Protease substrates and polypeptides containing protease cleavage sequences

Abstract

To provide protease substrates, protease-cleavable peptide sequences, polypeptides containing protease cleavage sequences, and methods for producing the same, as well as to provide pharmaceutical compositions containing polypeptides containing protease cleavage sequences, and methods for releasing antigen-binding domains or ligands by cleavage of a protease cleavage sequence contained in a polypeptide.SOLUTION: Compounds that can be used as protease substrates, particularly peptide sequences that can be used as protease substrates and can be cleaved by proteases are discovered. It is discovered that polypeptides containing protease-cleavable peptide sequences (abbreviated as protease cleavage sequences) are useful in treating diseases, including administering such polypeptides, and polypeptides containing such protease cleavage sequences are useful in the manufacture of pharmaceuticals for treating diseases. Also polypeptides containing such protease cleavage sequences and methods for producing the same are created.SELECTED DRAWING: None

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 the protease cleavage sequence contained in a polypeptide. [Background technology]

[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 in proteins. Proteases occur naturally in all living organisms and are involved in a variety of physiological reactions, 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] Thus, there is a need to identify new substrates for proteases and to use these substrates in a variety of therapeutic, diagnostic, and prophylactic indications. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO2018 / 097307 [Patent Document 2] WO2018 / 097308 Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure has been made in light of these circumstances, and one of its purposes is to provide 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 the protease cleavage sequence contained in a polypeptide. [Means for solving the problem]

[0006] The inventors of the present disclosure have conducted extensive research to achieve the above-mentioned object, and have found compounds that can be used as protease substrates, in particular peptide sequences that can be used as protease substrates / that can be cleaved by proteases, and have also found that polypeptides containing protease-cleavable peptide sequences (abbreviated as protease cleavage sequences) are useful in treating diseases, including administering such polypeptides, and that polypeptides containing such protease cleavage sequences are useful in the manufacture of pharmaceuticals for treating diseases. Furthermore, the inventors of the present disclosure have created polypeptides containing such protease cleavage sequences and methods for producing them, thereby completing the present disclosure.

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

[10] , comprising at least one sequence selected from the following: 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 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 to 11th amino acid at the N-terminus of a selected sequence, a sequence from the 3rd to 10th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993, a sequence from the 3rd to 14th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003, a sequence from the 5th to 12th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003, a sequence from the 5th to 10th 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.

[12] A protease substrate comprising at least one sequence selected from the following: 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 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 to 11th amino acid at the N-terminus of a selected sequence, a sequence from the 3rd to 10th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993, a sequence from the 3rd to 14th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003, a sequence from the 5th to 12th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003, a sequence from the 5th to 10th 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.

[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] A protease substrate according to any one of

[12] to

[14] , which has a higher cleavage rate by the protease than a protease substrate comprising any one of the sequences of SEQ ID NOs: 1, 2, and 3.

[16] A protease substrate according to any one of

[12] to

[15] , which has a higher cleavage rate by human uPA than a protease substrate comprising any one of the sequences of SEQ ID NOs: 1, 2, and 3.

[17] A protease substrate according to any one of

[12] to

[16] , which has a higher cleavage rate by human MT-SP1 than a protease substrate comprising any one of the sequences of SEQ ID NOs: 1, 2, and 3.

[18] A protease substrate according to any one of

[12] to

[17] , which has a higher cleavage rate by mouse uPA than a protease substrate comprising any one of the sequences of SEQ ID NOs: 1, 2, and 3.

[19] A protease substrate according to any one of

[12] to

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

[20] A protease substrate according to any one of

[12] to

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

[21] A protease substrate according to any one of

[12] to

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

[22] A protease substrate according to any one of

[12] to

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

[23] A protease substrate according to any one of

[12] to

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

[24] A protease substrate according to 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: 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 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 to 11th amino acid at the N-terminus of a selected sequence, a sequence from the 3rd to 10th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993, a sequence from the 3rd to 14th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003, a sequence from the 5th to 12th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003, a sequence from the 5th to 10th 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.

[26] A polypeptide comprising at least one sequence selected from the following: 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 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 to 11th amino acid at the N-terminus of a selected sequence, a sequence from the 3rd to 10th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993, a sequence from the 3rd to 14th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003, a sequence from the 5th to 12th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003, a sequence from the 5th to 10th 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.

[27] 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. the 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, the 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, the 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, the 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, or the sequence selected from the sequences represented by SEQ ID NOs: 5 to 18003, is a protease cleavage sequence that can be cleaved 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 comprising the polynucleotide described in

[29] .

[31] A host cell comprising 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 a culture supernatant.

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

[27] , comprising an antigen-binding domain and a transport moiety, wherein the transport moiety has an inhibition domain that inhibits the antigen-binding activity of the antigen-binding domain. [A-2] The polypeptide of [A-1], wherein the inhibition of the antigen-binding activity of the antigen-binding domain by the repression domain when the protease cleavage sequence is cleaved by a protease is weaker than the inhibition of the antigen-binding activity of the antigen-binding domain by the repression domain when the protease cleavage sequence is not cleaved. [A-3] The polypeptide according to [A-1] or [A-2], wherein the antigen-binding domain has a shorter half-life in blood than the uncleaved polypeptide. [A-4] The polypeptide according to any one of [A-1] to [A-3], wherein the antigen-binding domain has a shorter half-life in blood than the delivery moiety. [A-5] The polypeptide according to any one of [A-1] to [A-4], wherein the molecular weight of the antigen-binding domain is smaller than the molecular weight of the transport moiety. [A-6] The polypeptide according to any one of [A-1] to [A-5], wherein the molecular weight of the antigen-binding domain is 60 kDa or less. [A-7] The polypeptide according to any one of [A-1] to [A-6], wherein the transport moiety has FcRn-binding activity, and the antigen-binding domain has no FcRn-binding activity or weaker FcRn-binding activity than the transport moiety. [A-8] The polypeptide of any one of [A-1] to [A-7], wherein the antigen-binding domain is releasable from the polypeptide, and the antigen-binding activity of the antigen-binding domain when released from the polypeptide is higher than the antigen-binding activity of the antigen-binding domain when not released from the polypeptide. [A-9] The polypeptide according to any one of [A-1] to [A-8], wherein the antigen-binding activity of the antigen-binding domain is inhibited by association of the antigen-binding domain with the inhibition domain of the transport moiety. [A-10] The polypeptide according to [A-8], wherein the antigen-binding domain can be released from the polypeptide upon cleavage of the protease cleavage sequence by a protease. [A-11] The polypeptide according to [A-9], wherein the association between the antigen-binding domain and the repression domain of the transport moiety is dissolved by cleavage of the protease cleavage sequence by a protease. [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 type of protease selected from human MT-SP1, mouse MT-SP1, human uPA, and mouse uPA. [A-14] A polypeptide according to any one of [A-1] to [A-13], further comprising a first flexible linker attached 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 consisting of a glycine-serine polymer. [A-16] The polypeptide according to [A-14] or [A-15], further comprising a second flexible linker attached 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 consisting of a glycine-serine polymer. [A-18] The polypeptide described in any one of [A-1] to [A-17], wherein the antigen-binding domain comprises or is a single-domain antibody, and the inhibitory domain of the transporter inhibits the antigen-binding activity of the single-domain antibody. [A-19] The polypeptide according to [A-18], wherein the single-domain antibody is a VHH, or a VH having antigen-binding activity as a single domain, or a VL having antigen-binding activity as a single domain. [A-20] A polypeptide described in any one of [A-1] to [A-19], wherein the antigen-binding domain comprises a single-domain antibody, and the inhibitory domain of the transporter 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-21] A polypeptide described in any one of [A-1] to [A-20], wherein the antigen-binding domain comprises a single-domain antibody, the inhibitory domain of the transporter 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-22] The polypeptide described in any one of [A-18] to [A-21], wherein the single-domain antibody is a VHH or a VH having antigen-binding activity as a single domain, the inhibitory domain of the transporter is an antibody VL, and the antigen-binding activity of the VHH or the VH having antigen-binding activity as a single domain is inhibited by associating with the antibody VL. [A-23] A polypeptide described in any one of [A-18] to [A-22], 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-24] 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 amino acid selected from amino acids 37V, 44G, 45L, or 47W (all Kabat numbering). [A-25] 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 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-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 polypeptide according to [A-31], wherein the transporter comprises an antibody heavy chain constant region, and the antibody heavy chain constant region and the antigen-binding domain are fused with or without a linker. [A-34] The polypeptide according to [A-31], wherein the transporter comprises an antibody light chain constant region, and the antibody light chain constant region and the antigen-binding domain are fused with or without a linker. [A-35] The polypeptide according to [A-33], wherein the N-terminus of the antibody heavy chain constant region of the transporter is fused to the C-terminus of the antigen-binding domain with or without a linker, and the protease cleavage sequence is located within the sequence of the antigen-binding domain or closer to the antigen-binding domain than amino acid 122 (EU numbering) of the heavy chain antibody constant region. [A-36] The polypeptide according to [A-34], wherein the N-terminus of the antibody light chain constant region of the transporter is fused to the C-terminus of the antigen-binding domain with or without a linker, and the protease cleavage sequence is located within the sequence of the antigen-binding domain or closer to the antigen-binding domain than amino acid 113 (Kabat numbering) of the light chain antibody constant region. [A-37] The polypeptide according to any one of [A-32] to [A-35], wherein the N-terminus of the antibody constant region of the transporter moiety is fused to the C-terminus of the antigen-binding domain with or without a linker, the antigen-binding domain is a single-domain antibody prepared from VH or VHH, and the protease cleavage sequence is located in the sequence of the antibody constant region or closer to the antibody constant region than amino acid 109 (Kabat numbering) of the single-domain antibody of the antigen-binding domain. [A-38] The polypeptide according to [A-32], wherein the N-terminus of the antibody constant region of the transporter is fused to the C-terminus of the antigen-binding domain with or without a linker, and the protease cleavage sequence is located near the boundary between the antigen-binding domain and the antibody constant region. [A-39] The polypeptide according to [A-33], wherein the N-terminus of the antibody heavy chain constant region of the transporter is fused to the C-terminus of the antigen-binding domain with 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. [A-40] The polypeptide according to [A-34], wherein the N-terminus of the antibody light chain constant region of the transporter is fused to the C-terminus of the antigen-binding domain with 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. [A-41] The polypeptide according to [A-39], wherein the antigen-binding domain is a single-domain antibody or VHH prepared from VH, and the protease cleavage sequence is located between amino acid 109 (Kabat numbering) of the single-domain antibody of the antigen-binding domain and amino acid 122 (EU numbering) of the antibody heavy chain constant region. [A-42] The polypeptide described in [A-40], wherein the antigen-binding domain is a single-domain antibody or VHH prepared from VH, and the protease cleavage sequence is located between amino acid 109 (Kabat numbering) of the single-domain antibody of the antigen-binding domain and amino acid 113 (Kabat numbering) of the antibody light chain constant region. [A-43] The polypeptide according to [A-39], wherein the antigen-binding domain is a single-domain antibody prepared from a VL, and the protease cleavage sequence is located between amino acid 104 (Kabat numbering) of the single-domain antibody antigen-binding domain and amino acid 122 (EU numbering) of the antibody heavy chain constant region. [A-44] The polypeptide according to [A-40], wherein the antigen-binding domain is a single-domain antibody prepared from a VL, and the protease cleavage sequence is located between amino acid 109 (Kabat numbering) of the single-domain antibody of the antigen-binding domain and amino acid 113 (Kabat numbering) of the antibody light chain constant region. [A-45] The polypeptide according to any one of [A-31] to [A-44], wherein the antibody constant region of the polypeptide is an IgG antibody constant region. [A-46] The polypeptide according to any one of [A-1] to [A-45], wherein the polypeptide is an IgG antibody-like molecule. [A-47] The polypeptide according to any one of [A-1] to [A-46], wherein when the antigen-binding domain is not released and measurement is performed using the BLI (Bio-Layer Interferometry) method (Octet), no binding between the antigen-binding domain and the antigen is observed. [A-48] The polypeptide according to any one of [A-1] to [A-47], wherein a second antigen-binding domain is further linked to the antigen-binding domain. [A-49] The polypeptide according to [A-48], wherein the second antigen-binding domain has an antigen-binding specificity different from that of the antigen-binding domain. [A-50] The polypeptide described in [A-48] or [A-49], wherein the second antigen-binding domain comprises a second single-domain antibody. [A-51] The polypeptide of [A-50], wherein 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 the single-domain antibody and the second single-domain antibody form a bispecific antigen-binding molecule in the released state of the antigen-binding domain and the second antigen-binding domain. [A-52] The polypeptide according to any one of [A-1] to [A-51], wherein the polypeptide further comprises an antigen-binding domain other than the antigen-binding domain, and the antigen-binding activity of the other antigen-binding domain is also inhibited by linking the other antigen-binding domain to the transport moiety of the polypeptide. [A-53] The polypeptide according to [A-52], wherein the additional antigen-binding domain has an antigen-binding specificity different from that of the antigen-binding domain. [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-61] A polypeptide comprising an antigen-binding domain, comprising: a sequence from the 4th to 15th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; a sequence from the 4th to 13th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; a sequence from the 6th to 13th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; a sequence from the 1st to 12th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; a sequence from the 3rd to 12th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; or a sequence from the 3rd to 11th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993. a sequence from the 3rd to 10th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; a sequence from the 3rd to 14th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; a sequence from the 5th to 12th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; a sequence from the 5th to 10th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; or a sequence represented by SEQ ID NOs: 5 to 18003. [A-62] The method described in [A-61], wherein the polypeptide further comprises a transport moiety, which has an inhibition domain that inhibits the antigen-binding activity of the antigen-binding domain. [A-63] The method according to [A-62], wherein the inhibition of the antigen-binding activity of the antigen-binding domain by the repression domain when the protease cleavage sequence is cleaved by a protease is weaker than the inhibition of the antigen-binding activity of the antigen-binding domain when the protease cleavage sequence is not cleaved. [A-64] The method described in [A-62] or [A-63], wherein the antigen-binding domain has a shorter half-life in blood than the uncleaved polypeptide. [A-65] The method described in any one of [A-62] to [A-64], wherein the antigen-binding domain has a shorter half-life in blood than the delivery moiety. [A-66] 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. [A-67] The method described in any one of [A-62] to [A-66], wherein the molecular weight of the antigen-binding domain is 60 kDa or less. [A-68] The method described in 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 weaker FcRn-binding activity than the transport moiety. [A-69] The method described in any one of [A-62] to [A-68], wherein the antigen-binding activity of the antigen-binding domain when released from the polypeptide is higher than the antigen-binding activity when not released from the polypeptide. [A-70] The method according to any one of [A-62] to [A-69], wherein the antigen-binding activity of the antigen-binding domain is inhibited by association of the antigen-binding domain with the inhibition domain of the transport moiety. [A-71] The method according to [A-70], wherein the association between the antigen-binding domain and the repression domain of the transport moiety is dissolved by cleavage of the protease cleavage sequence by a protease. [A-72] The method according to any one of [A-62] to [A-71], wherein the protease is matriptase and / or urokinase. [A-73] 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. [A-74] 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 described in [A-74], wherein the first flexible linker is a flexible linker consisting of a glycine-serine polymer. [A-76] The method according to [A-74] or [A-75], wherein a second flexible linker is further added to the other end of the protease cleavage sequence. [A-77] The method described in [A-76], wherein the second flexible linker is a flexible linker consisting of a glycine-serine polymer. [A-78] The method described in any one of [A-62] to [A-77], wherein the antigen-binding domain comprises or is a single-domain antibody, and the inhibitory domain of the transporter inhibits the antigen-binding activity of the single-domain antibody. [A-79] The method described in [A-78], wherein the single-domain antibody is a VHH, or a VH having antigen-binding activity as a single domain, or a VL having antigen-binding activity as a single domain. [A-80] A method described in any one of [A-62] to [A-79], wherein the antigen-binding domain comprises a single-domain antibody, the inhibitory domain of the transporter 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] A method described in any one of [A-62] to [A-80], wherein the antigen-binding domain comprises a single-domain antibody, the inhibitory domain of the transporter 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] A 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 as a single domain, the inhibitory domain of the transporter is an antibody VL, and the antigen-binding activity of the VHH or the VH having antigen-binding activity as 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] 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 set of amino acid substitutions selected from F37V / R45L, F37V / G47W, R45L / G47W, and F37V / R45L / G47W (all Kabat numbering). [A-89] The method described in any one of [A-78] to [A-81], 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-90] The method described in any one of [A-62] to [A-89], wherein the transport moiety has an FcRn binding region. [A-91] The method according to any one of [A-62] to [A-90], wherein the transport moiety comprises an antibody constant region. [A-92] The method described in [A-91], wherein the antibody constant region of the transporter and the antigen-binding domain are fused via a linker or without a linker. [A-93] The method described in [A-91], wherein the transport moiety comprises an antibody heavy chain constant region, and the antibody heavy chain constant region and the antigen-binding domain are fused with or without a linker. [A-94] The method described in [A-91], wherein the transport moiety comprises an antibody light chain constant region, and the antibody light chain constant region and the antigen-binding domain are fused with or without a linker. [A-95] The method described in [A-93], wherein the polypeptide comprises a fusion between the N-terminus of the antibody heavy chain constant region of the transporter and the C-terminus of the antigen-binding domain, with or without a linker, and the protease cleavage sequence is located within the sequence of the antigen-binding domain or closer to the antigen-binding domain than amino acid 122 (EU numbering) of the heavy chain antibody constant region. [A-96] The method described in [A-94], wherein the polypeptide comprises a fusion between the N-terminus of the antibody light chain constant region of the transporter and the C-terminus of the antigen-binding domain, with or without a linker, and the protease cleavage sequence is located within the sequence of the antigen-binding domain or closer to the antigen-binding domain than amino acid 113 (Kabat numbering) of the light chain antibody constant region. [A-97] The method described in any one of [A-92] to [A-95], wherein the polypeptide comprises a fusion between the N-terminus of the antibody constant region of the transport moiety and the C-terminus of the antigen-binding domain, with or without a linker, the antigen-binding domain being 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 closer to the antibody constant region than amino acid 109 (Kabat numbering) of the single-domain antibody of the antigen-binding domain. [A-98] The method described in [A-92], wherein the polypeptide has the N-terminus of the antibody constant region of the transporter fused to the C-terminus of the antigen-binding domain with or without a linker, and the protease cleavage sequence is located near the boundary between the antigen-binding domain and the antibody constant region. [A-99] The polypeptide described in [A-93], wherein the N-terminus of the antibody heavy chain constant region of the transporter is fused to the C-terminus of the antigen-binding domain with 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. [A-100] The method described in [A-34], wherein the polypeptide has the N-terminus of the antibody light chain constant region of the transporter fused to the C-terminus of the antigen-binding domain with 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. [A-101] The method described in [A-99], wherein the antigen-binding domain is a single-domain antibody or VHH prepared from VH, and the protease cleavage sequence is located between amino acid 109 (Kabat numbering) of the single-domain antibody of the antigen-binding domain and amino acid 122 (EU numbering) of the antibody heavy chain constant region. [A-102] The method described in [A-100], wherein the antigen-binding domain is a single-domain antibody or VHH prepared from VH, and the protease cleavage sequence is located between amino acid 109 (Kabat numbering) of the single-domain antibody of the antigen-binding domain and amino acid 113 (Kabat numbering) of the antibody light chain constant region. [A-103] The method described in [A-99], wherein the antigen-binding domain is a single-domain antibody prepared from a VL, and the protease cleavage sequence is located between amino acid 104 (Kabat numbering) of the single-domain antibody antigen-binding domain and amino acid 122 (EU numbering) of the antibody heavy chain constant region. [A-104] The method described in [A-100], wherein the antigen-binding domain is a single-domain antibody prepared from a VL, and the protease cleavage sequence is located between amino acid 109 (Kabat numbering) of the single-domain antibody antigen-binding domain and amino acid 113 (Kabat numbering) of the antibody light chain constant region. [A-105] The method described in any one of [A-91] to [A-104], wherein the antibody constant region of the polypeptide is an IgG antibody constant region. [A-106] The method described in any one of [A-62] to [A-105], wherein the polypeptide is an IgG antibody-like molecule. [A-107] The method according to any one of [A-62] to [A-106], wherein when the antigen-binding domain is not released and measurement is performed using BLI (Bio-Layer Interferometry) (Octet), no binding between the antigen-binding domain and the antigen is observed. [A-108] The method described in 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 described in [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 described in [A-108] or [A-109], wherein the second antigen-binding domain comprises a second single-domain antibody. [A-111] The method according to [A-110], wherein 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 the single-domain antibody and the second single-domain antibody form a bispecific antigen-binding molecule in the released state of the antigen-binding domain and the second antigen-binding domain. [A-112] The method described in any one of [A-62] to [A-111], wherein the polypeptide further comprises an antigen-binding domain other than the antigen-binding domain, and the antigen-binding activity of the other antigen-binding domain is also inhibited by linking the other antigen-binding domain to the transport moiety of the polypeptide. [A-113] The method described in [A-112], wherein the additional 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 when the protease cleavage sequence is cleaved is weaker than the binding of the ligand-binding molecule to the ligand when the protease cleavage sequence is not cleaved. [B-2] The ligand-binding molecule according to [B-1], wherein the ligand is released from the ligand-binding molecule when the protease cleavage sequence is cleaved. [B-3] The ligand-binding molecule according to any one of [B-1] to [B-2], wherein the protease is matriptase and / or urokinase. [B-4] The ligand-binding molecule according to any one of [B-1] to [B-3], wherein the protease is at least one protease selected from human MT-SP1, mouse MT-SP1, human uPA, and mouse uPA. [B-5] The ligand-binding molecule according to any one of [B-1] to [B-4], further comprising a first flexible linker attached to one end of the protease cleavage sequence. [B-6] The ligand-binding molecule according to [B-5], wherein the first flexible linker is a flexible linker made of a glycine-serine polymer. [B-7] The ligand-binding molecule according to [B-5] or [B-6], further comprising a second flexible linker attached to the other end of the protease cleavage sequence. [B-8] The ligand-binding molecule according to [B-7], wherein the second flexible linker is a flexible linker made of a glycine-serine polymer. [B-9] The ligand-binding molecule according to any one of [B-1] to [B-8], which comprises an antibody VH, an antibody VL, and an antibody constant region. [B-10] The ligand-binding molecule according to [B-9], 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-11] The ligand-binding molecule according to [B-10], wherein the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are introduced at any position in the sequence from amino acid 118 (EU numbering) to amino acid 140 (EU numbering) of the antibody heavy chain constant region. [B-12] The ligand-binding molecule according to [B-10], wherein the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are introduced at any position in the sequence from amino acid 108 (Kabat numbering) to amino acid 131 (Kabat numbering) of the antibody light chain constant region. [B-13] The ligand-binding molecule according to [B-9], wherein the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are located within the antibody VH or the antibody VL. [B-14] The ligand-binding molecule according to [B-13], wherein the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker, are introduced at any position in a sequence selected from the group consisting of amino acids 7 (Kabat numbering) to 16 (Kabat numbering), 40 (Kabat numbering) to 47 (Kabat numbering), 55 (Kabat numbering) to 69 (Kabat numbering), 73 (Kabat numbering) to 79 (Kabat numbering), 83 (Kabat numbering) to 89 (Kabat numbering), 95 (Kabat numbering) to 99 (Kabat numbering), and 101 (Kabat numbering) to 113 (Kabat numbering). [B-15] The ligand-binding molecule according to [B-13], wherein the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are introduced at any position in a sequence selected from the group consisting of amino acids 7 (Kabat numbering) to 19 (Kabat numbering), 39 (Kabat numbering) to 46 (Kabat numbering), 49 (Kabat numbering) to 62 (Kabat numbering), and 96 (Kabat numbering) to 107 (Kabat numbering) of the antibody VL. [B-16] The ligand-binding molecule according to [B-9], 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-17] The ligand-binding molecule according to [B-17], wherein the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are introduced at any position in the sequence from amino acid 109 (Kabat numbering) of the antibody VH to amino acid 122 (EU numbering) of the antibody heavy chain constant region. [B-18] The ligand-binding molecule according to [B-16], wherein the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are introduced at any position in the sequence from amino acid 104 (Kabat numbering) of the antibody VL to amino acid 113 (Kabat numbering) of the antibody light chain constant region. [B-19] The ligand-binding molecule according to any one of [B-9] to [B-18], wherein the antibody VL and the antibody VH in the ligand-binding molecule are associated with each other, and the association is dissolved by cleavage of the protease cleavage sequence by a protease. [B-20] The ligand-binding molecule according to any one of [B-1] to [B-19], 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-21] The ligand-binding molecule according to any one of [B-1] to [B-20], wherein the ligand is a cytokine or a chemokine. [B-22] The ligand-binding molecule according to any one of [B-1] to [B-20], wherein the ligand is selected from interleukins, interferons, hematopoietic factors, the TNF superfamily, chemokines, cell growth factors, and the TGF-β family. [B-23] The ligand-binding molecule according to any one of [B-1] to [B-22], wherein the ligand-binding molecule is an IgG antibody. [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 to the ligand. [B-26] The ligand-binding molecule according to [B-25], which does not bind to another ligand when fused to a ligand. [B-27] The ligand-binding molecule according to [B-25] or [B-26], wherein the ligand-binding molecule is fused to 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] that 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 when fused to 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 a ligand-binding molecule according to any one of [B-1] to [B-28]. [B-40] The production method according to [B-39], which comprises 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], which comprises 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 described in [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 a step of culturing the host cell according to [B-44]. [B-46] A method for producing the ligand-binding molecule according to [B-45], comprising the step of isolating the polypeptide from a culture supernatant. [B-47] A polynucleotide encoding the fusion protein according to any one of [B-30] to [B-34]. [B-48] A vector comprising the polynucleotide described in [B-46]. [B-49] A host cell comprising the polynucleotide described in [B-46] or the vector described in [B-48]. [B-50] A method for producing the fusion protein described in any one of [B-30] to [B-34], which comprises a step of culturing the host cell described in [B-49]. [B-51] A method for producing the fusion protein according to [B-50], comprising a step of isolating the polypeptide from a culture supernatant. [B-52] A ligand-binding molecule capable of binding to a ligand, comprising 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, or 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 ligand-binding molecule in a fusion protein of a ligand and a ligand-binding molecule, which is a sequence selected from SEQ ID NOs: 5 to 17201 from the 4th amino acid at the N-terminus to the 15th amino acid at the N-terminus, a sequence selected from SEQ ID NOs: 5 to 17201 from the 4th amino acid at the N-terminus to the 13th amino acid at the N-terminus, a sequence selected from SEQ ID NOs: 5 to 17201 from the 6th amino acid at the N-terminus to the 13th amino acid at the N-terminus, a sequence selected from SEQ ID NOs: 17202 to 17993 from the 1st amino acid at the N-terminus to the 12th amino acid at the N-terminus, a sequence selected from SEQ ID NOs: 17202 to 17993 from the 3rd amino acid at the N-terminus, a sequence selected from SEQ ID NOs: 17202 to 17993 from the 3rd amino acid at the N-terminus 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 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-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 while 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 when the protease cleavage sequence is cleaved is weaker than the binding of the ligand-binding molecule to the ligand when the protease cleavage sequence is not cleaved. [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 described in [B-61], wherein the first flexible linker is a flexible linker consisting 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 consisting 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 described in [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 described in [B-66], wherein the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are introduced at any position in the sequence from amino acid 118 (EU numbering) to amino acid 140 (EU numbering) of the antibody heavy chain constant region. [B-68] The method described in [B-66], wherein the protease cleavage sequence, or the protease cleavage sequence and the first flexible linker, or the protease cleavage sequence, the first flexible linker, and the second flexible linker are introduced at any position in the sequence from amino acid 108 (Kabat numbering) to amino acid 131 (Kabat numbering) of the antibody light chain constant region. [B-69] The method described in [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 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, the first flexible linker, and the second flexible linker are introduced at any position in a sequence selected from the group consisting of amino acids 7 (Kabat numbering) to 16 (Kabat numbering), 40 (Kabat numbering) to 47 (Kabat numbering), 55 (Kabat numbering) to 69 (Kabat numbering), 73 (Kabat numbering) to 79 (Kabat numbering), 83 (Kabat numbering) to 89 (Kabat numbering), 95 (Kabat numbering) to 99 (Kabat numbering), and 101 (Kabat numbering) to 113 (Kabat numbering). [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 amino acids 7 (Kabat numbering) to 19 (Kabat numbering), 39 (Kabat numbering) to 46 (Kabat numbering), 49 (Kabat numbering) to 62 (Kabat numbering), and 96 (Kabat numbering) to 107 (Kabat numbering). [B-72] The method described in [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 described in [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 amino acid 109 (Kabat numbering) of the antibody VH to amino acid 122 (EU numbering) of the antibody heavy chain constant region. [B-74] The method described in [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 amino acid 104 (Kabat numbering) of the antibody VL to amino acid 113 (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 with each other, and the association is dissolved by cleavage of the protease cleavage sequence by a protease. [B-76] A 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 by 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 selected from interleukins, interferons, hematopoietic factors, the TNF superfamily, chemokines, cell growth factors, and the 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 comprising a single-domain antibody, the single-domain antibody being capable of binding to a ligand and having at least one protease cleavage sequence introduced therein, such that the binding of the ligand-binding molecule to the ligand in a state in which the protease cleavage sequence is cleaved is weakened compared to the binding of the ligand-binding molecule to the ligand in a state in which 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 when 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 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], further comprising a first flexible linker attached 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 consisting of a glycine-serine polymer. [C-7] The ligand-binding molecule according to [C-5] or [C-6], further comprising a second flexible linker attached 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 consisting 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, a single-domain VH antibody, or a single-domain VL antibody. [C-10] The ligand-binding molecule according to [C-9], wherein 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 a 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 of the single-domain antibody selected from the following sequences: Single-domain antibody sequence from amino acid 7 (Kabat numbering) to amino acid 17 (Kabat numbering), single-domain antibody sequence from amino acid 12 (Kabat numbering) to amino acid 17 (Kabat numbering), single-domain antibody sequence from amino acid 31 (Kabat numbering) to amino acid 35b (Kabat numbering), single-domain antibody sequence from amino acid 40 (Kabat numbering) to amino acid 47 (Kabat numbering), single-domain antibody sequence from amino acid 50 (Kabat numbering) to amino acid 65 (Kabat numbering), single-domain antibody sequence from amino acid 55 (Kabat numbering) to Sequence up to amino acid 69 (Kabat numbering), single-domain antibody sequence from amino acid 73 (Kabat numbering) to amino acid 79 (Kabat numbering), single-domain antibody sequence from amino acid 83 (Kabat numbering) to amino acid 89 (Kabat numbering), single-domain antibody sequence from amino acid 95 (Kabat numbering) to amino acid 99 (Kabat numbering), single-domain antibody sequence from amino acid 95 (Kabat numbering) to amino acid 102 (Kabat numbering), single-domain antibody sequence from amino acid 101 (Kabat numbering) to amino acid 113 (Kabat numbering). [C-11] The ligand-binding molecule according to [C-9], wherein 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 a 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 of the single-domain antibody selected from the following sequences: Single-domain antibody sequence from amino acid number 7 (Kabat numbering) to amino acid number 19 (Kabat numbering), single-domain antibody sequence from amino acid number 24 (Kabat numbering) to amino acid number 34 (Kabat numbering), single-domain antibody sequence from amino acid number 39 (Kabat numbering) to amino acid number 46 (Kabat numbering), single-domain antibody sequence from amino acid number 49 (Kabat numbering) to amino acid number 62 (Kabat numbering), single-domain antibody sequence from amino acid number 50 (Kabat numbering) to amino acid number 56 (Kabat numbering), single-domain antibody sequence from amino acid number 89 (Kabat numbering) to amino acid number 97 (Kabat numbering), single-domain antibody sequence from amino acid number 96 (Kabat numbering) to amino acid number 107 (Kabat numbering). [C-12] A ligand-binding molecule described in any one of [C-1] to [C-11], wherein 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. [C-13] A ligand-binding molecule according to any one of [C-1] to [C-12], wherein the ligand is a molecule having biological activity and the single domain antibody has neutralizing activity against the ligand. [C-14] The ligand-binding molecule according to any one of [C-1] to [C-13], wherein the ligand-binding molecule comprises only a single-domain antibody containing the cleavage site or protease cleavage sequence. [C-15] The ligand-binding molecule according to any one of [C-1] to [C-14], further comprising an antibody Fc region. [C-16] The ligand-binding molecule according to any one of [C-1] to [C-15], wherein the ligand-binding molecule comprises a continuous peptide chain consisting of a single-domain antibody-antibody Fc region from the N-terminus to the C-terminus. [C-17] The ligand-binding molecule according to any one of [C-1] to [C-15], wherein the ligand-binding molecule is a dimer comprising two consecutive peptide chains consisting of a single-domain antibody, an antibody hinge region, and an antibody Fc region. [C-18] The ligand-binding molecule according to any one of [C-15] to [C-17], wherein the antibody Fc region is an Fc region comprising one sequence selected from the amino acid sequences shown in SEQ ID NOs: 18004 to 18007, or an Fc region mutant obtained by modifying any of these Fc regions. [C-19] The ligand-binding molecule according to any one of [C-1] to [C-18], wherein the ligand is a cytokine or a chemokine. [C-20] A ligand-binding molecule according to any one of [C-1] to [C-19], wherein the ligand is selected from interleukins, interferons, hematopoietic factors, the TNF superfamily, chemokines, cell growth factors, and the TGF-β family. [C-21] A ligand-binding molecule according to any one of [C-1] to [C-20], which is bound to the ligand. [C-22] The ligand-binding molecule according to any one of [C-1] to [C-20], which is fused to the ligand. [C-23] The ligand-binding molecule according to [C-22], wherein when the ligand-binding molecule is fused to a ligand, the single domain antibody contained in the ligand-binding molecule does not bind to any other ligand. [C-24] The ligand-binding molecule according to [C-22] or [C-23], wherein the ligand-binding molecule is fused to the ligand via a linker. [C-25] The ligand-binding molecule according to [C-24], wherein the linker does not contain a protease cleavage sequence. [C-26] A complex formed from the ligand and the ligand-binding molecule according to any one of [C-1] to [C-20]. [C-27] A fusion protein in which the ligand is fused with the ligand-binding molecule according to any one of [C-1] to [C-20]. [C-28] The fusion protein according to [C-27], wherein the single domain antibody does not bind to any other ligand when the ligand-binding molecule is fused to the ligand. [C-29] The fusion protein according to [C-27] or [C-28], wherein the ligand-binding molecule is fused to 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], wherein the ligand-linker-ligand-binding molecule are fused in this order from the N-terminus to the C-terminus. [C-32] A pharmaceutical composition comprising the ligand-binding molecule according to any one of [C-1] to [C-22]. [C-33] A pharmaceutical composition comprising a ligand-binding molecule according to any one of [C-1] to [C-21] and a ligand. [C-34] A pharmaceutical composition comprising the complex according to [C-26]. [C-35] A pharmaceutical composition comprising the fusion protein according to any one of [C-27] to [C-31]. [C-36] A method for producing a ligand-binding molecule according to any one of [C-1] to [C-20]. [C-37] The production method according to [C-36], which comprises introducing a protease cleavage sequence into a single domain antibody in a ligand-binding molecule comprising a single domain antibody. [C-38] A method for producing the fusion protein described in any one of [C-27] to [C-31], which comprises fusing a single-domain antibody-containing ligand-binding molecule having a protease cleavage sequence introduced therein with a ligand capable of binding to the single-domain antibody. [C-39] A polynucleotide encoding the ligand-binding molecule according to any one of [C-1] to [C-20]. [C-40] A vector comprising the polynucleotide described in [C-39]. [C-41] A host cell comprising the polynucleotide described in [C-39] or the vector described in [C-40]. [C-42] A method for producing the ligand-binding molecule described in any one of [C-1] to [C-20], which comprises a step of culturing the host cell described in [C-41]. [C-43] A method for producing the ligand-binding molecule according to [C-42], comprising the step of isolating the polypeptide from a culture supernatant. [C-44] A polynucleotide encoding the fusion protein according to any one of [C-27] to [C-31]. [C-45] A vector comprising the polynucleotide described in [C-43]. [C-46] A host cell comprising the polynucleotide described in [C-43] or the vector described in [C-45]. [C-47] A method for producing a fusion protein described in any one of [C-27] to [C-31], comprising a step of culturing the host cell described in [C-46]. [C-48] A method for producing the ligand-binding molecule according to [C-47], comprising the step of isolating the polypeptide from a 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 single domain antibody in a ligand-binding molecule capable of binding to a ligand, comprising 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, or 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 to 10th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993, a sequence from the 3rd to 14th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003, a sequence from the 5th to 12th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003, a sequence from the 5th to 10th amino acids 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-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 while 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 when the protease cleavage sequence is cleaved is weakened compared to the binding of the ligand-binding molecule to the ligand when the protease cleavage sequence is not cleaved. [C-56] The method according to [C-55], wherein the ligand is released from the ligand-binding molecule when the protease cleavage sequence is cleaved. [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 described in [C-59], wherein the first flexible linker is a flexible linker consisting 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 described in [C-61], wherein the second flexible linker is a flexible linker consisting of a glycine-serine polymer. [C-63] The method described in any one of [C-55] to [C-62], wherein the single-domain antibody is a VHH, a single-domain VH antibody, or a single-domain VL antibody. [C-64] The method according to [C-63], wherein 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 into one or more positions contained in one or more sequences of the single-domain antibody selected from the following sequences: Single-domain antibody sequence from amino acid 7 (Kabat numbering) to amino acid 17 (Kabat numbering), single-domain antibody sequence from amino acid 12 (Kabat numbering) to amino acid 17 (Kabat numbering), single-domain antibody sequence from amino acid 31 (Kabat numbering) to amino acid 35b (Kabat numbering), single-domain antibody sequence from amino acid 40 (Kabat numbering) to amino acid 47 (Kabat numbering), single-domain antibody sequence from amino acid 50 (Kabat numbering) to amino acid 65 (Kabat numbering), single-domain antibody sequence from amino acid 55 (Kabat numbering) to Sequence up to amino acid 69 (Kabat numbering), single-domain antibody sequence from amino acid 73 (Kabat numbering) to amino acid 79 (Kabat numbering), single-domain antibody sequence from amino acid 83 (Kabat numbering) to amino acid 89 (Kabat numbering), single-domain antibody sequence from amino acid 95 (Kabat numbering) to amino acid 99 (Kabat numbering), single-domain antibody sequence from amino acid 95 (Kabat numbering) to amino acid 102 (Kabat numbering), single-domain antibody sequence from amino acid 101 (Kabat numbering) to amino acid 113 (Kabat numbering). [C-65] The method according to [C-63], wherein 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 into one or more positions contained in one or more sequences of the single-domain antibody selected from the following sequences: Single-domain antibody sequence from amino acid number 7 (Kabat numbering) to amino acid number 19 (Kabat numbering), single-domain antibody sequence from amino acid number 24 (Kabat numbering) to amino acid number 34 (Kabat numbering), single-domain antibody sequence from amino acid number 39 (Kabat numbering) to amino acid number 46 (Kabat numbering), single-domain antibody sequence from amino acid number 49 (Kabat numbering) to amino acid number 62 (Kabat numbering), single-domain antibody sequence from amino acid number 50 (Kabat numbering) to amino acid number 56 (Kabat numbering), single-domain antibody sequence from amino acid number 89 (Kabat numbering) to amino acid number 97 (Kabat numbering), single-domain antibody sequence from amino acid number 96 (Kabat numbering) to amino acid number 107 (Kabat numbering). [C-66] A method according to any one of [C-55] to [C-65], wherein 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. [C-67] ​​A method according to any one of [C-55] to [C-66], wherein the ligand is a molecule having biological activity and the single domain antibody has neutralizing activity against the ligand. [C-68] The method of any one of [C-55] to [C-67], wherein the ligand-binding molecule comprises only a single domain antibody containing the cleavage site or protease cleavage sequence. [C-69] The method described in any one of [C-55] to [C-68], wherein the ligand-binding molecule further contains an antibody Fc region. [C-70] A method described in any one of [C-55] to [C-69], wherein the ligand-binding molecule comprises a continuous peptide chain consisting of a single-domain antibody-antibody Fc region from the N-terminus to the C-terminus. [C-71] The method described in [C-55] to [C-69], wherein the ligand-binding molecule is a dimer comprising two consecutive peptide chains consisting of a single-domain antibody, an antibody hinge region, and an antibody Fc region. [C-72] The method described in [C-69] to [C-71], wherein the antibody Fc region is an Fc region comprising one sequence selected from the amino acid sequences shown in SEQ ID NOs: 18004 to 18007, or an Fc region mutant obtained by modifying these Fc regions. [C-73] The method according to any one of [C-55] to [C-72], wherein the ligand is a cytokine or a chemokine. [C-74] A method according to any one of [C-55] to [C-73], wherein the ligand is selected from interleukins, interferons, hematopoietic factors, the TNF superfamily, chemokines, cell growth factors, and the TGF-β family. [Brief explanation of the drawings]

[0011] [Figure 1]

[0033] Figure 1 shows an example of a polypeptide comprising a protease cleavage sequence of the present disclosure. Here, the polypeptide comprises an antigen-binding domain and a delivery moiety. (A) A polypeptide in which the antigen-binding domain and delivery moiety are linked has a long half-life and does not bind to antigen. (B) Upon cleavage of the protease cleavage sequence, the antigen-binding domain is released and binds to the antigen, and the released antigen-binding domain has a short half-life. [Figure 2] This figure shows one embodiment of a method for producing the polypeptide shown in Figure 1. In this embodiment, the polypeptide of interest is an IgG antibody-like molecule. (A) A single-domain antibody that binds to a target antigen is obtained. (B) The single-domain antibody is associated with a VL instead of a VH of an IgG antibody so that the antigen-binding activity of the single-domain antibody is suppressed. (C) A protease cleavage sequence is introduced into the IgG antibody-like molecule precursor into which the single-domain antibody has been introduced. [Figure 3]

[0023] Figure 1 shows an example of a polypeptide comprising a protease cleavage sequence of the present disclosure. In this example, the polypeptide is an IgG antibody-like molecule, and an antigen-binding domain is provided in each of the regions corresponding to the two variable regions of an IgG antibody. The two antigen-binding domains may have similar or different antigen-binding specificities. [Figure 4] 1A and 1B are diagrams showing an example of a polypeptide comprising a protease cleavage sequence of the present disclosure. In this example, the polypeptide comprises a linked antigen-binding domain and a second antigen-binding domain. In this example, the released antigen-binding domain and the second antigen-binding domain form a bispecific antigen-binding molecule. (A) A diagram showing the polypeptide in an unreleased state. The antigen-binding activity of the antigen-binding domain is suppressed. (B) A diagram showing the release of a bispecific antigen-binding molecule formed by the antigen-binding domain and the second antigen-binding domain. (C) A diagram showing an example of a bispecific antigen-binding molecule after release, for example, a bispecific antigen-binding molecule for a T cell surface antigen and a cancer cell surface antigen. [Figure 5] 1 shows an example of a polypeptide comprising a protease cleavage sequence of the present disclosure. In this example, the polypeptide is a fusion protein of a ligand and an anti-ligand antibody. (A) The protease cleavage sequence is contained within the anti-ligand antibody, and when the protease cleavage sequence is not cleaved, the ligand binds to the anti-ligand antibody. (B) When the protease cleavage sequence is cleaved, portions of the ligand and anti-ligand antibody are released from the polypeptide, allowing the ligand to bind to a receptor. [Figure 6] 1A and 1B are diagrams showing an example of a polypeptide comprising a protease cleavage sequence of the present disclosure. Here, the polypeptide is an anti-ligand antibody. (A) The protease cleavage sequence is contained within the anti-ligand antibody, and when the protease cleavage sequence is not cleaved, the anti-ligand antibody can bind to the ligand. (B) When the protease cleavage sequence is cleaved, the ligand-binding activity of the anti-ligand antibody is weakened, and the ligand dissociates from the anti-ligand antibody, becoming capable of binding to the receptor. [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] 1 shows an example of a polypeptide comprising a protease cleavage sequence of the present disclosure, where the polypeptide is a dimeric protein comprising a single domain antibody-antibody hinge region-antibody Fc region comprising a protease cleavage sequence. [Figure 10] 1 shows an example of a polypeptide comprising a protease cleavage sequence of the present disclosure, where the polypeptide is a fusion protein between a ligand and a dimeric protein comprising a single domain antibody-antibody hinge region-antibody Fc region comprising the protease cleavage sequence. [Figure 11] Figure 1 shows the results of SDS-PAGE of protease-treated and untreated ligand-binding molecules. While the control molecules (lanes 12 and 13) that did not contain a protease cleavage sequence showed bands at the same position regardless of whether they were treated with or without protease, the ligand-binding molecules into which each protease cleavage sequence had been introduced showed new bands that appeared only after protease treatment, indicating that the single-domain antibody-containing ligand-binding molecules into which each protease cleavage sequence had been introduced were cleaved by protease treatment. [Figure 12]These are real-time binding graphs evaluating the binding of protease-treated and protease-untreated ligand-binding molecules to IL-6R. The title of each graph is the name of the measured sample, the vertical axis shows the relative binding of the ligand-binding molecule to IL-6R, and the horizontal axis shows time (s). The gray line shows the data for the protease-untreated sample, and the black line shows the data for the protease-treated sample. [Figure 13] Figure 1 shows the results of SDS-PAGE of protease-treated and untreated fusion proteins. While the control molecule not containing a protease cleavage sequence showed bands at the same position regardless of whether it was treated with or untreated with protease, each fusion protein containing a single-domain antibody-containing ligand-binding molecule into which a protease cleavage sequence had been introduced showed a new band that appeared only after protease treatment, indicating that each fusion protein containing a single-domain antibody-containing ligand-binding molecule into which a protease cleavage sequence had been introduced was cleaved by protease treatment. [Figure 14] FIG. 14 is a diagram showing a continuation of FIG. [Figure 15] FIG. 15 is a diagram showing a continuation of FIG. [Figure 16] FIG. 16 is a diagram showing a continuation of FIG. [Figure 17] These are real-time graphs evaluating the binding of free IL-6R to IL6R90-bio present in solutions of protease-treated and protease-untreated fusion proteins. The title of each figure is the name of the measurement sample, the vertical axis shows the relative binding of IL-6R to a biotinylated anti-IL-6R single-domain antibody-containing molecule (IL6R90-bio), and the horizontal axis shows time (s). The gray line shows data for samples untreated with protease, and the black line shows data for samples treated with protease. [Figure 18] FIG. 18 is a diagram showing a continuation of FIG. 17. [Figure 19] FIG. 1 shows the results of SDS-PAGE of a protease-treated fusion protein and a protease-untreated fusion protein. [Figure 20]Figure 1 shows real-time graphs showing the binding of free human PD-1 present in solutions of protease-treated and protease-untreated fusion proteins to a biotinylated anti-PD-1 single-domain antibody-containing molecule (PD1-bio). The title of each figure is the name of the measured sample, the vertical axis shows the relative binding of PD-1 to PD1-bio, and the horizontal axis shows time (s). The gray line shows data for the protease-untreated sample, and the black line shows data for the protease-treated sample. DETAILED DESCRIPTION OF THE INVENTION

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

[0013] peptide As used herein, the term "peptide" refers to a compound in which two or more amino acid molecules are bonded together by removing one molecule of water from the amino group of one molecule and the carboxyl group of the other molecule. There is no limit to the number of amino acids contained in a peptide. Therefore, both oligopeptides and polypeptides are included in the term "peptide."

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

[0015] Isolated Polypeptides The polypeptides of the present disclosure can refer to isolated polypeptides. An "isolated" polypeptide is one that has been separated from the components of its original environment. In some embodiments, the polypeptide is purified to greater than 95% or 99% purity, for example, as measured by 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, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007) for a review of methods for assessing antibody purity.

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

[0017] Specific types of proteases include, but are not limited to, cysteine ​​proteases (including cathepsin family B, L, S, etc.), aspartyl proteases (cathepsin D, E, K, O, etc.), serine proteases (matriptase (including MT-SP1), cathepsin A and G, thrombin, plasmin, urokinase (uPA), tissue plasminogen activator (tPA), elastase, proteinase 3, thrombin, kallikrein, triglyceride, etc.), and the like. metalloproteases (including membrane-bound (MMP14-17 and MMP24-25) and secreted (MMP1-13, MMP18-23, and MMP26-28) metalloproteases (MMP1-28)), proteases A disintegrin and metalloprotease (ADAM), metalloproteases with A disintegrin or thrombospondin motifs (ADAMTS), meprin (meprin α alpha), meprin beta), CD10 (CALLA), as well as prostate-specific antigen (PSA), legumain, TMPRSS3, TMPRSS4, neutrophil elastase (HNE), beta-secretase (BACE), fibroblast activation protein alpha (FAP), granzyme B, guanidinobenzoatase (GB), hepsin, neprilysin, NS3 / 4A, HCV-NS3 / 4, calpain, ADAMDEC1, renin, cathepsin C, cathepsin V / L2, cathepsin X / Z / P, cruzipain, otubain 2, kallikrein-related peptidases (KLKs (KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14)), bone morphogenetic protein 1 (BMP-1), activated protein C, blood coagulation-related proteases (Factor VIIa, Factor IXa, Factor Xa, Factor XIa, Factor XIIa), HtrA1, lactoferrin, marapsin, PACE4, DESC1, dipeptidyl peptidase 4 (DPP-4), TMPRSS2, cathepsin F, cathepsin H, cathepsin L2, cathepsin O, cathepsin S, granzyme A, Gepsin calpain 2, glutamate carboxypeptidase 2, AMSH-LikeThese include proteases, AMSH, gamma secretase, antiplasmin cleaving enzyme (APCE), decysin 1, N-Acetylated Alpha-Linked Acidic Dipeptidase-Like 1 (NAALADL1), and furin.

[0018] The protease of the present disclosure can be a protease closely associated with diseased tissue. For example, (1) proteases that are expressed at higher levels in diseased tissues than in normal tissues; (2) proteases that have higher activity in diseased tissues than in normal tissues; (3) proteases that are expressed at higher levels 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; Examples of diseased tissue include cancerous tissue and inflamed tissue.

[0019] The term "cancer tissue" refers to tissue containing at least one cancer cell. Thus, it refers to all cell types that contribute to the formation of a tumor mass, including cancer cells and endothelial cells, such as cancer tissue containing cancer cells and blood vessels. As used herein, a tumor mass refers to a foci of tumor tissue. The term "tumor" is generally used to refer to benign or malignant neoplasms.

[0020] As used herein, "inflamed tissue" includes, for example, the following: Joints in rheumatoid arthritis and osteoarthritis Lungs (alveoli) in bronchial asthma and COPD Digestive tract in inflammatory bowel disease, Crohn's disease, and ulcerative colitis Fibrotic tissue in liver, kidney, and lung fibrosis -Tissues undergoing rejection in organ transplants Blood vessels and heart (myocardium) in arteriosclerosis and heart failure Visceral fat in metabolic syndrome Skin tissue in atopic dermatitis and other dermatitis Spinal nerve damage in herniated discs and chronic lower back pain

[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 naturally occurring, endogenously produced, and / or recombinant form of uPA, which may be derived from any suitable organism, so long as it possesses serine protease activity. For example, in one embodiment of the present disclosure, the uPA is a two-chain active form (tc-uPA). Urokinase also encompasses human uPA or uPA from another species, such as mammalian-derived uPA, such as uPA 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). The terms urokinase-type plasminogen activator and uPA also encompass recombinantly produced uPA. This includes any "recombinantly produced" uPA, 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 tissues, and peptide sequences cleavable by urokinase (uPA) are cleaved more frequently in cancer tissues than in normal tissues.

[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] Methods for confirming protease cleavage An example of a method for evaluating cleavage of a protease substrate or protease cleavage sequence described herein by a protease is the method described in Mol Cell Proteomics. 2014 Jun;13(6):1585-97. doi: 10.1074 / mcp.M113.033308. Epub 2014 Apr 4. The protease substrate or protease cleavage sequence to be evaluated is immobilized on a peptide array, and the peptide array is treated with a solution containing the protease to be evaluated. The cleavage rate can be calculated using the fluorescence value measured from the chip as follows: TIFF2025134810000001.tif18170The type and concentration of protease used for evaluation, as well as the treatment temperature and treatment time, can be selected as appropriate. For example, treatment can be carried out for 1 hour at 37°C using 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.

[0024] Alternatively, instead of using a protease-containing solution, serum (including human serum and mouse serum) can be used to treat the peptide array, and the cleavage rate can be calculated using the fluorescence measured from the chip as follows: TIFF2025134810000002.tif18170 The type and concentration of serum used for evaluation, as well as the treatment temperature and treatment time, can be selected as appropriate; for example, human serum diluted to 80% can be used as the treatment solution, and treatment can be carried out overnight at 37°C. As used herein, "not cleaved by serum" can mean that the cleavage rate by serum measured and calculated by the above method is 1.5 or less, or that 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] Whether a protease cleavage sequence contained in a polypeptide has been cleaved by a protease can be qualitatively confirmed by subjecting a solution containing the polypeptide containing the protease cleavage sequence to SDS-PAGE (polyacrylamide gel electrophoresis) and measuring the molecular weight of the fragments. Alternatively, confirmation can be achieved by comparing the molecular weights of the polypeptide untreated with that of the polypeptide treated with a protease.

[0026] As used herein, the term "cleaved" refers to a state in which a polypeptide is cleaved 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 "uncleaved" 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 reduction of the cysteine-cysteine ​​disulfide bond of the protease cleavage sequence.

[0027] Furthermore, by quantifying the amount of cleaved fragments separated by electrophoresis such as SDS-PAGE after protease treatment, it is possible to evaluate the protease cleavage sequence and the cleavage rate of the molecule into which the protease cleavage sequence has been introduced. A non-limiting example of a method for evaluating the cleavage rate of a molecule into which a protease cleavage sequence has been introduced is as follows. For example, when evaluating the cleavage rate of an antibody variant incorporating a protease cleavage sequence using recombinant human u-Plasminogen Activator / Urokinase (human uPA, huPA) (R&D Systems; 1310-SE-010) or recombinant human Matriptase / ST14 Catalytic Domain (human MT-SP1, hMT-SP1) (R&D Systems; 3946-SE-010), the antibody variant is incubated with 40 nM huPA or 3 nM hMT-SP1, 100 μg / mL PBS, and 37°C for 1 hour before being subjected to capillary electrophoresis immunoassay. Capillary electrophoresis immunoassays can be performed using Protein Simple (Wes), but are not limited to this method. Alternatively, detection can be performed by Western blotting after separation by SDS-PAGE or other methods. An anti-human lambda chain HRP-conjugated antibody (Abcam; ab9007) can be used to detect the light chain before and after cleavage, but any antibody capable of detecting cleaved fragments can be used. The area of ​​each peak obtained after protease treatment can be output using Wes-specific software (Compass for SW; Protein Simple) to calculate the cleavage rate (%) of the modified antibody using the formula: (cleaved light chain peak area) * 100 / (cleaved light chain peak area + uncleaved light chain peak area). The cleavage rate can be calculated as long as protein fragments can be detected before and after protease treatment. This calculation is possible for various proteins, not just modified antibodies, that incorporate a protease cleavage sequence.

[0028] After administering a molecule incorporating a protease cleavage sequence to an animal, the in vivo cleavage rate can be calculated by detecting the administered molecule in a blood sample. For example, after administering a modified antibody incorporating a protease cleavage sequence to a mouse, plasma is collected from the blood sample, and the antibody is purified using Dynabeads Protein A (Thermo; 10001D) by a method known to those skilled in the art. The protease cleavage rate of the modified antibody can be evaluated by subjecting it to capillary electrophoresis immunoassay. Capillary electrophoresis immunoassays can be performed using Protein Simple (Wes), but are not limited to this. Alternatively, Western blotting can be used after separation by SDS-PAGE or other methods. Light chains of modified antibodies recovered from mice can be detected using an anti-human lambda chain HRP-labeled antibody (abcam; ab9007), but any antibody capable of detecting cleavage fragments can be used. The area of ​​each peak obtained by capillary electrophoresis immunoassay was output using Wes-specific software (Compass for SW; Protein Simple), and the remaining light chain ratio (light chain peak area) / (heavy chain peak area) was calculated, allowing the proportion of full-length light chain remaining uncleaved in the mouse body to be calculated. Calculation of in vivo cleavage efficiency is possible as long as protein fragments recovered from the body can be detected. This method allows for the calculation of cleavage rates for various proteins, including those containing protease cleavage sequences, in addition to modified antibodies. Calculating the cleavage rate using the above-described method makes it possible to compare the in vivo cleavage rates of modified antibodies containing different cleavage sequences, for example, and also to compare the cleavage rates of the same modified antibody between different animal models, such as normal mouse models and tumor-bearing mouse models.

[0029] Protease Substrates 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, a protease substrate is a peptide about 5 to 15 amino acids in length. The protease substrate of the present disclosure can be used to produce a protease-specific antibody with a binding activity of approximately 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 x 10 4 M -1 S -1 can be specifically modified (cleaved) at a rate of

[0030] In certain embodiments, a protease substrate of the present disclosure has a higher rate of cleavage by a protease than a protease substrate comprising any one of SEQ ID NOs: 1, 2, and 3. In certain embodiments, a protease substrate of the present disclosure has a higher cleavage rate with human uPA than a protease substrate comprising any one of SEQ ID NOs: 1, 2, and 3. In certain embodiments, the cleavage rate of a protease substrate of the present disclosure with human uPA is 1.5 or greater, 1.6 or greater, 1.8 or greater, 2 or greater, 2.2 or greater, 2.4 or greater, 2.6 or greater, 2.8 or greater, 3 or greater, 3.2 or greater, 3.4 or greater, 3.6 or greater, 3.8 or greater, 3.9 or greater, or 4 or greater. In certain embodiments, a protease substrate of the present disclosure has a higher cleavage rate by human MT-SP1 than a protease substrate comprising any one of SEQ ID NOs: 1, 2, and 3. In certain embodiments, the cleavage rate by human MT-SP1 of a protease substrate of the present disclosure is 1.5 or greater, 1.6 or greater, 1.8 or greater, 2 or greater, 2.2 or greater, 2.4 or greater, 2.6 or greater, 2.8 or greater, 3 or greater, 3.2 or greater, 3.4 or greater, 3.6 or greater, 3.8 or greater, 3.9 or greater, or 4 or greater. In certain embodiments, a protease substrate of the present disclosure has a higher cleavage rate with mouse uPA than a protease substrate comprising any one of SEQ ID NOs: 1, 2, and 3. In certain embodiments, the cleavage rate of a protease substrate of the present disclosure with mouse uPA is 1 or greater, 1.1 or greater, 1.2 or greater, 1.3 or greater, 1.4 or greater, 1.5 or greater, 1.6 or greater, 1.7 or greater, 1.8 or greater, 1.9 or greater, 2 or greater, 2.5 or greater, 3 or greater, 3.5 or greater, or 4 or greater. In certain embodiments, a protease substrate of the present disclosure has a higher cleavage rate by mouse MT-SP1 than a protease substrate comprising any one of SEQ ID NOs: 1, 2, and 3. In certain embodiments, the cleavage rate by mouse MT-SP1 of a protease substrate of the present disclosure is 1.5 or greater, 1.6 or greater, 1.8 or greater, 2 or greater, 2.2 or greater, 2.4 or greater, 2.6 or greater, 2.8 or greater, 3 or greater, 3.2 or greater, 3.4 or greater, 3.6 or greater, 3.8 or greater, 3.9 or greater, or 4 or greater. In certain embodiments, a protease substrate of the present disclosure has a higher ratio of the cleavage rate with human uPA to the cleavage rate with human serum than a protease substrate comprising any one of SEQ ID NOs: 1, 2, and 3. In certain embodiments, the ratio of the cleavage rate with human uPA to the cleavage rate with human serum of a protease substrate of the present disclosure is 0.9 or greater, 0.95 or greater, 1 or greater, 1.2 or greater, 1.4 or greater, 1.6 or greater, 1.8 or greater, 2 or greater, 2.2 or greater, 2.4 or greater, 2.6 or greater, 2.8 or greater, 3 or greater, 3.2 or greater, 3.4 or greater, 3.6 or greater, 3.8 or greater, or 4 or greater. When the cleavage rate with human serum is 1 or less, it can be converted to 1 before being used to calculate the ratio of the cleavage rate with human uPA to the cleavage rate with human serum. In certain embodiments, a 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 a protease substrate comprising any one of SEQ ID NOs: 1, 2, and 3. In certain embodiments, the ratio of the cleavage rate by human MT-SP1 to the cleavage rate by human serum of a protease substrate of the present disclosure is 0.7 or greater, 0.8 or greater, 0.9 or greater, 0.95 or greater, 1 or greater, 1.2 or greater, 1.4 or greater, 1.6 or greater, 1.8 or greater, 2 or greater, 2.2 or greater, 2.4 or greater, 2.5 or greater, or 2.6 or greater. When the cleavage rate by human serum is 1 or less, it can be converted to 1 before being used to calculate the ratio of the cleavage rate by human MT-SP1 to the cleavage rate by human serum. In certain embodiments, a protease substrate of the present disclosure has a higher ratio of the cleavage rate with mouse uPA to the cleavage rate with human serum than a protease substrate comprising any one of SEQ ID NOs: 1, 2, and 3. In certain embodiments, the ratio of the cleavage rate with mouse uPA to the cleavage rate with human serum of a 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, if the cleavage rate with human serum is 1 or less, it can be converted to 1 before being used to calculate the ratio of the cleavage rate with mouse uPA to the cleavage rate with human serum. In certain embodiments, a protease substrate of the present disclosure has a higher ratio of the cleavage rate with mouse MT-SP1 to the cleavage rate with human serum than a protease substrate comprising any one of SEQ ID NOs: 1, 2, and 3. In certain embodiments, the ratio of the cleavage rate with mouse MT-SP1 to the cleavage rate with human serum of a protease substrate of the present disclosure is 0.7 or greater, 0.8 or greater, 0.9 or greater, 0.95 or greater, 1 or greater, 1.2 or greater, 1.4 or greater, 1.6 or greater, 1.8 or greater, 2 or greater, 2.2 or greater, 2.4 or greater, 2.5 or greater, or 2.6 or greater. When the cleavage rate with human serum is 1 or less, it can be converted to 1 before being used to calculate the ratio of the cleavage rate with mouse MT-SP1 to the cleavage rate with human serum. In certain embodiments, the protease substrates of the present disclosure are not cleaved by human serum. As a specific example, the cleavage rate of the protease substrates of the present disclosure by human serum is 1.5 or less. As another specific example, the cleavage rate of the protease substrates of the present disclosure by human serum is lower than the cleavage rate of the peptide set forth in SEQ ID NO: 4 by human serum.

[0031] More specific examples include the 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, the 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, the 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, the 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, the 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, and the sequence from the 3rd amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993. A peptide represented by any of the sequences of SEQ ID NOs: 5 to 18003 is useful as a protease substrate that is hydrolyzed by the action of a protease. As another specific example, a peptide having a sequence consisting of, from the N-terminus, "a sequence selected from Group A below - a sequence selected from Group B below" is also useful as a protease substrate that is hydrolyzed by the action of a protease: (Group A) A sequence from the first amino acid to the ninth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the second amino acid to the ninth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 3rd amino acid to the 9th 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 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 5th amino acid to the 9th 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 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 7th amino acid to the 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 8th amino acid to the 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the first amino acid to the sixth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the second amino acid to the sixth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the third amino acid to the sixth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 4th amino acid to the 6th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 5th amino acid to the 6th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the first amino acid to the eighth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the second amino acid to the eighth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the third amino acid to the eighth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 4th amino acid to the 8th 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 8th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 6th amino acid to the 8th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 7th to 8th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; (Group B) A sequence from the 10th 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 10th amino acid to the 14th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 10th 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 10th amino acid to the 12th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 10th amino acid to the 11th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 7th amino acid to the 15th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 7th amino acid to the 14th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 7th amino acid to the 13th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 7th 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 7th 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 7th 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 7th amino acid to the 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 7th amino acid to the 8th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 9th amino acid to the 15th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 9th 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 9th amino acid to the 13th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 9th 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 9th amino acid to the 11th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 9th to 10th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003.

[0032] The protease substrates of the present disclosure can be used as a library for selecting those with properties suited to the purpose when incorporated into a polypeptide, for example. Specifically, the protease susceptibility of a polypeptide can be evaluated in order to selectively cleave the polypeptide with a protease localized in the lesion. After administration to the living body, a polypeptide may reach the lesion through contact with various proteases. Therefore, it is desirable for the polypeptide to be sensitive to the proteases localized in the lesion while being as resistant as possible to other proteases. To select a desired protease substrate according to the purpose, the protease resistance can be determined by comprehensively analyzing the sensitivity of each protease substrate to various proteases in advance. Based on the obtained protease resistance spectrum, a protease substrate with the required sensitivity and resistance can be found. Alternatively, a polypeptide incorporating a protease substrate may reach the lesion not only through the enzymatic action of the protease but also through various environmental stresses such as changes in pH, temperature, redox stress, etc. Based on information comparing the resistance of each protease substrate to such external factors, a protease substrate with desirable properties for a particular purpose can be selected.

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

[0034] The protease cleavage sequence of the present disclosure is capable of cleaving approximately 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 x 10 4 M -1 S -1 can be specifically modified (cleaved) at a rate of

[0035] For example, the 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, the 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, the 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, the 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, the 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, Any of the following sequences are useful as protease cleavage sequences: the sequence from the third to the eleventh amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the third to the tenth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003, the sequence from the fifth to the twelfth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003, the sequence from the fifth to the tenth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003, and the sequences represented by SEQ ID NOs: 5 to 18003. The present disclosure also relates to the use of these sequences as protease cleavage sequences. For example, any of the sequences consisting of "a sequence selected from Group A below - a sequence selected from Group B below" from the N-terminus is useful as a protease cleavage sequence. The present disclosure also relates to the use of these sequences as protease cleavage sequences: (Group A) A sequence from the first amino acid to the ninth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the second amino acid to the ninth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 3rd amino acid to the 9th 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 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 5th amino acid to the 9th 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 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 7th amino acid to the 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 8th amino acid to the 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the first amino acid to the sixth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the second amino acid to the sixth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the third amino acid to the sixth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 4th amino acid to the 6th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 5th amino acid to the 6th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the first amino acid to the eighth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the second amino acid to the eighth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the third amino acid to the eighth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 4th amino acid to the 8th 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 8th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 6th amino acid to the 8th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 7th to 8th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; (Group B) A sequence from the 10th 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 10th amino acid to the 14th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 10th 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 10th amino acid to the 12th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 10th amino acid to the 11th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 7th amino acid to the 15th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 7th amino acid to the 14th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 7th amino acid to the 13th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 7th 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 7th 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 7th 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 7th amino acid to the 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 7th amino acid to the 8th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 9th amino acid to the 15th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 9th 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 9th amino acid to the 13th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 9th 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 9th amino acid to the 11th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 9th to 10th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003.

[0036] In some embodiments, a protease cleavage sequence is linked or otherwise attached to an antibody, for example, a protease cleavage sequence is used to link one or more agents to an antibody that binds to a predetermined target, such that upon exposure to proteases, i.e., matriptase and / or uPA, the protease cleavage sequence is cleaved and the agent(s) are released from the antibody.

[0037] In certain embodiments, the protease cleavage sequences of the present disclosure have a higher rate of cleavage by a protease than a protease substrate comprising any one of SEQ ID NOs: 1, 2, and 3. In certain embodiments, a protease cleavage sequence of the present disclosure has a higher cleavage rate by human uPA than a protease substrate comprising any one of SEQ ID NOs: 1, 2, and 3. In certain embodiments, the cleavage rate by human uPA of a protease cleavage sequence of the present disclosure is 1.5 or greater, 1.6 or greater, 1.8 or greater, 2 or greater, 2.2 or greater, 2.4 or greater, 2.6 or greater, 2.8 or greater, 3 or greater, 3.2 or greater, 3.4 or greater, 3.6 or greater, 3.8 or greater, 3.9 or greater, or 4 or greater. In certain embodiments, a protease cleavage sequence of the present disclosure has a higher cleavage rate by human MT-SP1 than a protease substrate comprising any one of SEQ ID NOs: 1, 2, and 3. In certain embodiments, the cleavage rate by human MT-SP1 of a protease cleavage sequence of the present disclosure is 1.5 or greater, 1.6 or greater, 1.8 or greater, 2 or greater, 2.2 or greater, 2.4 or greater, 2.6 or greater, 2.8 or greater, 3 or greater, 3.2 or greater, 3.4 or greater, 3.6 or greater, 3.8 or greater, 3.9 or greater, or 4 or greater. In certain embodiments, the protease cleavage sequences of the present disclosure have a higher cleavage rate with mouse uPA than a protease substrate comprising any one of SEQ ID NOs: 1, 2, and 3. In certain embodiments, the cleavage rate of the protease cleavage sequences of the present disclosure with 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, a protease cleavage sequence of the present disclosure has a higher cleavage rate with mouse MT-SP1 than a protease substrate comprising any one of SEQ ID NOs: 1, 2, and 3. In certain embodiments, the cleavage rate of a protease cleavage sequence of the present disclosure with 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, a protease cleavage sequence of the present disclosure has a higher ratio of the cleavage rate with human uPA to the cleavage rate with human serum than a protease substrate comprising any one of SEQ ID NOs: 1, 2, and 3. In certain embodiments, the ratio of the cleavage rate with human uPA to the cleavage rate with human serum of a protease cleavage sequence of the present disclosure 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 with human serum is 1 or less, it can be converted to 1 and used to calculate the ratio of the cleavage rate with human uPA to the cleavage rate with human serum. In certain embodiments, a 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 than a protease substrate comprising any one of SEQ ID NOs: 1, 2, and 3. In certain embodiments, the ratio of the cleavage rate by human MT-SP1 to the cleavage rate by human serum of a protease cleavage sequence of the present disclosure is 0.7 or greater, 0.8 or greater, 0.9 or greater, 0.95 or greater, 1 or greater, 1.2 or greater, 1.4 or greater, 1.6 or greater, 1.8 or greater, 2 or greater, 2.2 or greater, 2.4 or greater, 2.5 or greater, or 2.6 or greater. Here, if the cleavage rate by human serum is 1 or less, it can be converted to 1 before being used to calculate the ratio of the cleavage rate by human MT-SP1 to the cleavage rate by human serum. In certain embodiments, a protease cleavage sequence of the present disclosure has a higher ratio of cleavage rate with mouse uPA to cleavage rate with human serum than a protease substrate comprising any one of SEQ ID NOs: 1, 2, and 3. In certain embodiments, the ratio of the cleavage rate with mouse uPA to cleavage rate with human serum of a 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, if the cleavage rate with human serum is 1 or less, it can be converted to 1 and used to calculate the ratio of the cleavage rate with mouse uPA to the cleavage rate with human serum. In certain embodiments, a protease cleavage sequence of the present disclosure has a higher ratio of the cleavage rate with mouse MT-SP1 to the cleavage rate with human serum than a protease substrate comprising any one of SEQ ID NOs: 1, 2, and 3. In certain embodiments, the ratio of the cleavage rate with mouse MT-SP1 to the cleavage rate with human serum of a 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, if the cleavage rate with human serum is 1 or less, it can be converted to 1 and used to calculate the ratio of the cleavage rate with mouse MT-SP1 to the cleavage rate with human serum. In one embodiment, the protease cleavage sequences of the present disclosure are not cleaved by human serum. As a specific example, the cleavage rate of the protease cleavage sequences 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 set forth in SEQ ID NO: 4 by human serum.

[0038] The protease cleavage sequences of the present disclosure can be used as a library for selecting polypeptides with properties suited to the intended purpose, for example, when incorporated into a polypeptide. Specifically, the protease sensitivity of a polypeptide can be evaluated in order to selectively cleave the polypeptide with a protease localized in the lesion. After administration to a living body, a polypeptide may reach the lesion through contact with various proteases. Therefore, it is desirable for the polypeptide to be sensitive to the proteases localized in the lesion while being as resistant as possible to other proteases. To select a desired protease cleavage sequence according to the intended purpose, the protease resistance can be determined 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 with the required sensitivity and resistance can be found. Alternatively, a polypeptide incorporating a protease cleavage sequence may reach the lesion not only through the enzymatic action of a protease but also through various environmental stresses such as changes in pH, temperature, redox stress, etc. Based on information comparing the resistance of each protease cleavage sequence to such external factors, a protease cleavage sequence with desired properties according to the purpose can be selected.

[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 that is resistant to cleavage by at least another protease. In some embodiments, the protease cleavage sequence is a substrate for matriptase and / or uPA that is resistant to cleavage by at least plasmin. In some embodiments, the protease cleavage sequence is a substrate for matriptase and / or uPA that is 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 that is resistant to cleavage by at least human serum.

[0041] Amino acid modification To modify an amino acid in the amino acid sequence of a polypeptide, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR can be appropriately used. Furthermore, several known methods can also be used to modify amino acids by substituting amino acids other than natural amino acids (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing a tRNA in which a non-natural amino acid is bound to an amber suppressor tRNA complementary to the UAG codon (amber codon), which is a type of stop codon, can be suitably used.

[0042] As used herein, the meaning of the term "and / or" used to describe amino acid modification sites includes any combination of "and" and "or." Specifically, for example, "amino acids 37, 45, and / or 47 are substituted" includes the following amino acid modification variations: (a) No. 37, (b) No. 45, (c) No. 47, (d) No. 37 and No. 45, (e) No. 37 and No. 47, (f) No. 45 and No. 47, (g) No. 37, No. 45 and No. 47.

[0043] Herein, expressions for amino acid modifications may be appropriately expressed by listing the one-letter or three-letter codes for the amino acid before and after the modification before and after a number representing a specific position. For example, the modification F37V or Phe37Val used when substituting an amino acid in an antibody variable region represents a substitution of Phe at position 37 according to the Kabat numbering system with Val. That is, the number represents the amino acid position according to the Kabat numbering system, the one-letter or three-letter code preceding the number represents the amino acid before substitution, and the one-letter or three-letter code following the number represents the amino acid after substitution. Similarly, the modification P238A or Pro238Ala used when substituting an amino acid in the Fc region of an antibody constant region represents a substitution of Pro at position 238 according to the EU numbering system with Ala. That is, the number represents the amino acid position according to the EU numbering system, the one-letter or three-letter code preceding the number represents the amino acid before substitution, and the one-letter or three-letter code following the number represents the amino acid after substitution.

[0044] "Inserting" amino acid sequence A into amino acid sequence B means dividing amino acid sequence B into two parts without deleting it and connecting the two parts with amino acid sequence A (i.e., creating a new amino acid sequence such as "first half of amino acid sequence B-amino acid sequence A-second half of amino acid sequence B"). "Introducing" amino acid sequence A into amino acid sequence B means dividing amino acid sequence B into two parts and connecting the two parts with amino acid sequence A. In addition to "inserting" amino acid sequence A into amino acid sequence B, it is also possible to delete one or more amino acid residues, including amino acid residues in amino acid sequence B adjacent to amino acid sequence A, and then connect the two parts with amino acid sequence A (i.e., replacing part of amino acid sequence B with amino acid sequence A). Furthermore, as used herein, the term "near the boundary between part A and part B" refers to a portion before or after the site where part A and part B are linked in a polypeptide, which portion does not significantly affect the secondary structure of part A and / or part B.

[0045] Antibodies and antibody fragments The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

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

[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 a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0048] In the present specification, although there are embodiments in which the antibody contains a protease cleavage sequence, the antibody can be expressed as an "antibody" regardless of whether or not the antibody contains a protease cleavage sequence.

[0049] Variable region The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of an antibody (VH and VL, respectively) typically have a similar structure, with each domain containing four conserved framework regions (FR) and three complementarity-determining regions (CDR). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). One VH or VL domain may be sufficient to confer antigen-binding specificity.

[0050] CDR As used herein, the term "complementarity determining region" or "CDR" refers to each region of an antibody variable domain or single domain antibody that is hypervariable in sequence and / or forms structurally defined loops ("hypervariable loops") and / or antigen contact residues ("antigen contacts"). Typically, antibodies contain six CDRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary antibody CDRs herein include the following: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) A combination of (a), (b), and / or (c) comprising 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] Typically, a single domain antibody comprises three CDRs: CDR1, CDR2, and CDR3. When the single domain antibody is a VHH or single domain VH antibody, the CDRs of the single domain antibody illustratively comprise: (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) A combination of (a), (b), and / or (c), comprising 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 illustratively include: (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) A combination of (a), (b), and / or (c), comprising 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 (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0054] FR "Framework" or "FR" refers to the variable domain or single domain antibody residues other than the complementarity determining region (CDR) residues. The FR of a variable domain or single domain antibody usually consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR sequences usually appear in the VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. In single domain antibodies, the CDR and FR sequences usually appear in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0055] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a 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 embodiment, for VL, the subgroup is subgroup κI according to Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III according to Kabat et al., supra.

[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 disulfide-bonded light chains and two identical heavy chains. From the N-terminus to the C-terminus, each heavy chain contains a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by a heavy chain constant region (CH) containing a CH1 domain, hinge region, CH2 domain, and CH3 domain. Similarly, from the N-terminus to the C-terminus, each light chain contains a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. The light chain of a native antibody can be assigned to one of two types, called kappa (κ) or lambda (λ), based on the amino acid sequence of its constant domain. The term "comprising a constant region" can include the entire constant region or a portion of the constant region.

[0057] Unless otherwise specified herein, the numbering of amino acid residues in antibody variable regions and antibody light and heavy chain constant regions is according to 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 present in the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

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

[0060] Hinge Area The term "hinge region" or "antibody hinge region" can refer to a region consisting of amino acids 216 to 230 (EU numbering) in an antibody heavy chain, or a portion thereof.

[0061] Single Domain Antibodies As used herein, the term "single-domain antibody" refers to an antibody that can exhibit antigen-binding activity using that domain alone. The structure of a single-domain antibody is not limited as long as that domain alone can exhibit antigen-binding activity. Conventional antibodies, exemplified by IgG antibodies, exhibit antigen-binding activity when a variable region is formed by pairing VH and VL, whereas single-domain antibodies are known to be able to exhibit antigen-binding activity using only 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 the form of a monomer. In some embodiments, the form of a single-domain antibody is similar to that of an antibody heavy chain variable region or an antibody light chain variable region. Examples of single domain antibodies include, but are not limited to, camelid heavy chain antibody variable regions (VHH), shark VHH, and the like. NAR Examples of single-domain antibodies include antigen-binding molecules that inherently lack light chains, such as those described in U.S. Pat. No. 6,248,516 B1, and antibody fragments that contain all or part of the VH domain or all or part of the VL domain of an antibody. Examples of single-domain antibodies, which are antibody fragments that contain all or part of the VH / VL domains of an antibody, include, but are not limited to, single-domain antibodies (hereinafter referred to as single-domain VH antibodies and single-domain VL antibodies) artificially created starting from a human antibody VH or human antibody VL, such as those described in U.S. Pat. No. 6,248,516 B1.

[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, camelids and transgenic animals into which genes capable of producing single-domain antibodies have been introduced. Camelids include camels, llamas, alpacas, dromedaries, and guanacos. Examples of transgenic animals into which genes capable of producing single-domain antibodies have been introduced include, but are not limited to, the transgenic animals described in International Publication No. WO 2015 / 143414 and U.S. Patent Publication No. US 2011 / 0123527 A1. Humanized single-domain antibodies can also be obtained by substituting human germline sequences or sequences similar thereto for the framework sequences of single-domain antibodies obtained from animals. Humanized single-domain antibodies (e.g., humanized VHHs) are also an embodiment of the single-domain antibodies herein.

[0063] Alternatively, single domain antibodies can be obtained from a polypeptide library containing single domain antibodies by ELISA, panning, or the like. Examples of polypeptide libraries containing single domain antibodies include, but are not limited to, naive antibody libraries obtained from various animals or humans (e.g., Methods in Molecular Biology 2012 911 (65-78), Biochimica et Biophysica Acta - Proteins and Proteomics 2006 1764:8 (1307-1319)), antibody libraries obtained by immunizing various animals (e.g., Journal of Applied Microbiology 2014 117:2 (528-536)), or synthetic antibody libraries created from antibody genes of various animals or humans (e.g., Journal of Biomolecular Screening 2016 21:1 (35-43), Journal of Biological Chemistry 2016 291:24 (12641-12657), AIDS 2016 30:11 (1691-1701)).

[0064] In the present disclosure, single domain antibodies may include a protease cleavage sequence, but may be referred to as "single domain antibodies" regardless of whether they include a protease cleavage sequence.

[0065] The single domain antibodies herein, in some forms, generally comprise: a) an amino acid sequence consisting of four framework regions / sequences interspersed with three complementarity determining regions / sequences, wherein the amino acid residue at position 11 according to the 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 interspersed with three complementarity determining regions / sequences, wherein the amino acid residue at position 37 according to the 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 interspersed with three complementarity determining regions / sequences, wherein the amino acid residue at position 44 according to the 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 interspersed with three complementarity determining regions / sequences, wherein the amino acid residue at position 45 according to the 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 interspersed with three complementarity determining regions / sequences, wherein the amino acid residue at position 47 according to the Kabat numbering is selected from the group consisting of W, L, F, A, G, I, M, R, S, V, Y, preferably W, L, F or R, and / or f) an amino acid sequence consisting of four framework regions / sequences interspersed with three complementarity determining regions / sequences, wherein the amino acid residue at position 83 according to the Kabat numbering is selected from the group consisting of R, K, N, E, G, I, M, Q, and T, preferably K or R, more preferably K, and / or g) an amino acid sequence consisting of four framework regions / sequences interspersed with three complementarity determining regions / sequences, wherein the amino acid residue at position 84 according to the Kabat numbering is selected from the group consisting of P, A, L, R, S, T, D, V, preferably P, and / or h) an amino acid sequence consisting of four framework regions / sequences interspersed with three complementarity determining regions / sequences, wherein the amino acid residue at position 103 according to the Kabat numbering is selected from the group consisting of W, P, R, and S, preferably W; and / or i) an amino acid sequence consisting of four framework regions / sequences interspersed with three complementarity determining regions / sequences, wherein the amino acid residue at position 104 according to the 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 / sequences interspersed with three complementarity determining regions / sequences (the amino acid residue at position 108 according to the Kabat numbering is selected from the group consisting of Q, L, and 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 following amino acid sequences consisting of four framework regions / sequences interspersed with three complementarity determining regions / sequences: k) an amino acid sequence in which the amino acid residues at positions 43 to 46 according to the Kabat numbering system are KERE or KQRE; l) an amino acid sequence in which the amino acid residues at positions 44 to 47 according to the Kabat numbering system are GLEW; m) An amino acid sequence in which the amino acid residues at positions 83 and 84 according to the Kabat numbering system are KP or EP.

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

[0068] humanized antibodies A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In one embodiment, a humanized antibody comprises substantially all of at least one, typically two, variable domains, in which all or substantially all CDRs correspond to those of a non-human antibody and all or substantially all FRs correspond to those of a human antibody. A "humanized single-domain antibody" refers to a chimeric single-domain antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In one embodiment, a humanized single-domain antibody comprises all or substantially all CDRs corresponding to those of a non-human antibody and all or substantially all FRs corresponding to those of a human antibody. In a humanized antibody, even if some of the residues in the FRs do not correspond to those of a human antibody, this is considered an example in which substantially all FRs correspond to those of a human antibody. For example, when humanizing VHH, which is one embodiment of a single-domain antibody, some of the residues in the FR must be changed to residues that do not correspond to those in human antibodies (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. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0069] meeting As used herein, "association" can be rephrased as, for example, a state in which two or more polypeptide regions interact with each other. Generally, hydrophobic bonds, hydrogen bonds, ionic bonds, etc. are formed between the target polypeptide regions to form an association. As a commonly observed example of an association, it is known that in antibodies, such as natural antibodies, the heavy chain variable region (VH) and the light chain variable region (VL) maintain a paired structure through non-covalent bonds between them. Dissolution of the association can be expressed, for example, as dissolution of all or part of the interaction between two or more polypeptide domains. Dissolution of the association between VH and VL may mean dissolution of all or part of the interaction between VH and VL.

[0070] FcRn binding region As used herein, the term "FcRn-binding region" refers to a region that has the ability to bind to FcRn, and any structure can be used as long as it has the ability to bind to FcRn. Molecules containing the FcRn-binding domain can be taken up into cells via the FcRn salvage pathway and then return to the plasma. For example, the relatively long plasma retention (slow elimination) of IgG molecules is due to the function of FcRn, which is known as a salvage receptor for IgG molecules. IgG molecules taken up into endosomes by pinocytosis bind to FcRn expressed in endosomes under acidic conditions within the endosome. IgG molecules that cannot bind to FcRn proceed to lysosomes where they are degraded, while IgG molecules that bind to FcRn migrate to the cell surface and dissociate from FcRn under neutral plasma conditions, returning to the plasma. The FcRn-binding region is preferably a region that directly binds to FcRn. A preferred example of an FcRn-binding region is the Fc region of an antibody. However, since a region capable of binding to a polypeptide capable of binding to FcRn, such as albumin or IgG, can indirectly bind to FcRn via albumin, IgG, or the like, the FcRn-binding region of the present disclosure may also be a region that binds to such a polypeptide capable of binding to FcRn.

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

[0072] The conditions for measuring the FcRn-binding activity of the FcRn-binding region can be appropriately selected by those skilled in the art and are not particularly limited. For example, as described in WO2009 / 125825, measurements can be performed in MES buffer at 37°C. Furthermore, the FcRn-binding activity of the FcRn-binding region of the present disclosure can be measured by methods known to those skilled in the art, such as using a Biacore (GE Healthcare). The binding activity of the FcRn-binding region to FcRn can be assessed by passing FcRn, the FcRn-binding region, or a molecule containing the FcRn-binding region as an analyte through a chip onto which the FcRn-binding region or a transport moiety containing the FcRn-binding region is immobilized, or onto which FcRn is immobilized.

[0073] The binding affinity between the FcRn-binding region and FcRn may be evaluated at any pH between pH 4.0 and pH 6.5 as a measurement condition. Preferably, a pH between pH 5.8 and pH 6.0, which is close to the pH in early endosomes in vivo, is used to determine the binding affinity between the FcRn-binding region and human FcRn. The binding affinity between the FcRn-binding region and FcRn may be evaluated at any temperature between 10°C and 50°C as a measurement condition. Preferably, a temperature between 15°C and 40°C is used to determine the binding affinity between the FcRn-binding region and human FcRn. More preferably, any temperature between 20°C and 35°C, such as any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C, is also used to determine the binding affinity between the FcRn-binding region and FcRn. A temperature of 25°C is a non-limiting example of an embodiment of the present disclosure.

[0074] An example of an FcRn-binding region includes, but is not limited to, the Fc region of an IgG antibody. When an Fc region of an IgG antibody is used, the type is not limited, and Fc regions such as IgG1, IgG2, IgG3, and IgG4 can be used. For example, an Fc region containing one of the amino acid sequences shown in SEQ ID NOs: 18004, 18005, 18006, and 18007 can be used.

[0075] Furthermore, not only the Fc region of a natural IgG antibody, but also modified Fc regions in which one or more amino acids have been substituted can be used, as long as they have FcRn-binding ability. For example, in the Fc region of an IgG antibody, the following positions are identified: 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, and 314 (EU numbering) It is possible to use a modified Fc region comprising an amino acid sequence in which at least one amino acid selected from positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 315, 317, 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 has been substituted with another amino acid.

[0076] More specifically, the FcRn-binding region is a region of the Fc region of an IgG antibody, as shown in EU numbering. an amino acid substitution of Gly at position 237 with Met; Amino acid substitution of Pro at position 238 with Ala, an amino acid substitution of Ser at position 239 with Lys; an amino acid substitution of Lys at position 248 with Ile; an amino acid substitution of Thr at position 250 with Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr; an amino acid substitution of Met at position 252 with Phe, Trp, or Tyr; an amino acid substitution of Ser at position 254 with Thr; an amino acid substitution of Arg at position 255 with Glu; an amino acid substitution of Thr at position 256 with Asp, Glu, or Gln; an amino acid substitution of Pro at position 257 with Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val; an amino acid substitution of Glu at position 258 with His; an amino acid substitution of Asp at position 265 with Ala; an amino acid substitution of Asp at position 270 with Phe; an amino acid substitution of Asn at position 286 with Ala or Glu; an amino acid substitution of Thr at position 289 with His; an amino acid substitution of Asn at position 297 with Ala; an amino acid substitution of Ser at position 298 with Gly; an amino acid substitution of Val to Ala at position 303; an amino acid substitution of Val to Ala at position 305; an amino acid substitution of Thr at position 307 with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr; an amino acid substitution of Val at position 308 with Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr; an amino acid substitution of Leu or Val at position 309 with Ala, Asp, Glu, Pro, or Arg; an amino acid substitution of Gln at position 311 with Ala, His, or Ile; an amino acid substitution of Asp at position 312 with Ala or His; an amino acid substitution of Leu at position 314 with Lys or Arg; an amino acid substitution of Asn at position 315 with Ala or His; an amino acid substitution of Lys at position 317 with Ala; an amino acid substitution of Asn at position 325 with Gly; an amino acid substitution of Ile to Val at position 332; an amino acid substitution of Lys at position 334 with Leu; an amino acid substitution of Lys at position 360 with His; an amino acid substitution of Asp at position 376 with Ala; an amino acid substitution of Glu at position 380 with Ala; an amino acid substitution of Glu at position 382 with Ala; an amino acid substitution of Asn or Ser at position 384 with Ala; an amino acid substitution of Gly at position 385 with Asp or His; an amino acid substitution of Gln at position 386 with Pro; Amino acid substitution of Pro at position 387 with Glu; an amino acid substitution of Asn at position 389 with Ala or Ser; an amino acid substitution of Ser at position 424 with Ala; an amino acid substitution of Met at position 428 with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr; an amino acid substitution of His at position 433 with Lys; an amino acid substitution of Asn at position 434 with Ala, Phe, His, Ser, Trp, or Tyr; and Amino acid substitution of Tyr or Phe at position 436 with His It is possible to use a modified Fc region comprising at least one amino acid substitution selected from:

[0077] From another perspective, the FcRn binding region is the EU numbering in the Fc region of an IgG antibody. Met at amino acid 237, Ala at amino acid 238; Lys at amino acid 239, Ile at amino acid 248; Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr at amino acid position 250; Phe, Trp, or Tyr at amino acid 252; Thr at amino acid 254, Glu at amino acid 255, Asp, Glu, or Gln at amino acid 256; Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val at amino acid position 257; His at amino acid 258, Ala at amino acid 265, Phe at amino acid 270, Ala or Glu at amino acid 286; His at amino acid 289, Ala at amino acid position 297; Gly at amino acid 298; Ala at amino acid position 303; Ala at amino acid 305; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr at amino acid 307; Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr at amino acid 308; Ala, Asp, Glu, Pro, or Arg at amino acid position 309; Ala, His, or Ile at amino acid 311; Ala or His at amino acid 312; Lys or Arg at amino acid 314; Ala or His at amino acid 315; Ala at amino acid 317; Gly at amino acid 325; Val at amino acid 332; Leu at amino acid 334, His at amino acid 360, Ala at amino acid 376; Ala at amino acid 380, Ala at amino acid 382, Ala at amino acid position 384; Asp or His at amino acid 385; Pro at amino acid 386, Glu at amino acid 387, Ala or Ser at amino acid 389; Ala at amino acid 424; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr at amino acid 428; Lys at amino acid 433, Ala, Phe, His, Ser, Trp, or Tyr at amino acid 434, and His at amino acid 436 It is possible to use an Fc region comprising at least one amino acid selected from:

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

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

[0080] How to generate antibodies Methods for producing antibodies with the desired binding activity are known to those skilled in the art. Methods for producing antibodies that bind to IL-6R (anti-IL-6R antibodies) are exemplified below. Antibodies that bind to antigens other than IL-6R can also be produced as appropriate according to the examples below. When producing single-domain antibodies, they can be produced as appropriate according to the examples below, although differences may exist in the animals immunized.

[0081] Anti-IL-6R antibodies can be obtained as polyclonal or monoclonal antibodies using known methods. Monoclonal antibodies derived from mammals are preferably produced as anti-IL-6R antibodies. Mammalian-derived monoclonal antibodies include those produced by hybridomas and those produced by host cells transformed with expression vectors containing antibody genes by genetic engineering techniques. Antibodies referred to in this application include "humanized antibodies" and "chimeric antibodies."

[0082] Monoclonal antibody-producing hybridomas can be prepared using known techniques, for example, as follows: A mammal is immunized using an IL-6R protein as a sensitizing antigen according to a conventional immunization method. The resulting immune cells are fused with known parent cells by a conventional cell fusion method. Next, monoclonal antibody-producing cells can be screened using conventional screening methods to select hybridomas that produce anti-IL-6R antibodies.

[0083] Specifically, monoclonal antibodies can be produced, for example, as follows. First, the IL-6R gene can be expressed to obtain the IL-6R protein used as a sensitizing antigen for antibody production. Specifically, a suitable host cell is transformed by inserting a gene sequence encoding IL-6R into a known expression vector. The desired human IL-6R protein is purified from the host cell or culture supernatant by a known method. To obtain soluble IL-6R from the culture supernatant, for example, soluble IL-6R is expressed as described by Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968). Purified native IL-6R protein can also be used as a sensitizing antigen.

[0084] The purified IL-6R protein can be used as a sensitizing antigen for immunization of mammals. A partial peptide of IL-6R can also be used as a sensitizing antigen. In this case, the partial peptide can be obtained by chemical synthesis from the amino acid sequence of human IL-6R. Alternatively, it can be obtained by incorporating a portion of the IL-6R gene into an expression vector and expressing it. It can also be obtained by degrading the IL-6R protein using a protease, but the region and size of the IL-6R peptide used as a partial peptide are not particularly limited. The number of amino acids constituting the peptide used as a sensitizing antigen is preferably at least 5 or more, for example, 6 or more, or 7 or more. More specifically, a peptide of 8 to 50 residues, preferably 10 to 30 residues, can be used as a sensitizing antigen.

[0085] Alternatively, a fusion protein obtained by fusing a desired partial polypeptide or peptide of the IL-6R protein with a different polypeptide can be used as a sensitizing antigen. For example, an antibody Fc fragment or a peptide tag can be suitably used to produce a fusion protein used as a sensitizing antigen. A vector expressing a fusion protein can be prepared by fusing genes encoding two or more desired polypeptide fragments in frame and inserting the fusion gene into an expression vector as described above. Methods for producing fusion proteins are described in Molecular Cloning, 2nd ed. (Sambrook, J et al., Molecular Cloning, 2nd ed., pp. 9:47-9:58 (1989) Cold Spring Harbor Lab. Press). Methods for obtaining IL-6R to be used as a sensitizing antigen and immunization methods using the same are also specifically described in WO2003 / 000883, WO2004 / 022754, WO2006 / 006693, etc.

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

[0087] The above-mentioned animals are immunized with the sensitizing antigen according to known methods. For example, a common method for immunization is to administer the sensitizing antigen intraperitoneally or subcutaneously to a mammal. Specifically, the sensitizing antigen is diluted at an appropriate dilution ratio with PBS (Phosphate-Buffered Saline) or physiological saline, and optionally mixed with a conventional adjuvant, such as Freund's complete adjuvant, and emulsified. The sensitizing antigen is then administered to the mammal several times every 4 to 21 days. A suitable carrier can also be used during immunization with the sensitizing antigen. In particular, when a partial peptide with a small molecular weight is used as the sensitizing antigen, it may be desirable to immunize with the sensitizing antigen peptide bound to a carrier protein such as albumin or keyhole limpet hemocyanin.

[0088] Hybridomas producing the desired antibodies can also be prepared using DNA immunization as follows. DNA immunization is an immunization method in which a vector DNA constructed in such a manner that a gene encoding an antigen protein can be expressed in the immunized animal is administered to the immunized animal, and a sensitizing antigen is expressed in the immunized animal's body, thereby conferring immune stimulation. Compared to general immunization methods in which a protein antigen is administered to the immunized animal, DNA immunization is expected to have the following advantages: -Maintaining the structure of membrane proteins such as IL-6R can provide immune stimulation -No need to purify the immunogen

[0089] To obtain the monoclonal antibody of the present disclosure by DNA immunization, first, DNA expressing IL-6R protein is administered to an animal to be immunized. DNA encoding IL-6R can be synthesized by known methods such as PCR. The obtained DNA is inserted into an appropriate expression vector and administered to the animal to be immunized. Commercially available expression vectors, such as pcDNA3.1, can be suitably used as the expression vector. Commonly used methods can be used to administer the vector to a living body. For example, DNA immunization can be performed by introducing gold particles adsorbed with the expression vector into the cells of an animal to be immunized using a gene gun. Furthermore, antibodies that recognize IL-6R can also be produced using the method described in International Publication WO 2003 / 104453.

[0090] After a mammal is immunized in this manner and an increase in the titer of an antibody that binds to IL-6R is confirmed in the serum, immune cells are collected from the mammal and subjected to cell fusion. Splenocytes are particularly preferred as immune cells.

[0091] Mammalian myeloma cells are used as the cells to be fused with the immune cells. The myeloma cells preferably contain an appropriate selection marker for screening. A selection marker refers to a trait that allows (or prevents) survival under specific culture conditions. Known selection markers include hypoxanthine-guanine-phosphoribosyltransferase deficiency (hereinafter abbreviated as HGPRT deficiency) and thymidine kinase deficiency (hereinafter abbreviated as TK deficiency). Cells deficient in HGPRT or TK are hypoxanthine-aminopterin-thymidine sensitive (hereinafter abbreviated as HAT sensitive). HAT-sensitive cells cannot synthesize DNA in HAT selective medium and die, but when fused with normal cells, they can continue DNA synthesis by utilizing the salvage pathway of normal cells, allowing them to grow even in HAT selective medium.

[0092] HGPRT-deficient or TK-deficient cells can be selected on media containing 6-thioguanine, 8-azaguanine (hereafter abbreviated as 8AG), or 5'-bromodeoxyuridine, respectively. Normal cells that incorporate these pyrimidine analogs into their DNA die. On the other hand, cells lacking these enzymes and unable to incorporate these pyrimidine analogs can survive in selective media. Another selectable marker, called G418 resistance, confers resistance to 2-deoxystreptamine antibiotics (gentamicin analogs) via the neomycin resistance gene. Various myeloma cell lines suitable for cell fusion are known.

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

[0094] Basically, cell fusion between the immune cells and myeloma cells is carried out according to known methods, such as the method of Kohler and Milstein et al. (Methods Enzymol. (1981) 73, 3-46). More specifically, the cell fusion can be carried out in a conventional nutrient medium in the presence of a cell fusion promoter, such as polyethylene glycol (PEG) or Sendai virus (HVJ), with the addition of an adjuvant such as dimethyl sulfoxide, if desired, to further enhance the fusion efficiency.

[0095] The ratio of immune cells to myeloma cells can be set arbitrarily. For example, the ratio of immune cells to myeloma cells is preferably 1 to 10. The culture medium used for the cell fusion may be, for example, RPMI1640 culture medium, MEM culture medium, or other conventional culture medium suitable for growing the myeloma cell line, and may further be suitably supplemented with serum supplements such as fetal calf serum (FCS).

[0096] For cell fusion, predetermined amounts of the immune cells and myeloma cells are thoroughly mixed in the culture medium, and a PEG solution (e.g., an average molecular weight of approximately 1000 to 6000) preheated to approximately 37°C is added, usually at a concentration of 30 to 60% (w / v). The mixture is gently mixed to form the desired fused cells (hybridomas). Next, an appropriate culture medium such as those listed above is successively added, and the mixture is centrifuged and the supernatant is removed. This procedure is repeated to remove cell fusion agents and other substances that are undesirable for hybridoma growth.

[0097] The hybridomas thus obtained can be selected by culturing them in a conventional selective culture medium, such as HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Culture can be continued using the HAT culture medium for a sufficient period of time (usually several days to several weeks) for cells other than the desired hybridoma (unfused cells) to die. Hybridomas producing the desired antibody are then screened and single-cloned by the conventional limiting dilution method.

[0098] The hybridomas thus obtained can be selected using a selective medium corresponding to the selection marker possessed by the myeloma used in cell fusion. For example, cells lacking HGPRT or TK can be selected by culturing them in HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). That is, when HAT-sensitive myeloma cells are used for cell fusion, cells that have successfully fused with normal cells can selectively grow in HAT medium. Culture in the above HAT medium is continued for a period of time sufficient for cells other than the desired hybridoma (non-fused cells) to die. Specifically, the desired hybridoma can generally be selected by culturing for several days to several weeks. Hybridomas producing the desired antibody can then be screened and single-cell cloned by the conventional limiting dilution method.

[0099] Screening and monocloning of the desired antibody can be preferably carried out by known screening methods based on antigen-antibody reactions. For example, a monoclonal antibody that binds to IL-6R can bind to IL-6R expressed on the cell surface. Such monoclonal antibodies can be screened, for example, by FACS (fluorescence activated cell sorting). FACS is a system that analyzes cells contacted with a fluorescent antibody using laser light and measures the fluorescence emitted by individual cells, thereby enabling measurement of antibody binding to the cell surface.

[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 non-transformed mammalian cells as a control host cell, the binding activity of the antibody to IL-6R on the cell surface can be selectively detected. That is, hybridomas producing IL-6R monoclonal antibodies can be obtained by selecting hybridomas that produce antibodies that do not bind to host cells but bind to IL-6R-overexpressing cells.

[0101] Alternatively, the binding activity of an antibody to immobilized IL-6R-expressing cells can be evaluated based on the principles of ELISA. For example, IL-6R-expressing cells are immobilized in the wells of an ELISA plate. The hybridoma culture supernatant is contacted with the immobilized cells in the wells, and antibodies that bind to the immobilized cells are detected. If the monoclonal antibody is derived from a mouse, the antibody that binds to the cells can be detected with an anti-mouse immunoglobulin antibody. Hybridomas that produce the desired antibody capable of binding to the antigen and are selected by these screening methods can be cloned by limiting dilution or other methods.

[0102] The hybridomas producing the monoclonal antibodies thus prepared can be subcultured in a conventional culture medium and can be stored for a long period of time in liquid nitrogen.

[0103] The hybridomas are cultured according to conventional methods, and the desired monoclonal antibodies can be isolated from the culture supernatant. Alternatively, the hybridomas can be administered to a compatible mammal to grow, and the monoclonal antibodies can be isolated from the ascites. The former method is suitable for obtaining highly purified antibodies.

[0104] Antibodies encoded by antibody genes cloned from antibody-producing cells such as hybridomas can also be suitably used. The cloned antibody genes are incorporated into an appropriate vector and introduced into a host, whereby the antibodies encoded by the genes are expressed. Methods for isolating antibody genes, introducing them into vectors, and transforming host cells have already been established, for example, by Vandamme et al. (Eur. J. Biochem. (1990) 192 (3), 767-775). Methods for producing recombinant antibodies are also known, as described below.

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

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

[0107] The desired cDNA fragment is purified from the resulting PCR product and then ligated to vector DNA. The recombinant vector thus constructed is introduced into E. coli or other bacteria, and colonies are selected. The desired recombinant vector can then be prepared from the E. coli that formed the colonies. Whether the recombinant vector contains the nucleotide sequence of the desired cDNA is then confirmed by known methods, such as the dideoxynucleotide chain termination method.

[0108] A convenient way to obtain genes encoding variable regions is to use the 5'-RACE method, which uses primers specifically designed for amplifying variable region genes. First, cDNA is synthesized using RNA extracted from hybridoma cells as a template, and a 5'-RACE cDNA library is obtained. A commercially available kit, such as the SMART RACE cDNA Amplification Kit, can be used to synthesize the 5'-RACE cDNA library.

[0109] The resulting 5'-RACE cDNA library is used as a template for PCR amplification of antibody genes. Primers for amplifying mouse antibody genes can be designed based on known antibody gene sequences. These primers have different base sequences for each immunoglobulin subclass. Therefore, it is recommended that the subclass be determined in advance using a commercially available kit such as the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche Diagnostics).

[0110] Specifically, for example, when the goal is to obtain a gene encoding mouse IgG, primers capable of amplifying genes encoding γ1, γ2a, γ2b, and γ3 heavy chains and κ and λ light chains can be used. To amplify IgG variable region genes, the 3' primer generally anneals to a region corresponding to the constant region close to the variable region. Meanwhile, the 5' primer used is a primer included in the 5' RACE cDNA library construction kit.

[0111] The PCR products thus amplified can be used to reconstitute immunoglobulins consisting of a combination of heavy and light chains. The desired antibodies can be screened using the binding activity of the reconstituted immunoglobulins to IL-6R as an indicator. For example, when the goal is to obtain antibodies against IL-6R, it is more preferable that the antibodies bind to IL-6R specifically. Antibodies that bind to IL-6R can be screened, for example, as follows: (1) contacting an antibody containing a V region encoded by a cDNA obtained from a hybridoma with an IL-6R-expressing cell; (2) detecting the binding of the antibody to the IL-6R-expressing cells; and (3) A step of selecting an antibody that binds to IL-6R-expressing cells.

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

[0113] A panning method using a phage vector is also preferably used as a method for screening antibodies using binding activity as an index. When screening for single domain antibodies, screening can be carried out appropriately according to the following examples. When antibody genes are obtained as a library of heavy and light chain subclasses from a polyclonal antibody-expressing cell population, a screening method using a phage vector is advantageous. Genes encoding the heavy and light chain variable regions can be linked with an appropriate linker sequence to form a single-chain Fv (scFv). By inserting a gene encoding an scFv into a phage vector, a phage that expresses scFv on its surface can be obtained. After contacting this phage with a desired antigen, DNA encoding an scFv with the desired binding activity can be recovered by recovering the phage that binds to the antigen. By repeating this procedure as necessary, scFv with the desired binding activity can be enriched.

[0114] After obtaining cDNA encoding the V region of the desired anti-IL-6R antibody, the cDNA is digested with restriction enzymes that recognize restriction enzyme sites inserted at both ends of the cDNA. Preferred restriction enzymes recognize and digest nucleotide sequences that appear infrequently in the nucleotide sequence constituting the antibody gene. Furthermore, to insert one copy of the digested fragment into a vector in the correct orientation, it is preferable to insert a restriction enzyme that generates cohesive ends. An antibody expression vector can be obtained by inserting the cDNA encoding the V region of the anti-IL-6R antibody digested as described above into an appropriate expression vector. In this case, a chimeric antibody can be obtained by fusing a gene encoding the antibody constant region (C region) with a gene encoding the V region in frame. Here, a chimeric antibody refers to an antibody in which the constant region and variable region are derived from different sources. Therefore, in addition to heterogeneous chimeric antibodies such as mouse-human, human-human allogeneic chimeric antibodies are also included in the chimeric antibodies of the present disclosure. A chimeric antibody expression vector can be constructed by inserting the V region gene into an expression vector that already contains a constant region. Specifically, for example, a restriction enzyme recognition sequence for a restriction enzyme that digests the V region gene can be appropriately positioned at the 5' end of an expression vector carrying DNA encoding the desired antibody constant region (C region). A chimeric antibody expression vector is constructed by fusion in-frame of both DNAs digested with the same combination of restriction enzymes.

[0115] To produce an anti-IL-6R monoclonal antibody, the antibody gene is incorporated into an expression vector so that its expression is controlled by an expression control region. Expression control regions for antibody expression include, for example, enhancers and promoters. Furthermore, an appropriate signal sequence can be added to the amino terminus so that the expressed antibody is secreted extracellularly. For example, a peptide having the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 18008) can be used as a signal sequence, although other suitable signal sequences can also be added. The expressed polypeptide is cleaved at the carboxyl terminal of the sequence, and the cleaved polypeptide can be secreted extracellularly as a mature polypeptide. Next, appropriate host cells can be transformed with this expression vector to obtain recombinant cells expressing DNA encoding the anti-IL-6R antibody.

[0116] Polynucleotides (nucleic acids) 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. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can include modifications made after synthesis, such as conjugation to a label. Other types of modifications include, for example, "caps," substitutions of one or more naturally occurring nucleotides with analogs, internucleotide modifications, such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalating agents (e.g., acridine, psoralens, etc.), those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, modified linkages (e.g., alpha-anomeric nucleic acids, etc.), and unmodified forms of polynucleotides. Additionally, any hydroxyl groups normally present on the sugar can be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to generate additional linkages to additional nucleotides, or conjugated to solid or semi-solid supports. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups.Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars commonly known in the art, including, 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, sedoheptuloses, acyclic analogs, and basic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which 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. Not all linkages in a polynucleotide need 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 propagating another nucleic acid to which it is linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of effecting the expression of nucleic acids to which they are operatively linked. Such vectors are also referred to herein as "expression vectors."

[0118] host cells etc. The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived from that cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as that for which the original transformed cell was screened or selected are also included herein.

[0119] Polypeptides containing protease cleavage sequences One aspect of the present disclosure relates to polypeptides that include a protease cleavage sequence. Another aspect of the present disclosure also relates to 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 4th amino acid to the 13th 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 5th amino acid to the 15th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993, a sequence from the 6th amino acid to the 13th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993, a sequence from the 7th 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 8th amino acid to the 9th amino acid at the N-terminus of the sequence from the third amino acid at the N-terminus to the eleventh amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; the sequence from the third amino acid at the N-terminus to the tenth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; the sequence from the fifth amino acid at the N-terminus to the twelfth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; the sequence from the fifth amino acid at the N-terminus to the tenth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; and a polypeptide comprising at least one sequence selected from the sequences set forth in any of SEQ ID NOs: 5 to 18003. Another aspect of the present disclosure also relates to a polypeptide comprising any of the sequences consisting of "a sequence selected from Group A below - a sequence selected from Group B below" in this order from the N-terminus: (Group A) A sequence from the first amino acid to the ninth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the second amino acid to the ninth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 3rd amino acid to the 9th 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 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 5th amino acid to the 9th 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 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 7th amino acid to the 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 8th amino acid to the 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the first amino acid to the sixth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the second amino acid to the sixth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the third amino acid to the sixth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 4th amino acid to the 6th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 5th amino acid to the 6th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the first amino acid to the eighth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the second amino acid to the eighth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the third amino acid to the eighth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 4th amino acid to the 8th 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 8th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 6th amino acid to the 8th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 7th to 8th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; (Group B) A sequence from the 10th 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 10th amino acid to the 14th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 10th 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 10th amino acid to the 12th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 10th amino acid to the 11th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 7th amino acid to the 15th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 7th amino acid to the 14th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 7th amino acid to the 13th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 7th 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 7th 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 7th 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 7th amino acid to the 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 7th amino acid to the 8th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 9th amino acid to the 15th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 9th 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 9th amino acid to the 13th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 9th 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 9th amino acid to the 11th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 9th to 10th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003.

[0120] In one embodiment of the polypeptide of the present disclosure, the polypeptide is selected from SEQ ID NOs: 5 to 17201, the 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, the 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, the 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, the 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, the 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, the 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, The polypeptide contains at least one sequence selected from the sequence from the third amino acid at the N-terminus to the tenth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the third amino acid at the N-terminus to the fourteenth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003, the sequence from the fifth amino acid at the N-terminus to the twelfth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003, the sequence from the fifth amino acid at the N-terminus to the tenth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003, and any of the sequences shown in SEQ ID NOs: 5 to 18003, and the sequence in the polypeptide is cleavable by a protease, i.e., the sequence functions as a protease cleavage sequence in the polypeptide. In one embodiment of the polypeptide of the present disclosure, the polypeptide contains any one of sequences consisting of "a sequence selected from Group A below - a sequence selected from Group B below" in this order from the N-terminus, and the sequence in the polypeptide is cleavable by a protease, i.e., the sequence functions as a protease cleavage sequence in the polypeptide: (Group A) A sequence from the first amino acid to the ninth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the second amino acid to the ninth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 3rd amino acid to the 9th 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 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 5th amino acid to the 9th 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 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 7th amino acid to the 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 8th amino acid to the 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the first amino acid to the sixth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the second amino acid to the sixth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the third amino acid to the sixth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 4th amino acid to the 6th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 5th amino acid to the 6th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the first amino acid to the eighth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the second amino acid to the eighth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the third amino acid to the eighth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 4th amino acid to the 8th 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 8th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 6th amino acid to the 8th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 7th to 8th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; (Group B) A sequence from the 10th 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 10th amino acid to the 14th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 10th 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 10th amino acid to the 12th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 10th amino acid to the 11th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 7th amino acid to the 15th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 7th amino acid to the 14th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 7th amino acid to the 13th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 7th 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 7th 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 7th 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 7th amino acid to the 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 7th amino acid to the 8th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 9th amino acid to the 15th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 9th 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 9th amino acid to the 13th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 9th 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 9th amino acid to the 11th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 9th to 10th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003.

[0121] In some aspects of the polypeptides of the present disclosure, the protease cleavage sequence further comprises a flexible linker attached to either or both ends. The flexible linker at one end of the protease cleavage sequence can be referred to as a first flexible linker, and the flexible linker at the other end can be referred to as a second flexible linker. In certain embodiments, the protease cleavage sequence and flexible linker comprise one of the following formulas: (protease cleavage sequence) (first flexible linker)-(protease cleavage sequence) (protease cleavage sequence)-(second flexible linker) (first flexible linker)-(protease cleavage sequence)-(second flexible linker) In this embodiment, the flexible linker is preferably a peptide linker. The first and second flexible linkers are independently and optionally present and may be the same or different flexible linkers containing at least one flexible amino acid (e.g., Gly). For example, the linker may contain a sufficient number of residues to provide the desired protease accessibility to the protease cleavage sequence (amino acids selected from Arg, Ile, Gln, Glu, Cys, Tyr, Trp, Thr, Val, His, Phe, Pro, Met, Lys, Gly, Ser, Asp, Asn, Ala, etc., particularly Gly, Ser, Asp, Asn, Ala, especially Gly and Ser, particularly Gly, etc.).

[0122] Flexible linkers suitable for use on either end of a protease cleavage sequence typically improve protease access to the protease cleavage sequence and increase the cleavage efficiency of the protease. Suitable flexible linkers can be readily selected and can range in length from 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, or 3 to 12 amino acids, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids. In some embodiments of the present disclosure, the flexible linker is a peptide linker of 1 to 7 amino acids.

[0123] Examples of flexible linkers include, but are not limited to, glycine polymers (G)n, glycine-serine polymers (e.g., including (GS)n, (GSGGS: SEQ ID NO: 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 flexible linkers known in the art. Of these, glycine and glycine-serine polymers have attracted attention because these amino acids are relatively unstructured and therefore more likely to function as neutral tethers between components. Examples of flexible linkers made of glycine-serine polymers include, but are not limited to, Ser Gly·Ser(GS) Ser·Gly(SG) Gly Gly Ser (GGS) Gly·Ser·Gly (GSG) Ser Gly Gly (SGG) Gly·Ser·Ser (GSS) Ser·Ser·Gly (SSG) Ser Gly Ser (SGS) Gly·Gly·Gly·Ser (GGGS, SEQ ID NO: 18009) Gly·Gly·Ser·Gly (GGSG, SEQ ID NO: 18010) Gly·Ser·Gly·Gly (GSGG, SEQ ID NO: 18011) Ser·Gly·Gly·Gly (SGGG, SEQ ID NO: 18012) Gly·Ser·Ser·Gly (GSSG, SEQ ID NO: 18013) Gly·Gly·Gly·Gly·Ser (GGGGS, SEQ ID NO: 18014) Gly·Gly·Gly·Ser·Gly (GGGSG, SEQ ID NO: 18015) Gly·Gly·Ser·Gly·Gly (GGSGG, SEQ ID NO: 18016) Gly·Ser·Gly·Gly·Gly (GSGGG, SEQ ID NO: 18017) Gly·Ser·Gly·Gly·Ser (GSGGS, SEQ ID NO: 18018) Ser·Gly·Gly·Gly·Gly (SGGGG, SEQ ID NO: 18019) Gly·Ser·Ser·Gly·Gly (GSSGG, SEQ ID NO: 18020) Gly·Ser·Gly·Ser·Gly (GSGSG, SEQ ID NO: 18021) Ser·Gly·Gly·Ser·Gly (SGGSG, SEQ ID NO: 18022) Gly·Ser·Ser·Ser·Gly (GSSSG, SEQ ID NO: 18023) Gly·Gly·Gly·Gly·Gly·Ser (GGGGGS, SEQ ID NO: 18024) Ser·Gly·Gly·Gly·Gly·Gly·Gly (SGGGGG, SEQ ID NO: 18025) Gly·Gly·Gly·Gly·Gly·Gly·Ser (GGGGGGS, SEQ ID NO: 18026) Ser·Gly·Gly·Gly·Gly·Gly·Gly·Gly (SGGGGGG, SEQ ID NO: 18027) (Gly·Gly·Gly·Gly·Ser(GGGGS, SEQ ID NO: 18014))n (Ser·Gly·Gly·Gly·Gly (SGGGG, SEQ ID NO: 18019)) [n is an integer of 1 or more], etc. However, the length and sequence of the peptide linker can be appropriately selected by those skilled in the art depending on the purpose.

[0124] Polypeptides comprising a protease cleavage sequence of the present disclosure include 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, and a sequence selected from SEQ ID NOs: 17202 to 17993. the sequence from the 3rd to 11th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993, the sequence from the 3rd to 10th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003, the sequence from the 5th to 12th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003, the sequence from the 5th to 10th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003, or the sequence of any of SEQ ID NOs: 5 to 18003, other configurations are not important as long as it contains at least one sequence selected from the sequence shown in any of SEQ ID NOs: 5 to 18003. The polypeptides comprising the protease cleavage sequence of the present disclosure may have any other structure as long as they contain, from the N-terminus, a sequence selected from Group A below and a sequence selected from Group B below: (Group A) A sequence from the first amino acid to the ninth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the second amino acid to the ninth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 3rd amino acid to the 9th 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 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 5th amino acid to the 9th 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 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 7th amino acid to the 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 8th amino acid to the 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the first amino acid to the sixth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the second amino acid to the sixth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the third amino acid to the sixth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 4th amino acid to the 6th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 5th amino acid to the 6th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the first amino acid to the eighth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the second amino acid to the eighth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the third amino acid to the eighth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 4th amino acid to the 8th 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 8th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 6th amino acid to the 8th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 7th to 8th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; (Group B) A sequence from the 10th 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 10th amino acid to the 14th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 10th 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 10th amino acid to the 12th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 10th amino acid to the 11th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 7th amino acid to the 15th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 7th amino acid to the 14th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 7th amino acid to the 13th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 7th 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 7th 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 7th 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 7th amino acid to the 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 7th amino acid to the 8th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 9th amino acid to the 15th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 9th 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 9th amino acid to the 13th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 9th 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 9th amino acid to the 11th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 9th to 10th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003.

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

[0126] Polypeptides containing an antigen-binding domain and a transport moiety In one embodiment of a polypeptide of the present disclosure, the polypeptide comprises an antigen-binding domain and a transport moiety, wherein the transport moiety comprises a repression domain that represses the antigen-binding activity of the antigen-binding domain. The present disclosure also relates to a method of releasing an antigen-binding domain from a polypeptide comprising the antigen-binding domain and a transport moiety described herein, comprising cleaving a protease cleavage sequence in the polypeptide with a protease.

[0127] As used herein, the term "antigen-binding domain" is limited only to its ability to bind to a target antigen. Any antigen-binding domain may be used, provided that it binds to the target antigen. Examples of such domains include, but are not limited to, antibody heavy chain variable regions (VH) and light chain variable regions (VL), single domain antibodies (sdAb), modules called A domains of about 35 amino acids contained in Avimer, a cell membrane protein present in living organisms (International Publication Nos. WO2004 / 044011 and WO2005 / 040229), Adnectin containing the 10Fn3 domain, which is a domain that binds to proteins in fibronectin, a glycoprotein expressed on cell membranes (International Publication No. WO2002 / 032925), Affibody scaffolded by an IgG binding domain that constitutes a bundle of three helices consisting of 58 amino acids of Protein A (International Publication No. WO1995 / 001937), and ankyrin repeats having a structure in which subunits of a turn containing 33 amino acid residues, two antiparallel helices, and a loop are repeatedly stacked. Examples of such proteins include DARPins (Designed Ankyrin Repeat proteins), which are regions exposed on the molecular surface of ankyrin repeat (AR) molecules (International Publication WO 2002 / 020565); Anticalin, which is a four-loop region supporting one side of a barrel structure in which eight highly conserved antiparallel strands twist toward the center in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) (International Publication WO 2003 / 029462); and a concave region of a parallel sheet structure within a horseshoe-shaped structure formed by repeated stacking of leucine-rich-repeat (LRR) modules in the variable lymphocyte receptor (VLR), which does not have an immunoglobulin structure and is part of the adaptive immune system of jawless fish such as lampreys and hagfish (International Publication WO 2008 / 016854).

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

[0129] A suitable example of the antigen-binding domain of the present disclosure is an antigen-binding domain with 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'. Antigen-binding domains with a molecular weight of 60 kDa or less are generally 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). From another perspective, a preferred example of the antigen-binding domain of the present disclosure is an antigen-binding domain with 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', etc.

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

[0131] As used herein, the term "antigen" is limited only to the epitope to which the antigen-binding domain binds. Suitable examples of antigens include, but are not limited to, peptides, polypeptides, and proteins derived from animals or humans. Suitable examples of antigens include, but are not limited to, molecules expressed on the surface of target cells (e.g., cancer cells, inflammatory cells), molecules expressed on the surface of other cells in tissues containing target cells, molecules expressed on the surface of cells that play an immunological role in target cells and tissues containing target cells, and large molecules present in the interstitium of tissues containing 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, and 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, A RC, ART, Artemin, Anti-Id, ASPARTIC, Atrial Natriuretic Factor, av / b3 Integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulatory factor (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, CD 8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD3 3 (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, Clostridium perfringens 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, サイトケラチン tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, complement defense factor (Decay accelerating factor)factor), 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 / E phB4, 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, fractalcohol In, 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), GD F-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, 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 receptor 1 (IGFR), insulin-like growth factor receptor 2 (IGFR), insulin-like growth factor receptor 3 (IGFR), insulin-like growth factor receptor 4 (IGFR), insulin-like growth factor receptor 5 (IGFR), insulin-like growth factor receptor 6 (IGFR), insulin-like growth factor receptor 7 (IGFR), insulin-like growth factor receptor 8 (IGFR), insulin-like growth factor receptor 9 (IGFR), insulin-like growth factor receptor 1 ... Factor 1, integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin alpha 6, integrin beta 1, integrin beta 2, 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, Lefty, 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 inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, NC adherin, NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4, or -6, neurturin, 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, PDK-1, P ECAM, 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, 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-α connectin, 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-like receptor)receptor)1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expression 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, polymeric 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 include factor 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 also include receptors, but even when these receptors exist in a soluble form in biological fluids, they can be used as antigens to which the antigen-binding domains of the present disclosure bind.

[0134] Examples of the antigens include membrane molecules expressed on the cell membrane and soluble molecules secreted extracellularly from cells. When an antigen-binding domain of the present disclosure binds to a soluble molecule secreted from a cell, the antigen-binding domain preferably has neutralizing activity.

[0135] There is no limitation on the solution in which a soluble molecule exists, and the soluble molecule may exist in biological fluids, i.e., all fluids filling the vessels or the spaces between tissues and cells in a living body. In one non-limiting embodiment, a soluble molecule bound by an antigen-binding domain of the present disclosure may exist in extracellular fluid. In vertebrates, extracellular fluid refers collectively to components in bone and cartilage, such as plasma, interstitial fluid, lymph, dense connective tissue, cerebrospinal fluid, spinal fluid, aspirate, or synovial fluid, as well as transcellular fluids (fluids in various glandular cavities resulting from the active transport and secretion activity of cells, and fluids in the digestive tract and other body cavities), such as alveolar fluid (bronchoalveolar lavage fluid), ascites, pleural effusion, pericardial fluid, cystic fluid, or aqueous humor (aqueous humor).

[0136] As used herein, the term "transport moiety" refers to a portion of a polypeptide other than the antigen-binding domain. The transport moieties of the present disclosure are typically peptides or polypeptides composed of amino acids, and in a specific embodiment, the transport moiety in the polypeptide is linked to the antigen-binding domain via a protease cleavage sequence. The transport moieties 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 or hydrophobic interactions.

[0137] The delivery moiety of the present disclosure has a repression domain that suppresses the antigen-binding activity of the antigen-binding domain. As used herein, the term "repression domain" is limited only to the repression of the antigen-binding activity of the antigen-binding domain. The repression domain may have any structure as long as it can suppress the antigen-binding activity of the antigen-binding domain. Examples of such repression domains include, but are not limited to, antibody heavy chain variable regions (VH), antibody light chain variable regions (VL), pre-B cell receptors, and single-domain antibodies. The repression domain may comprise the entire delivery moiety or a portion of the delivery moiety.

[0138] When a polypeptide of the present disclosure comprises an antigen-binding domain and a transport moiety, in one embodiment, the inhibition of the antigen-binding activity of the antigen-binding domain by the repression domain when the protease cleavage sequence is cleaved by a protease is weaker than the inhibition of the antigen-binding activity of the antigen-binding domain when the protease cleavage sequence is uncleaved.

[0139] When a polypeptide of the disclosure comprises an antigen-binding domain and a delivery moiety, in some embodiments, the antigen-binding domain has a shorter serum half-life than the uncleaved polypeptide. When a polypeptide of the disclosure comprises an antigen-binding domain and a delivery moiety, in some embodiments, the antigen-binding domain has a shorter serum half-life than the delivery moiety.

[0140] Where a polypeptide of the disclosure comprises an antigen-binding domain and a transport moiety, in some embodiments, the molecular weight of the antigen-binding domain is less than the molecular weight of the transport moiety, hi some embodiments, the molecular weight of the antigen-binding domain can be 60 kDa or less.

[0141] When a polypeptide of the disclosure comprises an antigen-binding domain and a transport moiety, in one embodiment, 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 a polypeptide of the present disclosure comprises an antigen-binding domain and a delivery moiety, in some embodiments, the polypeptide comprising the antigen-binding domain and delivery moiety has a longer serum half-life than the antigen-binding domain present alone. To extend the half-life of the polypeptide, when a polypeptide of the present disclosure comprises an antigen-binding domain and a delivery moiety, in some embodiments, the delivery moiety is designed to have a longer serum half-life. Examples of embodiments that extend the serum half-life of the delivery moiety include, but are not limited to, a delivery moiety with a larger molecular weight, FcRn-binding activity, albumin-binding activity, or PEGylation. Furthermore, when a polypeptide of the present disclosure comprises an antigen-binding domain and a delivery moiety, in some embodiments, the delivery moiety has a longer serum half-life than the antigen-binding domain (i.e., the antigen-binding domain has a shorter serum half-life than the delivery moiety).

[0143] In the present disclosure, when comparing the half-life of an antigen-binding domain alone with that of a polypeptide, or the half-life of an antigen-binding domain with a delivery moiety, it is preferable to compare the half-life in humans. If it is difficult to measure the half-life in humans, the half-life in humans can be predicted based on the 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.).

[0144] In one embodiment, the serum half-life of the delivery moiety is increased by increasing the molecular weight of the delivery moiety. In one embodiment, the serum half-life of the delivery moiety is increased relative to the serum half-life of the antigen-binding domain by increasing the molecular weight of the delivery moiety relative to the molecular weight of the antigen-binding domain.

[0145] One embodiment for extending the blood half-life of a delivery moiety is to confer FcRn-binding ability to the delivery moiety, which can usually be achieved by incorporating an FcRn-binding region into the delivery moiety.

[0146] Having FcRn-binding ability in the delivery moiety does not mean that the antigen-binding domain does not have FcRn-binding ability. In an embodiment in which the blood half-life of the delivery moiety is made longer than that of the antigen-binding domain, the antigen-binding domain may not have FcRn-binding ability, or even if the antigen-binding domain has FcRn-binding ability, it may have weaker FcRn-binding ability than the delivery moiety.

[0147] One embodiment for extending the blood half-life of a delivery moiety is to conjugate the delivery moiety to albumin. Albumin is not excreted renally and has FcRn-binding activity, resulting in a long blood half-life of 17 to 19 days (J Clin Invest. 1953 Aug; 32(8): 746-768.). It has been reported that proteins bound to albumin become bulky and are able to indirectly bind to FcRn, thereby increasing their blood half-life (Antibodies 2015, 4(3), 141-156).

[0148] Furthermore, one embodiment for extending the blood half-life of a delivery moiety is to PEGylate the delivery moiety. PEGylation of a protein increases the protein's bulkiness and simultaneously inhibits its degradation by proteases in the blood, which is thought to extend the blood half-life of the protein (J Pharm Sci. 2008 Oct;97(10):4167-83.).

[0149] When a polypeptide of the present disclosure comprises an antigen-binding domain and a transport moiety, in some embodiments, the transport moiety comprises an antibody Fc region. In one specific embodiment, the transport moiety comprises the CH2 and CH3 domains of a human IgG antibody. In one specific embodiment, the transport moiety comprises a portion of a human IgG1 antibody heavy chain extending from Cys226 or Pro230 to the carboxyl terminus of the heavy chain, with the proviso that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region may or may not be present.

[0150] Where a polypeptide of the disclosure comprises an antigen-binding domain and a transport moiety, in one embodiment, the transport moiety comprises an antibody constant region. In a more preferred embodiment, the transport moiety comprises an IgG antibody constant region. In a more preferred embodiment, the transport moiety comprises a human IgG antibody constant region.

[0151] When a polypeptide of the present disclosure comprises an antigen-binding domain and a transport moiety, in one embodiment, the transport moiety comprises a region having a structure substantially similar to that of an antibody heavy chain constant region, and a region having a structure substantially similar to that of an antibody light chain, which is bound to the antigen-binding domain by covalent bonds, such as disulfide bonds, or non-covalent bonds, such as hydrogen bonds or hydrophobic interactions.

[0152] When a polypeptide of the present disclosure comprises an antigen-binding domain and a delivery moiety, in some embodiments, the antigen-binding domain is releasable from the polypeptide, and the antigen-binding activity of the antigen-binding domain when released from the polypeptide is higher than when not released from the polypeptide.

[0153] Where a polypeptide of the disclosure comprises an antigen-binding domain and a transport moiety, in some embodiments, the protease cleavage sequence is cleaved by a protease, allowing the antigen-binding domain to be released from the polypeptide.

[0154] When a polypeptide of the present disclosure comprises an antigen-binding domain and a delivery moiety, in certain embodiments, the antigen-binding activity of the antigen-binding domain increases when released from the polypeptide compared to before release. In other words, when the antigen-binding domain is not released from the polypeptide, its antigen-binding activity is inhibited by the inhibition domain. Methods for confirming that the antigen-binding activity of the antigen-binding domain is inhibited by the inhibition domain include fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), electrogenerated chemiluminescence (ECL), surface plasmon resonance (SPR) (Biacore), and bio-layer interferometry (BLI) (Octet). When a polypeptide of the present disclosure comprises an antigen-binding domain and a delivery moiety, in some embodiments, the antigen-binding activity of the antigen-binding domain when released 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 when the antigen-binding domain is not released from the polypeptide. When a polypeptide of the present disclosure comprises an antigen-binding domain and a delivery moiety, in some embodiments, no binding between the antigen-binding domain and the antigen is observed when the antigen-binding activity of the antigen-binding domain before release is measured by one of the methods described above. When a polypeptide of the present disclosure comprises an antigen-binding domain and a delivery moiety, in certain embodiments, the antigen-binding domain can be released from the polypeptide by cleavage of the protease cleavage sequence, and in such embodiments, the antigen-binding activity can be compared by comparing the antigen-binding activity of the polypeptide before and after cleavage. That is, the antigen-binding activity measured using the cleaved polypeptide will be 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 more specific embodiments, when the antigen-binding activity of the uncleaved polypeptide is measured by one of the above methods, no binding between the antigen-binding domain and the antigen is observed. When a polypeptide of the present disclosure comprises an antigen-binding domain and a delivery moiety, in certain embodiments, the protease cleavage sequence is cleaved by a protease, and therefore, in such embodiments, antigen-binding activity can be compared by comparing the antigen-binding activity of the polypeptide before and after protease treatment. That is, the antigen-binding activity measured using the protease-treated polypeptide will be 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 more specific embodiments, when the antigen-binding activity of a polypeptide that has not been treated with a protease is measured by a method selected from the above methods, no binding between the antigen-binding domain and the antigen is observed.

[0155] When a polypeptide of the disclosure comprises an antigen-binding domain and a delivery moiety, in some embodiments, the antigen-binding activity of the antigen-binding domain is inhibited by association of the antigen-binding domain with the delivery moiety's repression domain.

[0156] When a polypeptide of the disclosure comprises an antigen-binding domain and a transport moiety, in one embodiment, cleavage of the protease cleavage sequence by a protease disrupts the association of the antigen-binding domain with the repression domain of the transport moiety.

[0157] When a polypeptide of the present disclosure comprises an antigen-binding domain and a delivery moiety, in one embodiment, the antigen-binding domain comprises or is a single-domain antibody, and the repression domain of the delivery moiety represses the antigen-binding activity of the single-domain antibody. The single-domain antibody may be a VHH, a single-domain VH with antigen-binding activity, or a single-domain VL with antigen-binding activity.

[0158] When a polypeptide of the present disclosure comprises an antigen-binding domain and a delivery moiety, in some embodiments, the repression domain of the delivery moiety is associated with the antigen-binding domain. The repression domain may be part of the delivery moiety or the entire delivery moiety. From another perspective, the portion of the delivery moiety that is associated with the antigen-binding domain may be referred to as the repression domain. In a more specific embodiment, the antigen-binding domain of a single-domain antibody and the repression domain of VL, VH, or VHH form an association similar to that of antibody VH and antibody VL. In an even more specific embodiment, the antigen-binding domain of a single-domain antibody and the repression domain of VL, VH, or VHH form an association similar to that of antibody VH and antibody VL. Once this association is formed, the repression domain conformationally inhibits the binding of the antigen-binding domain to the antigen, or alters the conformation of the antigen-binding site of the antigen-binding domain, thereby inhibiting the antigen-binding activity of the single-domain antibody by the VL, VH, or VHH. In an embodiment using a VHH as the single-domain antibody, if the CDR3, the primary antigen-binding site of the VHH, or a site nearby it, is present at the interface where it associates with the repression domain, it is believed that the repression domain conformationally inhibits the binding of the VHH to the antigen. Furthermore, the association between the inhibitory domain and the antigen-binding domain can be dissolved, for example, by cleaving the cleavage site. Dissolution of the association can be expressed, for example, as dissolution of the interaction between two or more polypeptide regions. The interaction between the two or more polypeptide regions may be completely dissolved, or only part of the interaction between the two or more polypeptide regions may be dissolved.

[0159] As used herein, the term "interface" generally refers to the surface at which association (interaction) occurs, and the amino acid residues that form the interface generally refer to one or more amino acid residues contained in the polypeptide region involved in the association, more preferably amino acid residues that come close to each other during association and are involved in the interaction. Specific examples of such interactions include non-covalent bonds such as hydrogen bonds, electrostatic interactions, and salt bridges formed between amino acid residues that come close to each other during association.

[0160] As used herein, "amino acid residues forming an interface" refers, more specifically, to amino acid residues contained in a polypeptide region that constitutes the interface. The polypeptide region that constitutes the interface refers, for example, to a polypeptide region that is responsible for selective intramolecular or intermolecular binding in antibodies, ligands, receptors, substrates, etc. Specific examples of such regions include heavy chain variable regions and light chain variable regions in antibodies. When the polypeptide of the present disclosure comprises an antigen-binding domain and a transporter moiety, examples of such regions include an antigen-binding domain and an inhibitory domain in certain embodiments. Examples of amino acid residues that form an interface include, but are not limited to, amino acid residues that come close to each other during association. Amino acid residues that come close to each other during association can be found, for example, by analyzing the three-dimensional structure of a polypeptide and examining the amino acid sequence of the polypeptide region that forms an interface during association of the polypeptide.

[0161] When a polypeptide of the present disclosure comprises an antigen-binding domain and a delivery moiety, in certain embodiments, amino acid residues in the antigen-binding domain involved in the association or in the repression domain involved in the association can be modified to promote the association of the antigen-binding domain and the repression domain. In a more specific embodiment, amino acid residues in the antigen-binding domain that form an interface with the repression domain or amino acid residues in the repression domain that form an interface with the antigen-binding domain can be modified. In a preferred embodiment, the modification of amino acid residues that form the interface involves introducing mutations into the interface so that two or more amino acid residues forming the interface have different charges. Modifications of amino acid residues that result in different charges include modifying a positively charged amino acid residue to a negatively charged or uncharged amino acid residue, modifying a negatively charged amino acid residue to a positively charged or uncharged amino acid residue, and modifying an uncharged amino acid residue to a positively or negatively charged amino acid residue. Such amino acid modifications are intended to promote association, and the position and type of amino acid modified are not limited as long as the purpose of promoting association is achieved. Modifications include, but are not limited to, substitutions.

[0162] When a polypeptide of the present disclosure comprises an antigen-binding domain and a delivery moiety, in one embodiment, the antigen-binding domain, VHH, is associated with the inhibitory domain, VL. Examples of amino acid residues in VHH involved in the association with VL include amino acid residues that form the interface between VHH and VL. Examples of amino acid residues in VHH involved in the association with VL include, but are not limited to, amino acid residues at positions 37, 44, 45, and 47 (J. Mol. Biol. (2005) 350, 112-125). Promotion of the association between VHH and VL inhibits the activity of VHH. Similarly, examples of amino acid residues in VL involved in the association with VHH include amino acid residues that form the interface between VHH and VL.

[0163] To promote the association of VHH with VL, amino acid residues in the VHH involved 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, it is also possible to use a VHH that originally has amino acid residues 37V, 44G, 45L, and / or 47W without modifying any of the residues in the VHH. Furthermore, as long as the goal of promoting the association between VHH and VL is achieved, it is possible to modify the amino acid residues in VL involved in the association with VHH rather than the amino acids in VHH, and it is also possible to introduce amino acid modifications into both VHH and VL.

[0164] When a polypeptide of the present disclosure comprises an antigen-binding domain and a delivery moiety, in one embodiment, a VHH is used as the antigen-binding domain and a VH or VHH is used as the repression domain, allowing the antigen-binding domain and the repression domain to associate. To promote the association between the VHH antigen-binding domain and the VH or VHH repression domain, amino acid residues in the VHH antigen-binding domain that are involved in the association with the VH or VHH repression domain can be identified and modified. Alternatively, amino acid residues in the VH or VHH repression domain that are involved in the association with the VHH antigen-binding domain can be identified and modified.

[0165] Furthermore, when using a single-domain antibody other than VHH as the antigen-binding domain, it is also possible to identify amino acid residues in the antigen-binding domain or the inhibitory domain that are involved in the association and modify those amino acid residues.

[0166] When a polypeptide of the present disclosure comprises an antigen-binding domain and a delivery moiety, in one embodiment, the delivery moiety and the antigen-binding domain are fused via a linker. In a more specific embodiment, the delivery moiety and the antigen-binding domain are fused via a linker that comprises a protease cleavage sequence. In another specific embodiment, the delivery moiety and the antigen-binding domain are fused via a linker, and the resulting fusion protein comprises a protease cleavage sequence.

[0167] When a polypeptide of the present disclosure comprises an antigen-binding domain and a delivery moiety, in some embodiments, the delivery moiety and the antigen-binding domain are fused without a linker. In a more specific embodiment, an amino acid bond is formed between the N-terminal amino acid of the delivery moiety and the C-terminal amino acid of the antigen-binding domain to form a fusion protein. The resulting fusion protein contains a protease cleavage sequence. In certain embodiments, one to several amino acids at the N-terminus of the delivery moiety and / or one to several amino acids at the C-terminus of the antigen-binding domain are modified to fuse the N-terminus of the delivery moiety and the C-terminus of the antigen-binding domain, thereby forming a protease cleavage sequence near the fusion site. More specifically, for example, when LSGRSDNH (SEQ ID NO: 18031) is used as the protease cleavage sequence, the four C-terminal amino acids of the antigen-binding domain can be replaced with the LSGR sequence and the four N-terminal amino acids of the delivery moiety can be replaced with the SDNH sequence to form a protease cleavage sequence.

[0168] When a polypeptide of the present disclosure comprises an antigen-binding domain and a transport moiety, the protease cleavage sequence may be located anywhere in the polypeptide, as long as it releases the antigen-binding domain upon cleavage by a protease and does not lose the antigen-binding activity of the released antigen-binding domain.

[0169] When a polypeptide of the present disclosure comprises an antigen-binding domain and a transport moiety, in one embodiment, the transport moiety comprises an antibody constant region, and the N-terminus of the antibody constant region is fused to the C-terminus of the antigen-binding domain with or without a linker. In certain embodiments, the protease cleavage sequence is located within the antibody constant region comprised in the delivery moiety. In this case, the protease cleavage sequence may be located within the antibody constant region such that the antigen-binding domain can be released upon protease cleavage. In a specific embodiment, the protease cleavage sequence is located within the antibody heavy chain constant region comprised in the delivery moiety, more specifically, on the antigen-binding domain side of amino acid 140 (EU numbering) in the antibody heavy chain constant region, or on the antigen-binding domain side of amino acid 122 (EU numbering) in the antibody heavy chain constant region. In another specific embodiment, the protease cleavage sequence is located within the antibody light chain constant region comprised in the delivery moiety, more specifically, 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.

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

[0171] When a polypeptide of the present disclosure comprises an antigen-binding domain and a transport moiety, in one embodiment, the transport moiety comprises an antibody constant region, and the antigen-binding domain is a single-domain antibody, and the antibody constant region and the single-domain antibody are fused with or without a linker. In a more specific embodiment, the N-terminus of the antibody constant region is fused with or without a linker to the C-terminus of the single-domain antibody. In another specific embodiment, the C-terminus of the antibody constant region is fused with or without a linker to the N-terminus of the single-domain antibody. In certain embodiments, the protease cleavage sequence is located in the antibody constant region comprised in the delivery moiety. In more specific embodiments, the protease cleavage sequence is located in the antibody heavy chain constant region closer to the single domain antibody than amino acid 140 (EU numbering) or closer to the single domain antibody than amino acid 122 (EU numbering) in the antibody heavy chain constant region. In another specific embodiment, the protease cleavage sequence is located in the antibody light chain constant region closer to the antigen-binding domain than amino acid 130 (Kabat numbering) or closer to the antigen-binding domain than 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 or VHH constructed from a VH, and the protease cleavage sequence is located closer to the antibody constant region of the single-domain antibody than amino acid 35b (Kabat numbering), or closer to the antibody constant region of the single-domain antibody than amino acid 95 (Kabat numbering), or closer to the antibody constant region of the single-domain antibody than amino acid 109 (Kabat numbering). In another specific embodiment, the single-domain antibody is a single-domain antibody constructed from a VL, and the protease cleavage sequence is located closer to the antibody constant region of the single-domain antibody than amino acid 32 (Kabat numbering), or closer to the antibody constant region of the single-domain antibody than amino acid 91 (Kabat numbering), or closer to the antibody constant region of the single-domain antibody than amino acid 104 (Kabat numbering). In certain embodiments, the protease cleavage sequence is located near the interface between the antigen-binding domain and the transport moiety, which refers to the area around the site where the antigen-binding domain and the transport moiety are joined, that does not significantly affect the secondary structure of the antigen-binding domain. In more specific embodiments, the antigen-binding domain is linked to an antibody constant region contained in the transfer moiety, and the protease cleavage sequence is located near the interface between the antigen-binding domain and the antibody constant region. Near the interface between the antigen-binding domain and the antibody constant region can refer to near the interface between the antigen-binding domain and the antibody heavy chain constant region, or near the interface between the antigen-binding domain and the antibody light chain constant region. When the antigen-binding domain is a single-domain antibody or VHH constructed from a VH and is linked to an antibody heavy chain constant region, near the interface between the antigen-binding domain and the antibody constant region can refer to the region between amino acid 101 (Kabat numbering) of the single-domain antibody and amino acid 140 (EU numbering) of the antibody heavy chain constant region, or the region between amino acid 109 (Kabat numbering) of the single-domain antibody and amino acid 122 (EU numbering) of the antibody heavy chain constant region. When the antigen-binding domain is a single-domain antibody or VHH prepared from VH and is linked to an antibody light-chain constant region, the vicinity of the boundary between the antigen-binding domain and the antibody light-chain constant region can refer to the region between amino acid 101 (Kabat numbering) for single-domain antibodies and amino acid 130 (Kabat numbering) for antibody light-chain constant regions, or the region between amino acid 109 (Kabat numbering) for single-domain antibodies and amino acid 113 (Kabat numbering) for antibody light-chain constant regions. When 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 refers to the region from amino acid 96 (Kabat numbering) for single-domain antibodies or from amino acid 104 (Kabat numbering) for single-domain antibodies.

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

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

[0174] When a polypeptide of the present disclosure comprises an antigen-binding domain and a delivery moiety, the polypeptide may contain one or more antigen-binding domains. The repression domain that represses the antigen-binding activity of each of the multiple antigen-binding domains may also be one or more. Each of the multiple antigen-binding domains may be associated with a repression domain. Each of the multiple antigen-binding domains may be fused to a delivery moiety. Each of the multiple antigen-binding domains may be releasable from the polypeptide. There may be multiple protease cleavage sequences for releasing the multiple antigen-binding domains, each corresponding to one of the antigen-binding domains.

[0175] When the polypeptide is an IgG antibody-like molecule, an embodiment in which an antigen-binding domain is provided in each of the regions corresponding to the two variable regions of an IgG antibody, as shown in Figure 3, would be an embodiment that would be understood by a person skilled in the art after reading this disclosure. Whether the antigen-binding domains incorporated into both arms have the same antigen-binding specificity or different antigen-binding specificities, this is an embodiment that would naturally be understood by a person skilled in the art after reading this disclosure, and it is clear that this does not deviate from the scope of the present disclosure.

[0176] Where a polypeptide of the disclosure comprises 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, a single-domain antibody, an antibody fragment, a module called an A domain of about 35 amino acids contained in Avimer, a cell membrane protein present in vivo (International Publication Nos. WO2004 / 044011 and WO2005 / 040229), an Adnectin containing the 10Fn3 domain, which is a domain that binds to a protein in fibronectin, a glycoprotein expressed on the cell membrane (International Publication No. WO2002 / 032925), an Affibody using an IgG-binding domain composed of a 58-amino acid three-helix bundle of Protein A as a scaffold (International Publication No. WO1995 / 001937), and a DARPins (Designed Ankyrin Repeat (AR)) region exposed on the molecular surface of ankyrin repeats (AR) having a structure in which a 33-amino acid residue turn, two antiparallel helices, and a loop subunit are repeatedly stacked. Examples of such a domain include an anticalin domain, which is a four-loop region supporting one side of a barrel structure in which eight antiparallel strands are highly conserved in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) (International Publication WO 2003 / 029462), and a concave region of a parallel sheet structure within a horseshoe-shaped structure in which leucine-rich-repeat (LRR) modules are repeatedly stacked in the variable lymphocyte receptor (VLR) that does not have an immunoglobulin structure and is part of the adaptive immune system of jawless fish such as lampreys and hagfish (International Publication WO 2008 / 016854). 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 weight of the linked antigen-binding domain and the second antigen-binding domain is 60 kDa or less. In some more specific embodiments, the antigen-binding domain and the second antigen-binding domain are single-domain antibodies with different antigen-binding specificities, and the linked antigen-binding domain and the second antigen-binding domain can be released 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, bispecific antigen-binding molecules 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, bispecific antigen-binding molecules in which the antigen-binding domain and the second antigen-binding domain bind to different subunits of the same antigen, and bispecific antigen-binding molecules in which the antigen-binding domain and the second antigen-binding domain bind to different epitopes of the same antigen. Such bispecific antigen-binding molecules are thought to be useful in the treatment of diseases caused by target cells, as they can recruit immune cells to the vicinity of the target cells. The antigen-binding activity of the second antigen-binding domain may or may not be inhibited by the delivery moiety. Furthermore, the second antigen-binding domain may or may not associate with a partial structure of the delivery moiety. In particular, when the antigen-binding domain and the second antigen-binding domain have different antigen-binding specificities, for example, as shown in Figure 4, even if the antigen-binding activity of the second antigen-binding domain is not inhibited or even if the second antigen-binding domain does not associate with a partial structure of the delivery moiety, the antigen-binding activity of the antigen-binding domain cannot be exhibited in an unreleased state, and a bispecific antigen-binding molecule in which the antigen-binding domain and the second antigen-binding domain are linked cannot exhibit the function of bispecifically binding to two types of antigens. FIG. 4 illustrates one embodiment in which the antigen-binding domain is further linked to a second antigen-binding domain.

[0177] As used herein, the term "specificity" refers to the property of one of two specifically binding molecules not substantially binding to any other molecules than the one or more other molecules to which it binds. This term 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 particular epitope among multiple epitopes contained in an antigen. Here, "not substantially binding" is determined according to the method described in the section on binding activity, and refers to the binding activity of a specific binding molecule for molecules other than the other molecule being 80% or less, typically 50% or less, preferably 30% or less, and particularly preferably 15% or less of its binding activity for the other molecule.

[0178] A sequence represented by any one of SEQ ID NOs: 5 to 18003, or a partial sequence contained in such a sequence that can be used as a protease cleavage sequence (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, the sequence from the 3rd to 11th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; the sequence from the 3rd to 10th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; the sequence from the 3rd to 14th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; the sequence from the 5th to 12th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; and the sequence from the 5th to 10th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003) can be used as protease cleavage sites in the polypeptides exemplified in FIG. A sequence consisting of "a sequence selected from Group A below - a sequence selected from Group B below" in this order from the N-terminus can also be used as a protease cleavage site in the polypeptides exemplified in Figure 1: (Group A) A sequence from the first amino acid to the ninth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the second amino acid to the ninth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 3rd amino acid to the 9th 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 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 5th amino acid to the 9th 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 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 7th amino acid to the 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 8th amino acid to the 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the first amino acid to the sixth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the second amino acid to the sixth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the third amino acid to the sixth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 4th amino acid to the 6th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 5th amino acid to the 6th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the first amino acid to the eighth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the second amino acid to the eighth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the third amino acid to the eighth amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 4th amino acid to the 8th 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 8th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 6th amino acid to the 8th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 7th to 8th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; (Group B) A sequence from the 10th 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 10th amino acid to the 14th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 10th 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 10th amino acid to the 12th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 10th amino acid to the 11th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 5 to 17201; A sequence from the 7th amino acid to the 15th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 7th amino acid to the 14th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 7th amino acid to the 13th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 7th 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 7th 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 7th 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 7th amino acid to the 9th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 7th amino acid to the 8th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17202 to 17993; A sequence from the 9th amino acid to the 15th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 9th 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 9th amino acid to the 13th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 9th 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 9th amino acid to the 11th amino acid at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003; A sequence from the 9th to 10th amino acids at the N-terminus of a sequence selected from SEQ ID NOs: 17994 to 18003. The molecule shown in Figure 1 shows that the polypeptide containing the antigen-binding domain and the carrier moiety has a long half-life, the antigen-binding activity of the antigen-binding domain is suppressed, and it does not bind to antigen (A). After the antigen-binding domain is released, the antigen-binding activity is restored and the half-life is short (B).

[0179] The polypeptide shown in Figure 1 can be varied in many ways, but when using an IgG antibody-like molecule, it can be produced by the production method exemplified in Figure 2. First, a single-domain antibody (e.g., VH or VHH) that binds to the target antigen is obtained (A). The obtained single-domain antibody is swapped with one of the VH and VL of an IgG antibody having a germline sequence, and then associated with the other VH and VL to form an IgG antibody-like molecule (B). A protease cleavage sequence is introduced into the IgG antibody-like molecule (C). An example of the position for introduction is near the boundary between the introduced single-domain antibody (VH or VHH) and the constant region (CH1 or CL). Single-domain antibodies possess antigen-binding activity when present as a single domain, but lose this activity when forming a variable region with VL / VH / VHH or other components. Because VL / VH are naturally occurring human antibody sequences with germline sequences, the risk of immunogenicity is low, and the likelihood of anti-drug antibodies recognizing the VL / VH is extremely low. Furthermore, when forming a variable region with a single-domain antibody using VHH, humanizing the VHH reduces the risk of immunogenicity and the likelihood of anti-drug antibodies recognizing the humanized VHH. Single-domain antibodies are released by protease cleavage of the protease cleavage sequence inserted into the IgG antibody-like molecule. The released single-domain antibodies retain antigen-binding activity. IgG antibody-like molecules before protease cleavage have a structure similar to that of typical IgG molecules and therefore have a long circulation. However, single-domain antibodies released by protease cleavage lack an Fc region and have a molecular weight of approximately 13 kDa, resulting in rapid renally excretion. In fact, the half-life of full-length IgG is about 2-3 weeks (Blood. 2016 Mar 31;127(13):1633-41.), while the half-life of single-domain antibodies is about 2 hours (Antibodies 2015, 4(3), 141-156.) Therefore, antigen-binding molecules activated by proteases have a short half-life in the blood and are less likely to bind to antigens in normal tissues. When the single domain antibody is a VL, a similar concept can be achieved by introducing a protease cleavage sequence, for example, near the interface between the VL and CL.

[0180] The present disclosure also relates to methods of producing polypeptides where the polypeptides comprise a delivery moiety having a repression domain and an antigen-binding domain. One method for producing a polypeptide comprising an antigen-binding domain and a delivery moiety having a repression domain of the present disclosure is to obtain an antigen-binding domain with antigen-binding activity, link the antigen-binding domain to the delivery moiety to form a polypeptide precursor such that the antigen-binding activity of the antigen-binding domain is repressed by the repression domain, and then insert a protease cleavage sequence into the polypeptide precursor or modify a portion of the polypeptide precursor with a protease cleavage sequence. It is sufficient to introduce a protease cleavage sequence into the polypeptide precursor, and the method for introducing the protease cleavage sequence may be either by inserting a protease cleavage sequence or by modifying a portion of the polypeptide precursor. Furthermore, it will be clear to those skilled in the art from reading this specification that both methods can be combined to introduce a protease cleavage sequence into the polypeptide precursor, and this would not depart from the scope of the present disclosure. Another method for producing a polypeptide comprising an antigen-binding domain and a delivery moiety having a repression domain of the present disclosure involves obtaining an antigen-binding domain with antigen-binding activity and linking the antigen-binding domain and delivery moiety via a protease cleavage sequence to form a polypeptide such that the antigen-binding activity of the antigen-binding domain is repressed by the repression domain. When the antigen-binding domain and delivery moiety are linked via a protease cleavage sequence, the protease cleavage sequence may be sandwiched between the antigen-binding domain and delivery moiety, or a portion of the antigen-binding domain and / or a portion of the delivery moiety may be modified and used as part of the protease cleavage sequence.

[0181] For embodiments using a single domain antibody as the antigen binding domain, the following describes a method for producing a polypeptide comprising a delivery moiety having a repression domain and an antigen binding domain.

[0182] In one embodiment of the present disclosure, a method for producing a polypeptide comprising a delivery moiety having a repression domain and an antigen binding domain comprises the steps of: (a) obtaining a single domain antibody that binds to a target antigen; (b) linking the single domain antibody obtained in step (a) to the delivery moiety to form a polypeptide precursor such that the antigen-binding activity of the single domain antibody is inhibited by the inhibition domain of the delivery moiety; (c) introducing a protease cleavage sequence into the polypeptide precurs...

Claims

1. A method for producing a protease substrate, the method comprising the step of providing a protease substrate comprising an amino acid sequence represented by GGSXXXXXRSANPRG (SEQ ID NO: 18079), TSTSGRXXXXXGGGS (SEQ ID NO: 18080), or GGGSXXXRXXGGGSG (SEQ ID NO: 18081) (wherein X represents any of the 17 naturally occurring amino acids other than C, R, or K).

2. 10. The method of claim 1, (i) providing a plurality of protease substrates comprising the amino acid sequence GGSXXXXXRSANPRG (SEQ ID NO: 18079), where X is any of the 17 naturally occurring amino acids other than C, R, and K; (ii) treating some or all of the protease substrate with human matriptase (hMT-SP1); and (iii) treating some or all of the protease substrate with human urokinase (huPA); and (iv) selecting a protease substrate that has a higher cleavage rate with each protease than a protease substrate containing the amino acid sequence shown in SEQ ID NO: 2; A method comprising:

3. 3. The method of claim 2, (i) treating some or all of the protease substrate with mouse urokinase (muPA); and (ii) selecting a protease substrate that has a higher cleavage rate with muPA than a protease substrate comprising the amino acid sequence set forth in SEQ ID NO: 2; A method comprising:

4. 4. The method of claim 2 or 3, (i) treating some or all of the protease substrate with human serum; and (ii) selecting a protease substrate that is cleaved at a lower rate by human serum than a protease substrate comprising the amino acid sequence set forth in SEQ ID NO: 4; A method comprising:

5. 10. The method of claim 1, (i) providing a plurality of protease substrates comprising the amino acid sequence TSTSGRXXXXXGGGS (SEQ ID NO: 18080) or GGGSXXXRXXGGGSG (SEQ ID NO: 18081) (wherein X is any of the 17 naturally occurring amino acids other than C, R, or K); (ii) treating some or all of the protease substrate with human matriptase (hMT-SP1); and (iii) treating some or all of the protease substrate with human urokinase (huPA); and (iv) selecting a protease substrate that has a higher cleavage rate with each protease than the protease substrate containing the amino acid sequence shown in SEQ ID NO: 3; A method comprising:

6. 6. The method of claim 5, (i) treating some or all of the protease substrate with mouse urokinase (muPA); and (ii) selecting a protease substrate that has a higher cleavage rate by muPA than a protease substrate comprising the amino acid sequence set forth in SEQ ID NO: 3; A method comprising:

7. 7. The method of claim 5 or 6, (i) treating some or all of the protease substrate with human serum; and (ii) selecting a protease substrate that is cleaved at a lower rate by human serum than a protease substrate comprising the amino acid sequence set forth in SEQ ID NO: 4; A method comprising:

8. A protease substrate produced by the method of any one of claims 1 to 7.

9. A polypeptide comprising a protease substrate produced by the method of any one of claims 1 to 7.

10. 10. The polypeptide of claim 9, (i) a polypeptide comprising an antigen-binding domain and a transport moiety, the transport moiety having a repression domain that represses the antigen-binding activity of the antigen-binding domain; (ii) the polypeptide is a ligand-binding molecule capable of binding to a ligand, wherein the binding of the ligand-binding molecule to the ligand when the protease substrate is cleaved is weaker than the binding of the ligand-binding molecule to the ligand when the protease substrate is uncleaved; or (iii) The polypeptide is a ligand-binding molecule capable of binding to a ligand, the ligand-binding molecule comprising a single-domain antibody, the single-domain antibody capable of binding to a ligand, and having at least one protease substrate introduced therein, such that binding of the ligand-binding molecule to the ligand in a state in which the protease substrate is cleaved is weakened compared to binding of the ligand-binding molecule to the ligand in a state in which the protease substrate is not cleaved.

11. A fusion protein formed from a polypeptide described in claim 9 or 10 and a ligand, wherein the polypeptide is a ligand-binding molecule capable of binding to the ligand, and the ligand-binding molecule is fused to the ligand.

12. A complex formed by a polypeptide according to claim 9 or 10 and a ligand, wherein the polypeptide is a ligand-binding molecule capable of binding to the ligand, and the ligand-binding molecule is bound to the ligand.

13. A pharmaceutical composition comprising a polypeptide and a ligand according to claim 9 or 10, a fusion protein according to claim 11, or a complex according to claim 12.

14. 14. Use of the pharmaceutical composition of claim 13 in the manufacture of a medicament for treating a disease, wherein the disease is characterized in that the protease is expressed or activated in diseased tissue or cells in diseased tissue.

15. Use of a protease substrate described in claim 8, a polypeptide described in claim 9 or 10, a fusion protein described in claim 11, or a complex described in claim 12 in a method for detecting the presence or absence of a protease, comprising subjecting one or more of the protease substrates, polypeptides, fusion proteins, or complexes to protease treatment and detecting their cleavage.

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