Fc region of an antibody. However, since a region capable of binding to a polypeptide having a binding ability to FcRn such as albumin or IgG can indirectly bind to FcRn via albumin or IgG, the FcRn binding region in the present disclosure may be a region that binds to such a polypeptide having a binding ability to FcRn.

[0071] The binding activity of the FcRn binding region in the present disclosure to FcRn, particularly human FcRn, can be measured by methods known to those skilled in the art as described in the section on the binding activity, and those skilled in the art can appropriately determine the conditions. The binding activity to human FcRn can be evaluated as KD (Dissociation constant), apparent KD (Apparent dissociation constant), kd (Dissociation rate), or apparent kd (Apparent dissociation rate). These can be measured by methods known to those skilled in the art. For example, Biacore (GE healthcare), Scatchard plot, flow cytometer, etc. can be used.

[0072] 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, as described in WO2009 / 125825, it can be measured under the conditions of MES buffer and 37°C. In addition, the binding activity of the FcRn binding region of the present disclosure to FcRn can be measured by methods known to those skilled in the art. For example, it can be measured using Biacore (GE Healthcare). The measurement of the binding activity between the FcRn binding region and FcRn can be evaluated by flowing FcRn or a molecule containing the FcRn binding region or FcRn as an analyte to a chip immobilized with the FcRn binding region or a transport moiety containing the FcRn binding region or FcRn, respectively.

[0073] As the pH used for the measurement conditions, the binding affinity between the FcRn-binding region and FcRn may be evaluated at any pH from pH 4.0 to pH 6.5. Preferably, in order to determine the binding affinity between the FcRn-binding region and human FcRn, a pH of pH 5.8 to pH 6.0, which is close to the pH in early endosomes in vivo, is used. As the temperature used for the measurement conditions, the binding affinity between the FcRn-binding region and FcRn may be evaluated at any temperature from 10°C to 50°C. Preferably, in order to determine the binding affinity between the FcRn-binding region and human FcRn, a temperature of 15°C to 40°C is used. More preferably, any temperature from 20°C to 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 similarly used to determine the binding affinity between the FcRn-binding region and FcRn. The temperature of 25°C is a non-limiting example of the embodiments of the present disclosure.

[0074] As one example of the FcRn-binding region, but not limited thereto, for example, the Fc region of an IgG antibody can be mentioned. When using the Fc region of an IgG antibody, its type is not limited, and Fc regions such as IgG1, IgG2, IgG3, and IgG4 can be used. For example, it is possible to use an Fc region containing one sequence selected from the amino acid sequences represented by SEQ ID NOs: 18004, 18005, 18006, and 18007.

[0075] In addition to the Fc region of a natural IgG antibody, as long as it has FcRn-binding properties, a modified Fc region in which one or more amino acids are substituted can also be used. For example, it is possible to use a modified Fc region comprising an amino acid sequence in which at least one amino acid selected from the 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, 315th, 317th, 325th, 332nd, 334th, 360th, 376th, 380th, 382nd, 384th, 385th, 386th, 387th, 389th, 424th, 428th, 433rd, 434th, and 436th positions in the EU numbering of the Fc region of an IgG antibody is substituted with another amino acid.

[0076] More specifically, as the FcRn binding region, an amino acid substitution in which Gly at the 237th position in the EU numbering of the Fc region of an IgG antibody is substituted with Met, an amino acid substitution in which Pro at the 238th position is substituted with Ala, an amino acid substitution in which Ser at the 239th position is substituted with Lys, an amino acid substitution in which Lys at the 248th position is substituted with Ile, an amino acid substitution in which Thr at the 250th position is substituted with Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr, an amino acid substitution in which Met at the 252nd position is substituted with Phe, Trp, or Tyr, an amino acid substitution in which Ser at the 254th position is substituted with Thr, an amino acid substitution in which Arg at the 255th position is substituted with Glu, an amino acid substitution in which Thr at the 256th position is substituted with Asp, Glu, or Gln, an amino acid substitution in which Pro at the 257th position is substituted with Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val, an amino acid substitution in which Glu at the 258th position is substituted with His, an amino acid substitution in which Asp at the 265th position is substituted with Ala, ​An amino acid substitution that replaces the 270th Asp with Phe, An amino acid substitution that replaces the 286th Asn with Ala or Glu, An amino acid substitution that replaces the 289th Thr with His, An amino acid substitution that replaces the 297th Asn with Ala, An amino acid substitution that replaces the 298th Ser with Gly, An amino acid substitution that replaces the 303rd Val with Ala, An amino acid substitution that replaces the 305th Val with Ala, An amino acid substitution that replaces the 307th Thr with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr, An amino acid substitution that replaces the 308th Val with Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr, An amino acid substitution that replaces the 309th Leu or Val with Ala, Asp, Glu, Pro, or Arg, An amino acid substitution that replaces the 311th Gln with Ala, His, or Ile, An amino acid substitution that replaces the 312th Asp with Ala or His, An amino acid substitution that replaces the 314th Leu with Lys or Arg, An amino acid substitution that replaces the 315th Asn with Ala or His, An amino acid substitution that replaces the 317th Lys with Ala, An amino acid substitution that replaces the 325th Asn with Gly, An amino acid substitution that replaces the 332nd Ile with Val, An amino acid substitution that replaces the 334th Lys with Leu, An amino acid substitution that replaces the 360th Lys with His, An amino acid substitution that replaces the 376th Asp with Ala, An amino acid substitution that replaces the 380th Glu with Ala, An amino acid substitution that replaces the 382nd Glu with Ala, An amino acid substitution that replaces the 384th Asn or Ser with Ala, an amino acid substitution that replaces the 385th Gly with Asp or His, an amino acid substitution that replaces the 386th Gln with Pro, an amino acid substitution that replaces the 387th Pro with Glu, an amino acid substitution that replaces the 389th Asn with Ala or Ser, an amino acid substitution that replaces the 424th Ser with Ala, an amino acid substitution that replaces 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 that replaces the 433rd His with Lys, an amino acid substitution that replaces the 434th Asn with Ala, Phe, His, Ser, Trp, or Tyr, and an amino acid substitution that replaces the 436th Tyr or Phe with His It is possible to use a modified Fc region comprising at least one amino acid substitution selected from:

[0077] Viewed from another aspect, as the FcRn binding region, in the Fc region of an IgG antibody, according to the EU numbering Met at the 237th amino acid, 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 the 254th amino acid, 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 the 257th amino acid, His at the 258th amino acid, Ala at the 265th amino acid, Phe at the 270th amino acid, 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 the 303rd amino acid, Ala at the 305th amino acid, Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr at the 307th amino acid, Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr at the 308th amino acid, Ala, Asp, Glu, Pro, or Arg at the 309th amino acid, 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 the 317th amino acid, Gly at the 325th amino acid, Val at the 332nd amino acid, Leu at the 334th amino acid, His at the 360th amino acid, Ala at the 376th amino acid, Ala at the 380th amino acid, Ala at the 382nd amino acid, Ala at the 384th amino acid, Asp or His at the 385th amino acid, Pro at the 386th amino acid, Glu at the 387th amino acid, Ala or Ser at the 389th amino acid, Ala at the 424th amino acid, Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr at the 428th amino acid, Lys at the 433rd amino acid, Ala, Phe, His, Ser, Trp, or Tyr at the 434th amino acid, and His at the 436th amino acid It is possible to use an Fc region containing at least one amino acid selected from

[0078] Affinity "Affinity" refers to the total strength of non-covalent interactions between one binding site of a molecule (e.g., an antibody) and the binding partner of the molecule (e.g., an antigen). Unless otherwise indicated, the "binding affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific and exemplary embodiments for measuring binding affinity are described below.

[0079] Monoclonal antibody As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies that make up that population are identical and / or bind to the same epitope, except for variant antibodies that may occur (e.g., variant antibodies that include naturally occurring mutations, or variant antibodies that occur during the production of a monoclonal antibody preparation. Such variants are usually present in a small amount). In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the characteristic of an antibody that is obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of an antibody by any particular method. For example, monoclonal antibodies used in accordance with the present disclosure may be made by a variety of techniques, including, but not limited to, the hybridoma method, the recombinant DNA method, the phage display method, and methods that utilize transgenic animals that include all or part of the human immunoglobulin locus. Such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0080] Method for producing antibody Methods for making antibodies having a desired binding activity are known to those of skill in the art. The following is an example of a method for making an antibody that binds to IL-6R (anti-IL-6R antibody). Antibodies that bind to antigens other than IL-6R can also be appropriately made according to the following examples. When making single-domain antibodies, although there are differences in immunized animals, etc., they can be appropriately made according to the following examples.

[0081] Anti-IL-6R antibodies can be obtained as polyclonal or monoclonal antibodies using known means. As anti-IL-6R antibodies, monoclonal antibodies derived from mammals can be preferably produced. Monoclonal antibodies derived from mammals include those produced by hybridomas and those produced by host cells transformed with an expression vector containing an antibody gene by genetic engineering techniques. The antibodies mentioned in the present application include "humanized antibodies" and "chimeric antibodies".

[0082] Monoclonal antibody-producing hybridomas can be produced, for example, as follows by using known techniques. That is, using the IL-6R protein as an immunizing antigen, a mammal is immunized according to a normal immunization method. The resulting immune cells are fused with known parent cells by a normal cell fusion method. Next, by a normal screening method, hybridomas that produce anti-IL-6R antibodies can be selected by screening monoclonal antibody-producing cells.

[0083] Specifically, the production of monoclonal antibodies is carried out, for example, as shown below. First, by expressing the IL-6R gene, the IL-6R protein used as an immunizing antigen for antibody acquisition can be obtained. That is, a suitable host cell is transformed by inserting a gene sequence encoding IL-6R into a known expression vector. The desired human IL-6R protein is purified from the host cell or the culture supernatant by a known method. In order to obtain soluble IL-6R from the culture supernatant, for example, soluble IL-6R as described by Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968) is expressed. Also, the purified natural IL-6R protein can be used as an immunizing antigen in the same manner.

[0084] 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. At this time, the partial peptide can also 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 also be obtained by decomposing the IL-6R protein using a proteolytic enzyme, but the region and size of the IL-6R peptide used as a partial peptide are not particularly limited to a special mode. The number of amino acids constituting the peptide as a sensitizing antigen is preferably at least 5 or more, for example, 6 or more, or 7 or more. More specifically, peptides of 8 to 50, preferably 10 to 30 residues can be used as sensitizing antigens.

[0085] In addition, a desired partial polypeptide of the IL-6R protein or a fusion protein in which a peptide is fused with a different polypeptide can be used as a sensitizing antigen. To produce a fusion protein used as a sensitizing antigen, for example, an Fc fragment of an antibody, a peptide tag, etc. can be preferably used. A vector expressing the fusion protein can be prepared by fusing genes encoding two or more desired polypeptide fragments in-frame and inserting the fusion gene into the expression vector as described above. The method for producing the fusion protein is described in Molecular Cloning 2nd ed. (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47 - 9.58(1989) Cold Spring Harbor Lab. press). The method for obtaining IL- used as a sensitizing antigen and the immunization method using the same are also specifically described in WO2003 / 000883, WO2004 / 022754, WO2006 / 006693, etc.

[0086] The mammalian animals immunized with the sensitizing antigen are not limited to specific animals, but are preferably selected in consideration of compatibility with the parent cells used for cell fusion. Generally, rodent animals such as mice, rats, hamsters, or rabbits, monkeys, etc. are preferably used. When obtaining single-domain antibodies, camelid animals or transgenic animals into which a gene capable of producing single-domain antibodies has been introduced are preferably used.

[0087] The above animals are immunized with the sensitizing antigen according to known methods. For example, as a general method, immunization is carried out by administering the sensitizing antigen into the abdominal cavity or subcutaneously of the mammal. Specifically, the sensitizing antigen diluted at an appropriate dilution ratio with PBS (Phosphate-Buffered Saline), physiological saline, etc. is mixed with a normal adjuvant such as Freund's complete adjuvant, if desired, emulsified, and then the sensitizing antigen is administered to the mammal several times every 4 to 21 days. Also, an appropriate carrier can be used during the immunization with the sensitizing antigen. In particular, when a small molecular weight partial peptide is used as the sensitizing antigen, it may be desirable to immunize the sensitizing antigen peptide conjugated with a carrier protein such as albumin or keyhole limpet hemocyanin.

[0088] Also, hybridomas that produce the desired antibody can be prepared by using DNA immunization as follows. DNA immunization is an immunization method in which immunostimulation is given by expressing the sensitizing antigen in the immunized animal after administering vector DNA constructed in such a way that the gene encoding the antigen protein can be expressed in the immunized animal. Compared with the general immunization method in which a protein antigen is administered to an immunized animal, the following advantages are expected for DNA immunization. - Immunostimulation can be given while maintaining the structure of membrane proteins such as IL-6R - There is no need to purify the immunizing antigen

[0089] To obtain the monoclonal antibodies of the present disclosure 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 an appropriate expression vector and administered to the immunized animal. As the expression vector, commercially available expression vectors such as pcDNA3.1 can be preferably used. As a method for administering the vector to a living body, generally used methods can be used. For example, DNA immunization is performed by introducing gold particles adsorbed with the expression vector into the cells of an immunized animal individual with a gene gun. Furthermore, the antibody recognizing IL-6R can also be prepared using the method described in International Publication WO 2003 / 104453.

[0090] After the mammal is immunized in this way and an increase in the antibody titer binding to IL-6R in the serum is confirmed, immune cells are collected from the mammal and subjected to cell fusion. As the preferred immune cells, spleen cells can be particularly used.

[0091] As the cells to be fused with the immune cells, myeloma cells of a mammal are used. The myeloma cells preferably have an appropriate selection marker for screening. The selection marker refers to a trait that can (or cannot) survive under specific culture conditions. Known selection markers include hypoxanthine-guanine phosphoribosyl transferase deficiency (hereinafter abbreviated as HGPRT deficiency), or thymidine kinase deficiency (hereinafter abbreviated as TK deficiency). Cells having a deficiency in HGPRT or TK are hypoxanthine-aminopterin-thymidine sensitive (hereinafter abbreviated as HAT sensitive). HAT-sensitive cells cannot perform DNA synthesis in HAT selection medium and die, but when fused with normal cells, they can utilize the salvage pathway of normal cells to continue DNA synthesis and thus grow even in HAT selection medium.

[0092] Cells deficient in HGPRT or TK 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 DNA die. On the other hand, cells lacking these enzymes that cannot incorporate these pyrimidine analogs can survive in the selective medium. In addition, a selection marker called G418 resistance confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) by the neomycin resistance gene. Various myeloma cells suitable for cell fusion are known.

[0093] As such myeloma cells, for example, P3 (P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (C. Eur. J. Immunol. (1976) 6 (7), 511-519), MPC-11 (Cell (1976) 8 (3), 405-415), SP2 / 0 (Nature (1978) 276 (5685), 269-270), FO (J. Immunol. Methods (1980) 35 (1-2), 1-21), S194 / 5.XX0.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323), R210 (Nature (1979) 277 (5692), 131-133), etc. can be preferably used.

[0094] Basically, according to known methods, such as the methods of Keller and Milstein et al. (Methods Enzymol. (1981) 73, 3-46), etc., cell fusion of the immune cells and myeloma cells is performed. More specifically, for example, the cell fusion can be carried out in a normal nutrient culture medium in the presence of a cell fusion promoter. As the cell fusion promoter, for example, polyethylene glycol (PEG), Sendai virus (HVJ), etc. are used, and an auxiliary agent such as dimethyl sulfoxide is added and used as desired to further enhance the fusion efficiency.

[0095] The usage ratio of immune cells to myeloma cells can be arbitrarily set. For example, it is preferable to set the immune cells to be 1 to 10 times that of the myeloma cells. As the culture medium used for the cell fusion, for example, RPMI1640 culture medium, MEM culture medium, which are suitable for the growth of the myeloma cell line, and other normal culture media used for this type of cell culture can be used, and a serum supplement such as fetal bovine serum (FCS) can be preferably added.

[0096] For cell fusion, a predetermined amount of the above-mentioned immune cells and myeloma cells are well mixed in the culture medium, and a PEG solution (for example, with an average molecular weight of about 1000 to 6000) pre-warmed to about 37°C is usually added at a concentration of 30 to 60% (w / v). By gently mixing the mixture, the desired fused cells (hybridomas) are formed. Then, the appropriate culture medium mentioned above is sequentially added, and the operation of centrifuging to remove the supernatant is repeated to remove cell fusion agents and the like that are not favorable for the growth of hybridomas.

[0097] The hybridomas thus obtained can be selected by culturing them in a normal selection culture medium, for example, HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). The culture using the HAT culture medium can be continued for a time sufficient for cells other than the desired hybridomas (non-fused cells) to die (usually, such a sufficient time is several days to several weeks). Then, screening and single cloning of hybridomas that produce the desired antibody are carried out by the usual limiting dilution method.

[0098] The hybridomas thus obtained can be selected by using a selection culture medium corresponding to the selection marker possessed by the myeloma used for cell fusion. For example, cells having a deficiency in HGPRT or TK can be selected by culturing them in an 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 have successfully fused with normal cells can selectively proliferate in the HAT culture medium. The culture using the above HAT culture medium is continued for a time sufficient for cells other than the desired hybridomas (non-fused cells) to die. Specifically, generally, the desired hybridomas 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 carried out by the usual limiting dilution method.

[0099] Screening and single cloning of the desired antibody can be suitably carried out by a screening method based on a known antigen-antibody reaction. For example, a monoclonal antibody that binds to IL-6R can bind to IL-6R expressed on the cell surface. Such a monoclonal antibody can be screened, for example, by FACS (fluorescence activated cell sorting). FACS is a system that enables measurement of the binding of an antibody to the cell surface by analyzing cells contacted with a fluorescent antibody with a laser beam and measuring the fluorescence emitted by individual cells.

[0100] To screen for hybridomas producing the monoclonal antibodies of the present disclosure by FACS, first, cells expressing IL-6R are prepared. Preferred cells for screening are mammalian cells in which IL-6R is overexpressed. By using untransformed mammalian cells used as host cells as a control, the binding activity of the antibody to IL-6R on the cell surface can be selectively detected. That is, by selecting hybridomas that do not bind to host cells but bind to IL-6R overexpressing cells, hybridomas producing IL-6R monoclonal antibodies can be obtained.

[0101] Alternatively, the binding activity of the antibody to immobilized IL-6R expressing cells can be evaluated based on the principle of ELISA. For example, IL-6R expressing cells are immobilized in the wells of an ELISA plate. The culture supernatant of the hybridoma is contacted with the immobilized cells in the well, and the antibody that binds to the immobilized cells is detected. When the monoclonal antibody is derived from a mouse, the antibody bound to the cells can be detected by an anti-mouse immunoglobulin antibody. Hybridomas producing the desired antibody having the binding ability to the antigen selected by these screenings can be cloned by the limiting dilution method or the like.

[0102] Hybridomas producing the monoclonal antibodies thus prepared can be subcultured in a normal culture medium. Also, the hybridomas can be stored in liquid nitrogen for a long time.

[0103] The hybridomas are cultured according to a normal method, and the desired monoclonal antibody can be obtained from the culture supernatant. Alternatively, the hybridomas can be administered to a compatible mammalian animal to proliferate, and the monoclonal antibody can be obtained from the ascites. The former method is suitable for obtaining a high-purity antibody.

[0104] Antibodies encoded by antibody genes cloned from antibody-producing cells such as the hybridoma can also be suitably used. By incorporating the cloned antibody gene into an appropriate vector and introducing it into a host, the antibody encoded by the gene is expressed. Methods for the isolation of antibody genes, introduction into vectors, and transformation of 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, as described below, are also known.

[0105] For example, cDNA encoding the variable region (V region) of an anti-IL-6R antibody is obtained from hybridoma cells producing the anti-IL-6R antibody. For this purpose, usually, first, total RNA is extracted from the hybridoma. As a method for extracting mRNA from cells, for example, the following methods can be used. - Guanidine ultracentrifugation method (Biochemistry (1979) 18 (24), 5294-5299) - AGPC method (Anal. Biochem. (1987) 162 (1), 156-159)

[0106] The extracted mRNA can be purified using an mRNA Purification Kit (manufactured by GE Healthcare Biosciences) or the like. Alternatively, kits for directly extracting total mRNA from cells, such as the QuickPrep mRNA Purification Kit (manufactured by GE Healthcare Biosciences), are also commercially available. Using such a kit, mRNA can be obtained from a hybridoma. cDNA encoding the antibody V region can be synthesized from the obtained mRNA using reverse transcriptase. The cDNA can be synthesized by an AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (manufactured by Seikagaku Corporation) or the like. Also, for the synthesis and amplification of cDNA, the SMART RACE cDNA Amplification Kit (manufactured by Clontech) and the 5'-RACE method using PCR (Proc. Natl. Acad. Sci. USA (1988) 85 (23), 8998-9002, Nucleic Acids Res. (1989) 17 (8), 2919-2932) can be appropriately used. Furthermore, appropriate restriction enzyme sites described below can be introduced at both ends of the cDNA during the process of synthesizing such cDNA.

[0107] The target cDNA fragment is purified from the obtained PCR product and then ligated to vector DNA. In this way, a recombinant vector is prepared. After introducing it into Escherichia coli or the like and selecting colonies, the desired recombinant vector can be prepared from the Escherichia coli that formed the colonies. Then, whether the recombinant vector has the nucleotide sequence of the target cDNA is confirmed by a known method, for example, the dideoxynucleotide chain termination method or the like.

[0108] To obtain a gene encoding a variable region, it is convenient to use the 5'-RACE method with primers for amplifying the variable region gene. First, cDNA is synthesized using RNA extracted from hybridoma cells as a template, and a 5'-RACE cDNA library is obtained. Commercial kits such as the SMART RACE cDNA Amplification Kit are appropriately used for the synthesis of the 5'-RACE cDNA library.

[0109] Using the obtained 5'-RACE cDNA library as a template, the antibody gene is amplified by the PCR method. Primers for amplifying the mouse antibody gene can be designed based on the known antibody gene sequence. These primers have different base sequences for each subclass of immunoglobulin. Therefore, it is desirable to determine the subclass in advance using a commercial kit such as the Iso Strip Mouse Monoclonal Antibody Isotyping Kit (Roche Diagnostics).

[0110] Specifically, for example, when aiming to obtain a gene encoding mouse IgG, primers capable of amplifying genes encoding γ1, γ2a, γ2b, γ3 as the heavy chain and κ chain and λ chain as the light chain can be used. To amplify the variable region gene of IgG, generally, a primer that anneals to a portion corresponding to the constant region close to the variable region is used as the 3'-side primer. On the other hand, a primer attached to the 5' RACE cDNA library preparation kit is used as the 5'-side primer.

[0111] Using the PCR product amplified in this way, an immunoglobulin composed of a combination of the heavy chain and the light chain can be reconstituted. Using the binding activity of the reconstituted immunoglobulin to IL-6R as an index, the desired antibody can be screened. For example, when aiming to obtain an antibody against IL-6R, it is more preferable that the binding of the antibody to IL-6R is specific. An antibody that binds to IL-6R can be screened, for example, as follows; (1) A step of contacting an antibody containing a V region encoded by cDNA obtained from a hybridoma with IL-6R-expressing cells, (2) A step of detecting the binding between IL-6R-expressing cells and the antibody, and (3) A step of selecting an antibody that binds to IL-6R-expressing cells.

[0112] Methods for detecting the binding between an antibody (including single-domain antibodies) and IL-6R-expressing cells are known. Specifically, the binding between an antibody and IL-6R-expressing cells can be detected by techniques such as FACS described above. Fixed specimens of IL-6R-expressing cells can be appropriately used to evaluate the binding activity of the antibody.

[0113] As a method for screening an antibody using binding activity as an index, the panning method using a phage vector is also preferably used. Even when screening for single-domain antibodies, appropriate screening can be carried out according to the following examples. When an antibody gene is obtained as a library of heavy and light chain subclasses from a polyclonal antibody-expressing cell group, a screening method using a phage vector is advantageous. Genes encoding the variable regions of the heavy and light chains can be linked with an appropriate linker sequence to form a single-chain Fv (scFv). By inserting a gene encoding scFv into a phage vector, phages expressing scFv on the surface can be obtained. After contact between this phage and the desired antigen, DNA encoding scFv having the desired binding activity can be recovered by recovering the phage bound to the antigen. By repeating this operation as necessary, scFv having the desired binding activity can be concentrated.

[0114] After obtaining the cDNA encoding the V region of the target anti-IL-6R antibody, the cDNA is digested with a restriction enzyme that recognizes the restriction enzyme sites inserted at both ends of the cDNA. Preferred restriction enzymes recognize and digest a nucleotide sequence that appears with a low frequency in the nucleotide sequence constituting the antibody gene. Further, in order to insert one copy of the digested fragment into the vector in the correct orientation, it is preferable to insert a restriction enzyme that provides cohesive ends. An antibody expression vector can be obtained by inserting the cDNA encoding the V region of the anti-IL-6R antibody digested as described above into an appropriate expression vector. 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 origins of the constant region and the variable region are different. Therefore, in addition to heterologous chimeric antibodies such as mouse-human chimeric antibodies, human-human isologous chimeric antibodies are also included in the chimeric antibodies in the present disclosure. A chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector having a constant region in advance. Specifically, for example, a restriction enzyme recognition sequence of the restriction enzyme that digests the V region gene can be appropriately arranged on the 5' side of an expression vector holding DNA encoding a desired antibody constant region (C region). By fusing both digested with the same combination of restriction enzymes in-frame, a chimeric antibody expression vector is constructed.

[0115] To produce an anti-IL-6R monoclonal antibody, the antibody gene is incorporated into an expression vector so as to be expressed under the control of an expression control region. The expression control region for expressing the antibody includes, for example, an enhancer and a promoter. Further, an appropriate signal sequence may be added to the amino terminus so that the expressed antibody is secreted extracellularly. For example, as the signal sequence, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 18008) may be used, but other suitable signal sequences may also be added. The expressed polypeptide is cleaved at the carboxyl terminal portion of the above sequence, and the cleaved polypeptide may be secreted extracellularly as a mature polypeptide. Then, by transforming an appropriate host cell with this expression vector, a recombinant cell expressing the DNA encoding the anti-IL-6R antibody can be obtained.

[0116] Polynucleotide (nucleic acid) As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substance that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides can include modified nucleotides such as methylated nucleotides and their analogs. Non-nucleotide components may be interspersed within the nucleotide sequence. Polynucleotides can include post-synthetic modifications such as conjugation to a label. Other types of modifications include, for example, "caps", substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as those with uncharged linkages (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.) and charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.), those with pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalating agents (e.g., acridine, psoralen, etc.), those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha-anomer nucleic acids, etc.) and those with polynucleotides in unmodified form. Further, any hydroxyl group normally present on the sugar can be substituted, for example, with a phosphonate group, a phosphate group, protected with a standard protecting group, or activated to form a further linkage to an additional nucleotide, or conjugated to a solid or semi-solid support. The OH at the 5' and 3' termini can be phosphorylated or substituted with an amine or an organic cap group moiety of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized with standard protecting groups.Polynucleotides can also include analogous forms of ribose or deoxyribose sugars that are generally known in the art, such as, for example: 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose or xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and basic nucleoside analogs such as methyl riboside. One or more phosphodiester bonds can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by: P(O)S (“thioate”), P(S)S (“dithioate”), (O)NR2 (“amidate”), P(O)R, P(O)OR', CO, or CH2 (“formacetal”), where each R or R' is independently H, or substituted or unsubstituted alkyl (1-20C) optionally including an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. All linkages in the polynucleotide need not be identical. The above description applies to all polynucleotides referred to herein, including RNA and DNA.

[0117] Vector As used herein, the term “vector” refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is ligated. The term includes vectors as self-replicating nucleic acid structures, and vectors that are incorporated into the genome of a host cell into which they are introduced. A vector can be capable of effecting the expression of a nucleic acid to which it is operably linked. Such vectors are also referred to herein as “expression vectors”.

[0118] Host cell, etc. The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell into which foreign nucleic acid has been introduced (including progeny of such a cell). Host cells include "transformants" and "transformed cells", including the original transformed cells and progeny derived therefrom regardless of the number of passages. The progeny need not be identical in nucleic acid content to the parental cell and may include mutations. Mutant progeny having the same function or biological activity as that used when the original transformed cell was screened or selected are also included herein.

[0119] Polypeptide containing protease cleavage sequence One aspect of the present disclosure relates to a polypeptide comprising a protease cleavage sequence. Another aspect of the present disclosure also relates to a polypeptide comprising at least one sequence selected from the sequences from the 4th amino acid to the 15th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 5 to 17201, the sequences from the 4th amino acid to the 13th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 5 to 17201, the sequences from the 6th amino acid to the 13th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 5 to 17201, the sequences from the 1st amino acid to the 12th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 17202 to 17993, the sequences from the 3rd amino acid to the 12th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 17202 to 17993, the sequences from the 3rd amino acid to the 11th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 17202 to 17993, the sequences from the 3rd amino acid to the 10th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 17202 to 17993, the sequences from the 3rd amino acid to the 14th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 17994 to 18003, the sequences from the 5th amino acid to the 12th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 17994 to 18003, the sequences from the 5th amino acid to the 10th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 17994 to 18003, and at least one sequence selected from the sequences represented by any of SEQ ID NOs: 5 to 18003. Another aspect of the present disclosure also relates to a polypeptide comprising any of the sequences composed in the order of "a sequence selected from the following Group A - a sequence selected from the following Group B" from the N-terminus: (Group A) The sequence from the 1st amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 2nd amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 3rd amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 4th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 5th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 6th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 7th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 8th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 1st amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 2nd amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 3rd amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 4th amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 5th amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 1st amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, The sequence from the second amino acid to the eighth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the third amino acid to the eighth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the fourth amino acid to the eighth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the fifth amino acid to the eighth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the sixth amino acid to the eighth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the seventh amino acid to the eighth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003; (Group B) The sequence from the tenth amino acid to the fifteenth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201 The sequence from the tenth amino acid to the fourteenth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201 The sequence from the tenth amino acid to the thirteenth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201 The sequence from the tenth amino acid to the twelfth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201 The sequence from the tenth amino acid to the eleventh amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201 The sequence from the seventh amino acid to the fifteenth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993 The sequence from the seventh amino acid to the fourteenth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993 The sequence from the seventh amino acid to the thirteenth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993 The sequence from the seventh amino acid to the twelfth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993 The sequence from the 7th amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 9th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, The sequence from the 9th amino acid to the 14th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, The sequence from the 9th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, The sequence from the 9th amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, The sequence from the 9th amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, The sequence from the 9th amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003.

[0120] In certain embodiments of the polypeptide of the present disclosure, the sequence from the 4th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 4th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 6th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, the sequence from the 1st amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd amino acid to the 14th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, the sequence from the 5th amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, the sequence from the 5th amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, at least one sequence selected from the sequences represented by any of SEQ ID NOs: 5 to 18003 is included in the polypeptide, and the sequence in the polypeptide is cleavable by a protease, that is, the sequence functions as a protease cleavage sequence in the polypeptide. In certain embodiments of the polypeptide of the present disclosure, also, any of the sequences composed in the order of "sequence selected from the following Group A - sequence selected from the following Group B" from the N-terminus is included in the polypeptide, and the sequence in the polypeptide is cleavable by a protease, that is, the sequence functions as a protease cleavage sequence in the polypeptide: (Group A) The sequence from the 1st amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 2nd amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 3rd amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201 The sequence from the 4th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201 The sequence from the 5th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201 The sequence from the 6th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201 The sequence from the 7th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201 The sequence from the 8th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201 The sequence from the 1st amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993 The sequence from the 2nd amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993 The sequence from the 3rd amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993 The sequence from the 4th amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993 The sequence from the 5th amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993 The sequence from the 1st amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 2nd amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 3rd amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 4th amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 5th amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, The sequence from the 6th amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, The sequence from the 7th amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003; (Group B) The sequence from the 10th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 10th amino acid to the 14th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 10th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 10th amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 10th amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 7th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 14th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993 The sequence from the 9th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 9th amino acid to the 14th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 9th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 9th amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 9th amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 9th amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003

[0121] In certain embodiments of the polypeptides of the present disclosure, a flexible linker is further added to one or both ends of the protease cleavage sequence. The flexible linker at one end of the protease cleavage sequence can be referred to as the first flexible linker, and the flexible linker at the other end can be referred to as the second flexible linker. In certain embodiments, the protease cleavage sequence and the flexible linker comprise one of the following formulas. (Protease cleavage sequence) (First flexible linker)-(Protease cleavage sequence) (Protease cleavage sequence)-(Second flexible linker) (First flexible linker)-(Protease cleavage sequence)-(Second flexible linker) The movable linker in this embodiment is preferably a peptide linker. The first movable linker and the second movable linker are each independently and optionally present and are the same or different movable linkers containing at least one flexible amino acid (such as Gly). For example, the protease cleavage sequence has 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.) to obtain the desired protease accessibility.

[0122] The movable linkers suitable for use at both ends of the protease cleavage sequence usually improve the protease's access to the protease cleavage sequence and increase the protease cleavage efficiency. Suitable movable linkers can be easily selected, and suitable ones can be selected from 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, starting from 3 amino acids to 12 amino acids, etc. In some embodiments of the present disclosure, the movable linker is a peptide linker of 1 to 7 amino acids.

[0123] Examples of movable linkers include, but are not limited to, for example, glycine polymers (G)n, glycine - serine polymers (for example, including (GS)n, (GSGGS: SEQ ID NO: 18018)n, and (GGGS: SEQ ID NO: 18009)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 been noted because these amino acids are relatively unstructured and are likely to function as neutral tethers between components. Examples of movable linkers composed of glycine - serine polymers include, but are not limited to, for example, 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 No.: 18009) Gly·Gly·Ser·Gly(GGSG, Sequence No.: 18010) Gly·Ser·Gly·Gly(GSGG, Sequence No.: 18011) Ser·Gly·Gly·Gly(SGGG, Sequence No.: 18012) Gly·Ser·Ser·Gly(GSSG, Sequence No.: 18013) Gly·Gly·Gly·Gly·Ser(GGGGS, Sequence No.: 18014) Gly·Gly·Gly·Ser·Gly(GGGSG, Sequence No.: 18015) Gly·Gly·Ser·Gly·Gly(GGSGG, Sequence No.: 18016) Gly·Ser·Gly·Gly·Gly(GSGGG, Sequence No.: 18017) Gly·Ser·Gly·Gly·Ser(GSGGS, Sequence No.: 18018) Ser·Gly·Gly·Gly·Gly(SGGGG, Sequence No.: 18019) Gly·Ser·Ser·Gly·Gly(GSSGG, Sequence No.: 18020) Gly·Ser·Gly·Ser·Gly(GSGSG, Sequence No.: 18021) Ser·Gly·Gly·Ser·Gly(SGGSG, Sequence No.: 18022) Gly·Ser·Ser·Ser·Gly(GSSSG, Sequence No.: 18023) Gly·Gly·Gly·Gly·Gly·Ser (GGGGGS, SEQ ID NO: 18024) Ser·Gly·Gly·Gly·Gly·Gly (SGGGGG, SEQ ID NO: 18025) Gly·Gly·Gly·Gly·Gly·Gly·Ser (GGGGGGGS, SEQ ID NO: 18026) Ser·Gly·Gly·Gly·Gly·Gly·Gly (SGGGGGG, SEQ ID NO: 18027) (Gly·Gly·Gly·Gly·Ser (GGGGGS, SEQ ID NO: 18014))n (Ser·Gly·Gly·Gly·Gly (SGGGG, SEQ ID NO: 18019))n [n is an integer of 1 or more] and the like can be mentioned. However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art according to the purpose.

[0124] A polypeptide containing the protease cleavage sequence of the present disclosure may have any other configuration as long as it contains at least one sequence selected from the following sequences: the sequence from the 4th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201; the sequence from the 4th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201; the sequence from the 6th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201; the sequence from the 1st amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993; the sequence from the 3rd amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993; the sequence from the 3rd amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993; the sequence from the 3rd amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993; the sequence from the 3rd amino acid to the 14th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003; the sequence from the 5th amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003; the sequence from the 5th amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003; any sequence shown by SEQ ID NOs: 5 to 18003. A polypeptide containing the protease cleavage sequence of the present disclosure may also have any other configuration as long as it contains any one of the sequences configured in the order of "sequence selected from the following Group A - sequence selected from the following Group B" from the N-terminus: (Group A) The sequence from the 1st amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 2nd amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 3rd amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 4th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 5th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201 The sequence from the 6th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201 The sequence from the 7th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201 The sequence from the 8th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201 The sequence from the 1st amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993 The sequence from the 2nd amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993 The sequence from the 3rd amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993 The sequence from the 4th amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993 The sequence from the 5th amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993 The sequence from the 1st amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 2nd amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 3rd amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 4th amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 5th amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 6th amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 7th amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003; (Group B) The sequence from the 10th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 10th amino acid to the 14th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 10th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 10th amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 10th amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 7th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 14th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 7th amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 9th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, The sequence from the 9th amino acid to the 14th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 9th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 9th amino acid to the 12th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 9th amino acid to the 11th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the 9th amino acid to the 10th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003

[0125] The polypeptide containing the protease cleavage sequence of the present disclosure may be an activatable antibody. For example, the activatable antibody includes: (i) an antibody that specifically binds to a target; (ii) a masking moiety that inhibits the binding of the antibody to the target when the protease cleavage sequence is in an uncleaved state; and (iii) a cleavable moiety coupled to the antibody, where the cleavable moiety is at least one sequence selected from the protease cleavage sequences of the present disclosure

[0126] Polypeptide containing antigen-binding domain and transport portion In one embodiment of the polypeptide of the present disclosure, the polypeptide includes an antigen-binding domain and a transport moiety, and the transport moiety has a suppression domain that suppresses the antigen-binding activity of the antigen-binding domain The present disclosure also relates to a method for releasing an antigen-binding domain from a polypeptide, which includes cleavage of the protease cleavage sequence in the polypeptide containing the antigen-binding domain and the transport moiety described in the specification by a protease

[0127] As used herein, an "antigen-binding domain" is limited to only binding to a target antigen. Any domain structure can be used as long as it binds to the target antigen. Examples of such domains include, but are not limited to, the heavy-chain variable region (VH) and the light-chain variable region (VL) of an antibody, single-domain antibody (sdAb), a module called the A domain of about 35 amino acids contained in Avimer, which is a cell membrane protein present in vivo (International Publication WO2004 / 044011, WO2005 / 040229), Adnectin containing a 10Fn3 domain, which is a domain that binds to a protein in fibronectin, which is a glycoprotein expressed on the cell membrane (International Publication WO2002 / 032925), Affibody using an IgG-binding domain consisting of a bundle of three helices of 58 amino acids of Protein A as a scaffold (International Publication WO1995 / 001937), DARPins (Designed Ankyrin Repeat proteins), which are regions exposed on the molecular surface of ankyrin repeats (AR) having a structure in which a subunit of a turn containing 33 amino acid residues, two antiparallel helices, and loops are repeatedly stacked (International Publication WO2002 / 020565), Anticalin, etc., which are four loop regions that support one side of a barrel structure in which eight antiparallel strands highly conserved in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) are twisted in the central direction (International Publication WO2003 / 029462), and a sunken region of a parallel sheet structure inside a saddle-shaped structure in which leucine-rich-repeat (LRR) modules of a variable lymphocyte receptor (VLR), which has an immunoglobulin structure as an acquired immune system of agnathans such as lampreys and hagfishes, are repeatedly stacked (International Publication WO2008 / 016854).

[0128] Suitable examples of the antigen-binding domain of the present disclosure include an antigen-binding domain that can exhibit an antigen-binding function with a molecule composed only of the antigen-binding domain, an antigen-binding domain that can exhibit an antigen-binding function alone after being released from other linked peptides, and the like. Examples of such antigen-binding domains include, but are not limited to, single-domain antibodies, scFv, Fv, Fab, Fab', F(ab')2, and the like.

[0129] One suitable example of the antigen-binding domain of the present disclosure is an antigen-binding domain having a molecular weight of 60 kDa or less. Examples of such antigen-binding domains include, but are not limited to, single-domain antibodies, scFv, Fab, and Fab'. An antigen-binding domain having a molecular weight of 60 kDa or less is likely to be cleared by the kidney when present in the blood as a monomer (see J Biol Chem. 1988 Oct 15;263(29):15064-70). Viewed from another aspect, one suitable example of the antigen-binding domain of the present disclosure is an antigen-binding domain having a blood half-life of 12 hours or less. Examples of such antigen-binding domains include, but are not limited to, single-domain antibodies, scFv, Fab, Fab', and the like.

[0130] One suitable example of the antigen-binding domain of the present disclosure is a single-domain antibody (sdAb).

[0131] As used herein, "antigen" is limited only to include the epitope to which the antigen-binding domain binds. Suitable examples of antigens include, but are not limited to, for example, peptides, polypeptides, and proteins derived from animals or humans. Suitable examples of antigens include, but are not limited to, for example, molecules expressed on the surface of target cells (e.g., cancer cells, inflammatory cells), molecules expressed on the surface of other cells in the tissue containing the target cells, molecules expressed on the surface of cells having an immunological role with respect to the target cells and the tissue containing the target cells, and macromolecules present in the stroma of the tissue containing the target cells.

[0132] Antigens include the following molecules: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, welchii toxin, CKb8-1, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, PD-1, PD-L1, LAG3, TIM3, galectin-9, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, Cytokeratin tumor-related antigen, DAN, DCC, DcR3, DC-SIGN, Complement control factor (Decay acceleratingfactor), des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor IIa, factor VII, factor VIIIc, factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha 1, GFR-alpha 2, GFR-alpha 3, GITR, glucagon, Glut4, glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, growth hormone releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFGPEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-21, IL-23, IL-27, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, integrin alpha2, integrin alpha3, integrin alpha4, integrin alpha4 / beta1, integrin alpha4 / beta7, integrin alpha5 (alphaV), integrin alpha5 / beta1, integrin alpha5 / beta3, integrin alpha6, integrin beta1, integrin beta2, interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1bp1, LBP, LDGF, LECT2, Leftin, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Müllerian inhibiting substance, Mug, MuSK, NAIP, NAP, NCAD, N-Cadherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurulin, nerve growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-Cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSVFgp, Ret, Rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, Serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (Tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, Testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, Thrombin, Thymic Ck-1, Thyroid-stimulating hormone, Tie, TIMP, TIQ, Tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60), TNFRSF1B (TNF RIICD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-a connectin (Conectin), DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TLR (Toll-likereceptor) 1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen-expressing Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1(flt-1), VEGF, VEGFR, VEGFR-3(flt-4), VEGI, VIM, viral antigen, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, Aβ, CD81, CD97, CD98, DDR1, DKK1, EREG, Hsp90, IL-17 / IL-17R, IL-20 / IL-20R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F, SOD1, Chromogranin A, Chromogranin B, tau, VAP1, high molecular weight kininogen, IL-31, IL-31R, Nav1.1, Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9, EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, factor H, properdin, sclerostin, fibrinogen, fibrin, prothrombin, thrombin, tissue factor, factor V, factor Va, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factorExamples of the above antigens may include Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, Syndecan-1, Syndecan-2, Syndecan-3, Syndecan-4, LPA, S1P, and receptors for hormones and growth factors.

[0133] The above examples of antigens include receptors, and even when these receptors exist in a soluble form in a biological fluid, they can be used as antigens to which the antigen-binding domains of the present disclosure bind.

[0134] The above examples of antigens include membrane-type molecules expressed on the cell membrane and soluble-type molecules secreted extracellularly from cells. When the antigen-binding domain of the present disclosure binds to a soluble-type molecule secreted from a cell, it is preferable that the antigen-binding domain has neutralizing activity.

[0135] There is no limitation to the solution in which the soluble-type molecule exists, and the soluble-type molecule can exist in a biological fluid, that is, all liquids filling the vessels or between tissues and cells in vivo. In a non-limiting aspect, the soluble-type molecule to which the antigen-binding domain of the present disclosure binds can exist in the extracellular fluid. Extracellular fluid refers to the collective term for components in bone and cartilage such as plasma, interstitial fluid, lymph, dense connective tissue, cerebrospinal fluid, myeloid fluid, puncture fluid, or synovial fluid in vertebrates, alveolar fluid (bronchoalveolar lavage fluid), ascites, pleural effusion, pericardial effusion, cyst fluid, or aqueous humor (aqueous humor) and other cell-permeable fluids (fluids in various glandular lumens resulting from the active transport and secretion activities of cells, and luminal fluids in the digestive tract and other body cavities).

[0136] As used herein, the term "carrier portion" refers to the portion of a polypeptide other than the antigen-binding domain. The carrier portion of the present disclosure is typically a peptide or polypeptide composed of amino acids. As a specific embodiment, the carrier portion in a polypeptide is linked to the antigen-binding domain via a protease cleavage sequence. The carrier portion of the present disclosure may be a series of peptides or polypeptides linked by amide bonds, or a complex formed by multiple peptides or polypeptides through covalent bonds such as disulfide bonds or non-covalent bonds such as hydrogen bonds and hydrophobic interactions.

[0137] The carrier portion of the present disclosure has a suppression domain that suppresses the antigen-binding activity of the antigen-binding domain. As used herein, the term "suppression domain" is limited only to suppressing the antigen-binding activity of the antigen-binding domain. Any domain structure can be used as long as it can suppress the antigen-binding activity of the antigen-binding domain. Examples of such suppression domains include, but are not limited to, the variable heavy chain (VH) of an antibody, the variable light chain (VL) of an antibody, the pre-B cell receptor, and single-domain antibodies. The suppression domain may be composed of the entire carrier portion or a part of the carrier portion.

[0138] When the polypeptide of the present disclosure contains an antigen-binding domain and a carrier portion, in certain embodiments, the suppression of the antigen-binding activity of the antigen-binding domain by the suppression domain in the state where the protease cleavage sequence is cleaved by a protease is weaker than the suppression of the antigen-binding activity of the antigen-binding domain by the suppression domain in the state where the protease cleavage sequence is uncleaved.

[0139] When the polypeptide of the present disclosure contains an antigen-binding domain and a carrier portion, in certain embodiments, the antigen-binding domain has a shorter half-life in blood than the uncleaved polypeptide. When the polypeptide of the present disclosure contains an antigen-binding domain and a carrier portion, in certain embodiments, the antigen-binding domain has a shorter half-life in blood than the carrier portion.

[0140] When the polypeptide of the present disclosure includes an antigen-binding domain and a transport moiety, in certain embodiments, the molecular weight of the antigen-binding domain is less than that of the transport moiety. In certain embodiments, the molecular weight of the antigen-binding domain can be 60 kDa or less.

[0141] When the polypeptide of the present disclosure includes an antigen-binding domain and a transport moiety, in certain embodiments, the transport moiety has FcRn-binding activity, and the antigen-binding domain has no FcRn-binding activity or has weaker FcRn-binding activity than the transport moiety.

[0142] When the polypeptide of the present disclosure includes an antigen-binding domain and a transport moiety, in certain embodiments, the polypeptide comprising the antigen-binding domain and the transport moiety has a longer blood half-life compared to the antigen-binding domain alone. To make the half-life of the polypeptide longer, when the polypeptide of the present disclosure includes an antigen-binding domain and a transport moiety, in certain embodiments, the transport moiety is designed to have a longer blood half-life. Examples of embodiments for extending the blood half-life of the transport moiety include, but are not limited to, the transport moiety having a large molecular weight, or the transport moiety having FcRn-binding property, or the transport moiety having albumin-binding property, or the transport moiety being PEGylated. Also, when the polypeptide of the present disclosure includes an antigen-binding domain and a transport moiety, in certain embodiments, the transport moiety has a longer blood half-life than the antigen-binding domain (in other words, the antigen-binding domain has a shorter blood half-life than the transport moiety).

[0143] In the present disclosure, for comparing the half-life of the antigen-binding domain alone and the polypeptide, or for comparing the blood half-life of the antigen-binding domain and the transport moiety, it is preferable to compare the blood half-life in humans. When it is difficult to measure the blood half-life in humans, 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.), the blood half-life in humans can be predicted.

[0144] As one embodiment of extending the blood half-life of the transport moiety, the molecular weight of the transport moiety is large. As one embodiment of making the blood half-life of the transport moiety longer than that of the antigen-binding domain, the molecular weight of the transport moiety is made larger than that of the antigen-binding domain.

[0145] As one embodiment of extending the blood half-life of the transport moiety, making the transport moiety have FcRn-binding property can be mentioned. To make the transport moiety have FcRn-binding property, usually, there is a method of providing an FcRn-binding region in the transport moiety.

[0146] Making the transport moiety have FcRn-binding property does not mean that the antigen-binding domain does not have FcRn-binding property. As an embodiment of making the blood half-life of the transport moiety longer than that of the antigen-binding domain, not only that the antigen-binding domain does not have FcRn-binding property, but also even if the antigen-binding domain has FcRn-binding property, it may have a weaker FcRn-binding property than the transport moiety.

[0147] Also, as one embodiment of extending the blood half-life of the transport moiety, there is a method of binding the transport moiety to albumin. Since albumin is not subject to renal excretion and has FcRn-binding property, its blood half-life is as long as 17 - 19 days (J Clin Invest. 1953 Aug; 32(8): 746 - 768.). Therefore, the protein bound to albumin becomes bulky and can indirectly bind to FcRn, so it has been reported that the blood half-life increases (Antibodies 2015, 4(3), 141 - 156).

[0148] Furthermore, as one embodiment of extending the blood half-life of the transport moiety, there is a method of PEGylating the transport moiety. It is considered that PEGylating a protein makes the protein bulky and at the same time suppresses degradation by proteases in the blood, thereby extending the blood half-life of the protein (J Pharm Sci. 2008 Oct;97(10):4167 - 83.).

[0149] When the polypeptide of the present disclosure includes an antigen-binding domain and a carrier portion, in certain embodiments, the carrier portion includes an antibody Fc region. As a specific embodiment, the carrier portion includes the CH2 domain and the CH3 domain of a human IgG antibody. As a specific embodiment, the carrier portion includes a portion extending from Cys226 or Pro230 of the heavy chain of a human IgG1 antibody 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.

[0150] When the polypeptide of the present disclosure includes an antigen-binding domain and a carrier portion, in certain embodiments, the carrier portion includes an antibody constant region. In a more preferred embodiment, the carrier portion includes an IgG antibody constant region. In a more preferred embodiment, the carrier portion includes a human IgG antibody constant region.

[0151] When the polypeptide of the present disclosure includes an antigen-binding domain and a carrier portion, in certain embodiments, the carrier portion includes a region having a structure substantially similar to an antibody heavy chain constant region and a region having a structure substantially similar to an antibody light chain, which is bound to the region by a covalent bond such as a disulfide bond or a non-covalent bond such as a hydrogen bond or a hydrophobic interaction.

[0152] When the polypeptide of the present disclosure includes an antigen-binding domain and a carrier portion, in certain embodiments, the antigen-binding domain is detachable from the polypeptide, and the antigen-binding activity of the antigen-binding domain in the state of being detached from the polypeptide is higher than the antigen-binding activity in the state of not being detached from the polypeptide.

[0153] When the polypeptide of the present disclosure includes an antigen-binding domain and a carrier portion, in certain embodiments, the antigen-binding domain becomes detachable from the polypeptide by cleavage of a protease cleavage sequence by a protease.

[0154] When the polypeptide of the present disclosure includes an antigen-binding domain and a carrier portion, in certain embodiments, the antigen-binding domain dissociates from the polypeptide, resulting in a higher antigen-binding activity than before dissociation. In other words, when the antigen-binding domain is not dissociated from the polypeptide, its antigen-binding activity is in a state of being suppressed by the inhibitory domain. As methods for confirming that the antigen-binding activity of the antigen-binding domain is suppressed by the inhibitory domain, there are methods such as FACS (fluorescence activated cell sorting), ELISA (Enzyme-Linked ImmunoSorbent Assay), ECL (electrogenerated chemiluminescence), SPR (Surface Plasmon Resonance) method (Biacore), and BLI (Bio-Layer Interferometry) method (Octet). When the polypeptide of the present disclosure includes an antigen-binding domain and a carrier portion, in certain embodiments, the antigen-binding activity of the antigen-binding domain in the state of being dissociated from the polypeptide is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, or 3000-fold or more compared to the binding activity in the state where the antigen-binding domain is not dissociated from the polypeptide. When the polypeptide of the present disclosure includes an antigen-binding domain and a carrier portion, in certain embodiments, when measuring the antigen-binding activity of the antigen-binding domain by one method selected from the above methods for the antigen-binding domain before dissociation, no binding between the antigen-binding domain and the antigen is observed. When the polypeptide of the present disclosure includes an antigen-binding domain and a carrier portion, in certain embodiments, since the protease cleavage sequence is cleaved and the antigen-binding domain can be released from the polypeptide, the comparison of antigen-binding activity in such embodiments can be carried out by comparing the antigen-binding activities before and after cleavage of the polypeptide. That is, compared with the antigen-binding activity measured using the uncleaved polypeptide, the antigen-binding activity measured using the cleaved polypeptide is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, or 3000-fold or more. In some more specific embodiments, when measuring the antigen-binding activity of the uncleaved polypeptide by one method selected from the above methods, no binding between the antigen-binding domain and the antigen is observed. When the polypeptide of the present disclosure includes an antigen-binding domain and a carrier portion, in certain embodiments, since the protease cleavage sequence is cleaved by a protease, the comparison of antigen-binding activity in such embodiments can be carried out by comparing the antigen-binding activities before and after protease treatment of the polypeptide. That is, compared with the antigen-binding activity measured using the polypeptide without protease treatment, the antigen-binding activity measured using the polypeptide after protease treatment is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, or 3000-fold or more. In some more specific embodiments, when measuring the antigen-binding activity of the polypeptide without protease treatment by one method selected from the above methods, no binding between the antigen-binding domain and the antigen is observed.

[0155] When the polypeptide of the present disclosure includes an antigen-binding domain and a carrier portion, in certain embodiments, the antigen-binding activity of the antigen-binding domain is suppressed by the association of the antigen-binding domain and the inhibitory domain of the carrier portion.

[0156] When the polypeptide of the present disclosure includes an antigen-binding domain and a carrier portion, in certain embodiments, the association between the antigen-binding domain and the inhibitory domain of the carrier portion is eliminated by the protease cleavage sequence being cleaved by a protease.

[0157] When the polypeptide of the present disclosure includes an antigen-binding domain and a carrier portion, in certain embodiments, the antigen-binding domain includes or is a single-domain antibody, and the inhibitory domain of the carrier portion suppresses the antigen-binding activity of the single-domain antibody. The single-domain antibody may be a VHH, a VH having antigen-binding activity with a single domain, or a VL having antigen-binding activity with a single domain.

[0158] When the polypeptide of the present disclosure includes an antigen-binding domain and a carrier portion, in certain embodiments, the inhibitory domain of the carrier portion associates with the antigen-binding domain. The inhibitory domain may be part of the carrier portion or the entire carrier portion. From another perspective, the portion in the carrier portion that associates with the antigen-binding domain can also be referred to as the inhibitory domain. As a more specific embodiment, an antigen-binding domain that is a single-domain antibody and a inhibitory domain that is VL or VH or VHH form an association like that between an antibody VH and an antibody VL. As an even more specific embodiment, an antigen-binding domain that is a single-domain antibody and a inhibitory domain that is VL or VH or VHH form an association like that between an antibody VH and an antibody VL, and in the state where such an association is formed, the inhibitory domain sterically inhibits the binding between the antigen-binding domain and the antigen, or changes the three-dimensional structure of the antigen-binding site of the antigen-binding domain, whereby the antigen-binding activity of the single-domain antibody is inhibited by the VL or VH or VHH. In an embodiment where VHH is used as the single-domain antibody, when the CDR3, which is the main antigen-binding site of VHH, or a site in the vicinity thereof is present at the interface that associates with the inhibitory domain, it is considered that the binding between VHH and the antigen is sterically inhibited by the inhibitory domain. Also, the association between the inhibitory domain and the antigen-binding domain can be eliminated, for example, by cleaving a cleavage site. The elimination of the association can be paraphrased, for example, as the interaction state of two or more polypeptide regions being eliminated. Even if the interaction of two or more polypeptide regions is completely eliminated, or even if only a part of the interaction of two or more polypeptide regions is eliminated, it may be acceptable.

[0159] As used herein, the "interface" generally refers to the association surface during association (interaction), and the amino acid residues forming the interface are generally one or more amino acid residues contained in the polypeptide region used for the association, and more preferably, the amino acid residues that approach and are involved in the interaction during the association. Specifically, such an interaction includes non-covalent bonds such as when amino acid residues that approach each other during the association form hydrogen bonds, electrostatic interactions, or salt bridges.

[0160] As used herein, the "amino acid residue forming an interface", in detail, refers to an amino acid residue contained in a polypeptide region constituting the interface in the polypeptide region constituting the interface. The polypeptide region constituting the interface, for example, refers to a polypeptide region responsible for selective binding within or between molecules in an antibody, ligand, receptor, substrate, etc. Specifically, in an antibody, examples thereof include a heavy chain variable region, a light chain variable region, etc. When the polypeptide of the present disclosure contains an antigen-binding domain and a carrier portion, in certain embodiments, examples thereof include an antigen-binding domain and a suppression domain. Examples of the amino acid residue forming an interface include, but are not limited to, for example, amino acid residues that approach each other during association. Amino acid residues that approach each other during association can be found, for example, by analyzing the three-dimensional structure of the polypeptide and examining the amino acid sequence of the polypeptide region that forms an interface during the association of the polypeptide.

[0161] When the polypeptide of the present disclosure includes an antigen-binding domain and a carrier portion, in certain embodiments, in order to promote the association of the antigen-binding domain with the inhibitory domain, amino acid residues involved in the association in the antigen-binding domain, or amino acid residues involved in the association in the inhibitory domain can be modified. As a more specific embodiment, amino acid residues in the antigen-binding domain that form an interface with the inhibitory domain, or amino acid residues in the inhibitory domain that form an interface with the antigen-binding domain can be modified. In a preferred embodiment, the modification of the amino acid residues forming the interface is a method of introducing a mutation of an amino acid residue into the interface such that two or more amino acid residues forming the interface have different charges. The modification of the amino acid residues to have different charges includes modification from an amino acid residue having a positive charge to an amino acid residue having a negative charge or an amino acid residue having no charge, modification from an amino acid residue having a negative charge to an amino acid residue having a positive charge or an amino acid residue having no charge, and modification from an amino acid residue having no charge to an amino acid residue having a positive or negative charge. Such amino acid modifications are for promoting the association, and the position of the amino acid modification and the type of amino acid are not limited as long as the purpose of promoting the association can be achieved. The modification includes, but is not limited to, substitution.

[0162] When the polypeptide of the present disclosure includes an antigen-binding domain and a carrier portion, in certain embodiments, the VHH that is the antigen-binding domain associates with the VL that is the inhibitory domain. As an example of the amino acid residues in the VHH involved in the association with the VL, amino acid residues that form an interface between the VHH and the VL can be referred to. Also, as an example of the amino acid residues in the VHH involved in the association with the VL, not limited thereto, for example, the amino acid residues at positions 37, 44, 45, and 47 can be mentioned (J. Mol. Biol. (2005) 350, 112-125.). By promoting the association between the VHH and the VL, the activity of the VHH is inhibited. At the same time, as an example of the amino acid residues in the VL involved in the association with the VHH, amino acid residues that form an interface between the VHH and the VL can be referred to.

[0163] To promote the association of VHH and VL, the amino acid residues in VHH that participate in the association with VL can be modified. Examples of such amino acid substitutions include, but are not limited to, F37V, Y37V, E44G, Q44G, R45L, H45L, G47W, F47W, L47W, T47W, and / or S47W. Furthermore, without modifying each residue in VHH, VHH having amino acid residues of 37V, 44G, 45L, and / or 47W from the beginning can also be used. Furthermore, as long as the purpose of promoting the association of VHH and VL can be achieved, it is possible to modify the amino acid residues in VL that participate in the association with VHH instead of the amino acids in VHH, and it is also possible to introduce amino acid modifications into both VHH and VL.

[0164] When the polypeptide of the present disclosure contains an antigen-binding domain and a carrier portion, in certain embodiments, VHH is used as the antigen-binding domain, VH or VHH is used as the inhibitory domain, and the antigen-binding domain and the inhibitory domain can be associated. To promote the association between VHH, which is the antigen-binding domain, and VH or VHH, which is the inhibitory domain, the amino acid residues in VHH, which is the antigen-binding domain, that participate in the association with VH or VHH, which is the inhibitory domain, can be identified and those amino acid residues can be modified. Also, the amino acid residues in VH or VHH, which is the inhibitory domain, that participate in the association with VHH, which is the antigen-binding domain, can be identified and those amino acid residues can be modified.

[0165] Also, when using a single-domain antibody other than VHH as the antigen-binding domain, in the same way, the amino acid residues in the antigen-binding domain or the inhibitory domain that participate in the association can be identified and those amino acid residues can be modified.

[0166] When the polypeptide of the present disclosure includes an antigen-binding domain and a carrier portion, in certain embodiments, the carrier portion and the antigen-binding domain are fused via a linker. In a more specific embodiment, the carrier portion and the antigen-binding domain are fused via a linker that includes a protease cleavage sequence. In another specific embodiment, the carrier portion and the antigen-binding domain are fused via a linker, and the resulting fusion protein includes a protease cleavage sequence.

[0167] When the polypeptide of the present disclosure includes an antigen-binding domain and a carrier portion, in certain embodiments, the carrier portion and the antigen-binding domain are fused without a linker. In a more specific embodiment, an amino bond is formed between the N-terminal amino acid of the carrier portion and the C-terminal amino acid of the antigen-binding domain to form a fusion protein. The resulting fusion protein includes a protease cleavage sequence. In certain embodiments, 1 to several amino acids at the N-terminus of the carrier portion and / or 1 to several amino acids at the C-terminus of the antigen-binding domain are modified, and the N-terminus of the carrier portion and the C-terminus of the antigen-binding domain are fused to form a protease cleavage sequence near the fusion position. More specifically, for example, when LSGRSDNH (SEQ ID NO: 18031) is used as the protease cleavage sequence, the 4 amino acids at the C-terminus of the antigen-binding domain are made into the LSGR sequence, and the 4 amino acids at the N-terminus of the carrier portion are made into the SDNH sequence to form a protease cleavage sequence.

[0168] When the polypeptide of the present disclosure includes an antigen-binding domain and a carrier portion, the protease cleavage sequence may be located in any part of the polypeptide as long as it can release the antigen-binding domain upon protease cleavage and does not lose the antigen-binding activity of the released antigen-binding domain.

[0169] When the polypeptide of the present disclosure includes an antigen-binding domain and a carrier portion, in certain embodiments, the carrier portion includes an antibody constant region, and the N-terminus of the antibody constant region and the C-terminus of the antigen-binding domain are fused via a linker or without a linker. In certain embodiments, the protease cleavage sequence is located within the antibody constant region included in the transport moiety. In this case, the protease cleavage sequence may be located within the antibody constant region such that when subjected to cleavage by the protease, the antigen-binding domain is released. In a specific embodiment, the protease cleavage sequence is located within the heavy chain constant region of the antibody included in the transport moiety, and more specifically, on the antigen-binding domain side of amino acid 140 (EU numbering) in the heavy chain constant region of the antibody, or on the antigen-binding domain side of amino acid 122 (EU numbering) in the heavy chain constant region of the antibody. In another specific embodiment, the protease cleavage sequence is located within the light chain constant region of the antibody included in the transport moiety, and more specifically, on the antigen-binding domain side of amino acid 130 (Kabat numbering) in the light chain constant region of the antibody, or on the antigen-binding domain side of amino acid 113 (Kabat numbering) in the light chain constant region of the antibody.

[0170] When the polypeptide of the present disclosure includes an antigen-binding domain and a transport moiety, in certain embodiments, the antigen-binding domain is a single-domain antibody, and the C-terminus of the single-domain antibody and the N-terminus of the transport moiety are fused via a linker or without a linker. In certain embodiments, the protease cleavage sequence is located within the single-domain antibody. In a more specific embodiment, the single-domain antibody is a single-domain antibody prepared from VH or VHH, and the protease cleavage sequence is on the transport moiety side of amino acid 35b (Kabat numbering) of the single-domain antibody, or on the transport moiety side of amino acid 95 (Kabat numbering) of the single-domain antibody, or on the transport moiety side of amino acid 109 (Kabat numbering) of the single-domain antibody. In another specific embodiment, the single-domain antibody is a single-domain antibody prepared from VL, and the protease cleavage sequence is on the transport moiety side of amino acid 32 (Kabat numbering) of the single-domain antibody, or on the transport moiety side of amino acid 91 (Kabat numbering) of the single-domain antibody, or on the transport moiety side of amino acid 104 (Kabat numbering) of the single-domain antibody.

[0171] When the polypeptide of the present disclosure comprises an antigen-binding domain and a carrier moiety, in certain embodiments, the carrier moiety comprises an antibody constant region, the antigen-binding domain is a single-domain antibody, and the antibody constant region and the single-domain antibody are fused either via a linker or without a linker. In a more specific embodiment, the N-terminus of the antibody constant region and the C-terminus of the single-domain antibody are fused either via a linker or without a linker. In another specific embodiment, the C-terminus of the antibody constant region and the N-terminus of the single-domain antibody are fused either via a linker or without a linker. In certain embodiments, the protease cleavage sequence is located within the antibody constant region contained in the carrier moiety. In a more specific embodiment, the protease cleavage sequence is located on the single-domain antibody side of amino acid 140 (EU numbering) in the antibody heavy chain constant region, or on the single-domain antibody side of amino acid 122 (EU numbering) in the antibody heavy chain constant region. In another specific embodiment, the protease cleavage sequence is located on the antigen-binding domain side of amino acid 130 (Kabat numbering) in the antibody light chain constant region, or on the antigen-binding domain side of amino acid 113 (Kabat numbering) in the antibody light chain constant region. In certain embodiments, the protease cleavage sequence is located within a single-domain antibody. In a more specific embodiment, the single-domain antibody is a single-domain antibody made from VH or VHH, and the protease cleavage sequence is on the antibody constant region side of the 35b (Kabat numbering) amino acid of the single-domain antibody, or on the antibody constant region side of the 95 (Kabat numbering) amino acid of the single-domain antibody, or on the antibody constant region side of the 109 (Kabat numbering) amino acid of the single-domain antibody. In another specific embodiment, the single-domain antibody is a single-domain antibody made from VL, and the protease cleavage sequence is on the antibody constant region side of the 32 (Kabat numbering) amino acid of the single-domain antibody, or on the antibody constant region side of the 91 (Kabat numbering) amino acid of the single-domain antibody, or on the antibody constant region side of the 104 (Kabat numbering) amino acid of the single-domain antibody. In certain embodiments, the protease cleavage sequence is located near the boundary between the antigen-binding domain and the carrier moiety. The vicinity of the boundary between the antigen-binding domain and the carrier moiety refers to the portion before and after the site where the antigen-binding domain and the carrier moiety are linked and that does not significantly affect the secondary structure of the antigen-binding domain. In a more specific embodiment, the antigen-binding domain is linked to an antibody constant region contained within the transport moiety, and the protease cleavage sequence is located near the boundary between the antigen-binding domain and the antibody constant region. The vicinity of the boundary between the antigen-binding domain and the antibody constant region can refer to the vicinity of the boundary between the antigen-binding domain and the heavy chain constant region of the antibody, or the vicinity of the boundary between the antigen-binding domain and the light chain constant region of the antibody. When the antigen-binding domain is a single-domain antibody or VHH prepared from VH and is linked to the heavy chain constant region of the antibody, the vicinity of the boundary between the antigen-binding domain and the antibody constant region can refer to the region between the amino acid at position 101 (Kabat numbering) of the single-domain antibody and the amino acid at position 140 (EU numbering) of the heavy chain constant region of the antibody, or the region between the amino acid at position 109 (Kabat numbering) of the single-domain antibody and the amino acid at position 122 (EU numbering) of the heavy chain constant region of the antibody. When the antigen-binding domain is a single-domain antibody or VHH prepared from VH and is linked to the light chain constant region of the antibody, the vicinity of the boundary between the antigen-binding domain and the antibody light chain constant region can refer to the region between the amino acid at position 101 (Kabat numbering) of the single-domain antibody and the amino acid at position 130 (Kabat numbering) of the light chain constant region of the antibody, or the region between the amino acid at position 109 (Kabat numbering) of the single-domain antibody and the amino acid at position 113 (Kabat numbering) of the light chain constant region of the antibody. In the case where the antigen-binding domain is a single-domain antibody prepared from VL, the vicinity of the boundary between the antigen-binding domain and the antibody constant region is from the amino acid at position 96 (Kabat numbering) of the single-domain antibody or from the amino acid at position 104 (Kabat numbering) of the single-domain antibody.

[0172] When the polypeptide of the present disclosure includes an antigen-binding domain and a transport portion, in certain embodiments, the polypeptide is an IgG antibody-like molecule. Examples of such embodiments include, but are not limited to, for example, embodiments in which the transport portion includes an IgG antibody constant region, a single-domain antibody that is the antigen-binding domain replaces the VH of the IgG antibody, and the antigen-binding activity is suppressed by the VL; or embodiments in which the transport portion includes an IgG antibody constant region, a single-domain antibody that is the antigen-binding domain replaces the VL of the IgG antibody, and the antigen-binding activity is suppressed by the VH; or embodiments in which the transport portion includes an IgG antibody constant region, a single-domain antibody that is the antigen-binding domain replaces one of the VH / VL of the IgG antibody, and another single-domain antibody that suppresses the antigen-binding activity of the antigen-binding domain replaces the other of the VH / VL of the IgG antibody, and the like.

[0173] As used herein, the term "IgG antibody-like molecule" is used to define a molecule having a portion that is substantially similar to the structure of the constant domain or constant region such as an IgG antibody and a portion that is substantially similar to the structure of the variable domain or variable region such as an IgG antibody, and having a three-dimensional structure substantially similar to that of an IgG antibody. The domain similar to antibody CH1 and the domain similar to CL in the IgG antibody-like molecule can be used interchangeably with each other, that is, as long as the two domains have an interaction like CH1 and CL of the IgG antibody, the domain linked to the portion similar to the antibody hinge region can be either the antibody CH1 domain or the antibody CL domain. However, the "IgG antibody-like molecule" herein is not limited to exhibiting antigen-binding activity while maintaining a structure similar to that of an IgG antibody.

[0174] When the polypeptide of the present disclosure contains an antigen-binding domain and a carrier portion, the antigen-binding domain contained in the polypeptide may be one or plural. The inhibitory domain that inhibits the antigen-binding activity of each of the plural antigen-binding domains may also be one or plural. Each of the plural antigen-binding domains may form an association with an inhibitory domain. Each of the plural antigen-binding domains may be fused with a carrier portion. Each of the plural antigen-binding domains may be capable of dissociating from the polypeptide. The protease cleavage sequences for dissociating the plural antigen-binding domains may be plural corresponding to each antigen-binding domain.

[0175] When the polypeptide is an IgG antibody-like molecule, an embodiment in which antigen-binding domains are provided in portions corresponding to the two variable regions of the IgG antibody as shown in FIG. 3 would be an embodiment understandable to those skilled in the art who are familiar with the present disclosure. Whether the antigen-binding domains incorporated in both arms have the same antigen-binding specificity or different antigen-binding specificities is an embodiment that can be naturally understood by those skilled in the art who are familiar with the present disclosure, and it is obvious that it does not deviate from the scope of the present disclosure.

[0176] When the polypeptide of the present disclosure includes an antigen-binding domain and a transport moiety, in certain embodiments, the antigen-binding domain is further linked to a second antigen-binding domain. Examples of the second antigen-binding domain include, but are not limited to, for example, single-domain antibodies, antibody fragments, a module called the A domain of about 35 amino acids contained in Avimer, which is a cell membrane protein present in vivo (International Publication WO2004 / 044011, WO2005 / 040229), Adnectin containing a 10Fn3 domain, which is a domain that binds to a protein in fibronectin, a glycoprotein expressed on the cell membrane (International Publication WO2002 / 032925), Affibody with an IgG-binding domain composed of a three-helix bundle of 58 amino acids of ProteinA as a scaffold (International Publication WO1995 / 001937), DARPins (Designed Ankyrin Repeat proteins), which are regions exposed on the molecular surface of ankyrin repeats (AR) having a structure in which a subunit of a turn containing 33 amino acid residues and two antiparallel helices and loops are repeatedly stacked (International Publication WO2002 / 020565), Anticalin, etc., which are four loop regions that support one side of a barrel structure in which eight antiparallel strands highly conserved in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) are twisted in the central direction (International Publication WO2003 / 029462), a sunken region of a parallel sheet structure inside a saddle-shaped structure in which leucine-rich-repeat (LRR) modules of a variable lymphocyte receptor (VLR), which has an immunoglobulin structure as an acquired immune system of agnathans such as lampreys and hagfishes, are repeatedly stacked (International Publication WO2008 / 016854), and the like. In a preferred embodiment, the second antigen-binding domain has an antigen-binding specificity different from that of the antigen-binding domain.In a preferred embodiment, the molecular weights of the linked antigen-binding domain and the second antigen-binding domain are 60 kDa or less. In some more specific embodiments, the antigen-binding domain and the second antigen-binding domain are single-domain antibodies each having a different antigen-binding specificity, the linked antigen-binding domain and the second antigen-binding domain are releasable from the polypeptide, and the released antigen-binding domain and the second antigen-binding domain form a bispecific antigen-binding molecule. Examples of such bispecific antigen-binding molecules include, but are not limited to, for example, a bispecific antigen-binding molecule in which the antigen-binding domain specifically binds to a target cell surface antigen and the second antigen-binding domain specifically binds to an immune cell surface antigen, a bispecific antigen-binding molecule in which the antigen-binding domain and the second antigen-binding domain bind to different subunits of the same antigen, a bispecific antigen-binding molecule in which the antigen-binding domain and the second antigen-binding domain bind to different epitopes in the same antigen, and the like. Such bispecific antigen-binding molecules can recruit immune cells to the vicinity of target cells in the treatment of diseases caused by target cells and are considered useful. The antigen-binding activity of the second antigen-binding domain may or may not be suppressed by the transport moiety. Also, the second antigen-binding domain may or may not form an association with a partial structure of the transport moiety. In particular, when the antigen-binding domain and the second antigen-binding domain have different antigen-binding specificities, for example, as shown in FIG. 4, even if the antigen-binding activity of the second antigen-binding domain is not suppressed and the second antigen-binding domain does not form an association with a partial structure of the transport moiety, the antigen-binding activity of the antigen-binding domain cannot be exerted in a state where the antigen-binding domain does not dissociate, and the bispecific antigen-binding molecule in which the antigen-binding domain and the second antigen-binding domain are linked cannot exert the function of binding to two types of antigens bispecifically. In FIG. 4, one embodiment in which the antigen-binding domain is further linked to the second antigen-binding domain is illustrated.

[0177] As used herein, the term "specificity" refers to the property that one of the molecules that specifically binds does not substantially bind to molecules other than the one or more molecules to which it binds. It is also used when an antigen-binding domain has specificity for an epitope contained in a specific antigen. It is also used when an antigen-binding domain has specificity for a specific epitope among a plurality of epitopes contained in a certain antigen. Here, not substantially binding is determined according to the method described in the section of binding activity, and the binding activity of the specific binding molecule to molecules other than the partner molecule is 80% or less, usually 50% or less, preferably 30% or less, particularly preferably 15% or less of the binding activity to the partner molecule.

[0178] The sequences represented by any of SEQ ID NOs: 5 to 18003, or partial sequences that can be used as protease cleavage sequences contained in those sequences (sequences from the 4th amino acid to the 15th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 5 to 17201, sequences from the 4th amino acid to the 13th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 5 to 17201, sequences from the 6th amino acid to the 13th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 5 to 17201, sequences from the 1st amino acid to the 12th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 17202 to 17993, sequences from the 3rd amino acid to the 12th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 17202 to 17993, sequences from the 3rd amino acid to the 11th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 17202 to 17993, sequences from the 3rd amino acid to the 10th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 17202 to 17993, sequences from the 3rd amino acid to the 14th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 17994 to 18003, sequences from the 5th amino acid to the 12th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 17994 to 18003, sequences from the 5th amino acid to the 10th amino acid at the N-terminus of the sequences selected from SEQ ID NOs: 17994 to 18003) can be used as protease cleavage sites in the polypeptide exemplified in FIG. 1. An array composed in the order of "an array selected from the following Group A - an array selected from the following Group B" from the N-terminus can also be used as a protease cleavage site in the polypeptide exemplified in FIG. 1: (Group A) The sequence from the 1st amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 2nd amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 3rd amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 4th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 5th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 6th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 7th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 8th amino acid to the 9th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 5 to 17201, The sequence from the 1st amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 2nd amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 3rd amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 4th amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 5th amino acid to the 6th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17202 to 17993, The sequence from the 1st amino acid to the 8th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003, The sequence from the second amino acid to the eighth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 The sequence from the third amino acid to the eighth amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 17994 to 18003 ...

Claims

1. A protease substrate, (1) having a cleavage rate by human matriptase (MT-SP1) higher than that of a protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3, and / or (2) having a cleavage rate by human urokinase (uPA) higher than that of a protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3, wherein the protease substrate is a protease substrate selected from at least one of the following sequences: The sequence from the 4th amino acid to the 15th amino acid at the N-terminus of a sequence selected from SEQ ID NO: 430, 400, 8, 26, 107, 114, 117, 130, 133, 135, 143, 149, 155, 156, 170, 172, 179, 184, 191, 203, 225, 265, 268, 271, 339, 359, and 425, The sequence from the 4th amino acid to the 13th amino acid at the N-terminus of a sequence selected from SEQ ID NO: 430, 400, 8, 26, 107, 114, 117, 130, 133, 135, 143, 149, 155, 156, 170, 172, 179, 184, 191, 203, 225, 265, 268, 271, 339, 359, and 425, The sequence from the 6th amino acid to the 13th amino acid at the N-terminus of a sequence selected from SEQ ID NO: 430, 400, 8, 26, 107, 114, 117, 130, 133, 135, 143, 149, 155, 156, 170, 172, 179, 184, 191, 203, 225, 265, 268, 271, 339, 359, and 425, and A sequence represented by any of SEQ ID NO: 430, 400, 8, 26, 107, 114, 117, 130, 133, 135, 143, 149, 155, 156, 170, 172, 179, 184, 191, 203, 225, 265, 268, 271, 339, 359, and 425, comprising (i) having a higher ratio of the cleavage rate by human MT-SP1 or human uPA to the cleavage rate by human serum compared to a protease substrate containing any one of the sequences of SEQ ID NO: 1, 2, or 3, and / or (ii) having a lower cleavage rate by human serum compared to the peptide represented by SEQ ID NO: 4, a protease substrate.

2. Use of at least one sequence selected from the following as a protease cleavage sequence: The sequence from the 4th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 430, 400, 8, 26, 107, 114, 117, 130, 133, 135, 143, 149, 155, 156, 170, 172, 179, 184, 191, 203, 225, 265, 268, 271, 339, 359, and 425 The sequence from the 4th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 430, 400, 8, 26, 107, 114, 117, 130, 133, 135, 143, 149, 155, 156, 170, 172, 179, 184, 191, 203, 225, 265, 268, 271, 339, 359, and 425 The sequence from the 6th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 430, 400, 8, 26, 107, 114, 117, 130, 133, 135, 143, 149, 155, 156, 170, 172, 179, 184, 191, 203, 225, 265, 268, 271, 339, 359, and 425, and The sequence represented by any of SEQ ID NOs: 430, 400, 8, 26, 107, 114, 117, 130, 133, 135, 143, 149, 155, 156, 170, 172, 179, 184, 191, 203, 225, 265, 268, 271, 339, 359, and 425 [

3. ] A polypeptide comprising at least one protease cleavage sequence selected from the following: The sequence from the 4th amino acid to the 15th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 430, 400, 8, 26, 107, 114, 117, 130, 133, 135, 143, 149, 155, 156, 170, 172, 179, 184, 191, 203, 225, 265, 268, 271, 339, 359, and 425 The sequence from the 4th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 430, 400, 8, 26, 107, 114, 117, 130, 133, 135, 143, 149, 155, 156, 170, 172, 179, 184, 191, 203, 225, 265, 268, 271, 339, 359, and 425 The sequence from the 6th amino acid to the 13th amino acid at the N-terminus of the sequence selected from SEQ ID NOs: 430, 400, 8, 26, 107, 114, 117, 130, 133, 135, 143, 149, 155, 156, 170, 172, 179, 184, 191, 203, 225, 265, 268, 271, 339, 359, and 425, and The sequence represented by any of SEQ ID NOs: 430, 400, 8, 26, 107, 114, 117, 130, 133, 135, 143, 149, 155, 156, 170, 172, 179, 184, 191, 203, 225, 265, 268, 271, 339, 359, and 425. **Claim 4** The polypeptide according to claim 3, wherein the polypeptide comprises an antigen-binding domain and a transport portion, and the transport portion has a suppression domain that suppresses the antigen-binding activity of the antigen-binding domain. **Claim 5** The polypeptide according to claim 3, wherein the polypeptide is a ligand-binding molecule capable of binding to a ligand, and the binding of the ligand-binding molecule to the ligand in the state where the protease cleavage sequence is cleaved is weaker than the binding of the ligand-binding molecule to the ligand in the state where the protease cleavage sequence is uncleaved. **Claim 6** The polypeptide according to claim 3, wherein the polypeptide is a ligand-binding molecule capable of binding to a ligand, the ligand-binding molecule comprises a single-domain antibody, the single-domain antibody is capable of binding to the ligand and at least one protease cleavage sequence is introduced, 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 protease cleavage sequence is uncleaved. **Claim 7** A fusion protein in which the ligand is fused with the polypeptide according to claim 5 or claim 6. **Claim 8** A pharmaceutical composition comprising the polypeptide according to any one of claims 3 to 6, or the fusion protein according to claim 7. **Claim 9** A pharmaceutical composition comprising the polypeptide according to claim 5 or claim 6 and the ligand. **Claim 10** A polynucleotide encoding the polypeptide according to any one of claims 3 to 6, or the fusion protein according to claim 7.

11. A method for producing the polypeptide according to any one of Claims 3 to 6, or the fusion protein according to Claim 7.

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