Polypeptides Comprising an Antigen-Binding Domain and a Transport Moiety

Abstract

To provide pharmaceutical compositions and active ingredients that have reduced side effects and are suitable for the treatment of disease, and methods for producing them.SOLUTION: The present invention relates to polypeptides containing an antigen-binding domain and a carrying moiety having an inhibiting domain that inhibits the antigen-binding activity of the antigen-binding domain, where the polypeptides comprise a protease cleavage sequence comprising one or more sequences selected from a specific sequence group, and where the polypeptides have a longer half-life than that of the antigen-binding domain existing alone; methods for producing and screening for the polypeptides; pharmaceutical compositions containing the polypeptide; methods for producing and screening for a single-domain antibody whose antigen-binding activity is inhibited by its association with specific VL, VH or VHH; and fusion polypeptide libraries including a single-domain antibody whose antigen-binding activity is inhibited by its association with specific VL, VH or VHH.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present invention relates to polypeptides that comprise an antigen-binding domain and a delivery moiety having an inhibition domain that inhibits the antigen-binding activity of the antigen-binding domain, and that have a longer half-life than the antigen-binding domain present alone; methods for producing and screening for such polypeptides; pharmaceutical compositions comprising such polypeptides; methods for producing and screening for single-domain antibodies whose antigen-binding activity is inhibited upon association with a specific VL / VH / VHH; and libraries of fusion polypeptides comprising single-domain antibodies whose antigen-binding activity is inhibited upon association with a specific VL / VH / VHH. [Background technology]

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

[0003] Approved antibody-based cancer therapeutics include Rituxan, which targets the CD20 antigen, cetuximab, which targets the EGFR antigen, and Herceptin, which targets the HER2 antigen (Non-Patent Document 3). These antibody molecules bind to antigens expressed on cancer cells and exert cytotoxic activity against cancer cells through ADCC activity and other means. It is known that cytotoxic activity through ADCC and other means depends on the number of antigens expressed on the target cells of the therapeutic antibody (Non-Patent Document 4). Therefore, a high expression level of the target antigen is preferable from the perspective of the efficacy of the therapeutic antibody. However, even if the expression level of the antigen is high, if the antigen is expressed in normal tissues, ADCC and other cytotoxic activity will be exerted against normal cells, resulting in serious side effects. Therefore, it is preferable that the antigen targeted by a therapeutic antibody as a cancer therapeutic drug is specifically expressed on cancer cells. For example, antibody molecules against the EpCAM antigen, which is known as a cancer antigen, were thought to be promising as cancer treatment drugs, but the EpCAM antigen is also known to be expressed in the pancreas, and in fact, in clinical trials, it has been reported that administration of anti-EpCAM antibodies causes the side effect of pancreatitis due to cytotoxic activity against the pancreas (Non-Patent Document 5).

[0004] Following the success of antibody pharmaceuticals that exert cytotoxic activity through ADCC activity, second-generation improved antibody molecules that exert potent cytotoxic activity have been reported, such as by enhancing ADCC activity by removing fucose from the N-glycosylation chains in the Fc region of native human IgG1 (Non-Patent Document 6) and by enhancing binding to FcγRIIIa through amino acid substitution in the Fc region of native human IgG1 (Non-Patent Document 7). As antibody pharmaceuticals that exert cytotoxic activity against cancer cells by mechanisms other than the above-mentioned NK cell-mediated ADCC activity, improved antibody molecules that exert even more potent cytotoxic activity have also been reported, such as antibody drug conjugates (ADCs) in which antibodies are conjugated with drugs that have potent cytotoxic activity (Non-Patent Document 8), and small molecule antibodies that exert cytotoxic activity against cancer cells by recruiting T cells to the cancer cells (Non-Patent Document 9).

[0005] While these antibody molecules exhibit more potent cytotoxic activity, they can also exert cytotoxic activity against cancer cells with low antigen expression, they also exert cytotoxic activity against normal tissues with low antigen expression, similar to that observed in cancer cells. In fact, compared to cetuximab, a natural human IgG1 directed against the EGFR antigen, EGFR-BiTE, a bispecific antibody directed against CD3 and EGFR, exerts potent cytotoxic activity against cancer cells by recruiting T cells to cancer cells, thereby exerting antitumor effects. However, because EGFR is also expressed in normal tissues, serious side effects have been observed when EGFR-BiTE was administered to cynomolgus monkeys (Non-Patent Document 10). Furthermore, bivatuzumab mertansine, an ADC consisting of mertansine conjugated to an antibody directed against CD44v6, which is highly expressed in cancer cells, has been shown to cause severe skin and liver toxicity in clinical trials, due to the CD44v6 expression in normal tissues (Non-Patent Document 11).

[0006] When using an antibody that can exert strong cytotoxic activity even against cancer cells with low antigen expression, the target antigen must be expressed in an extremely cancer-specific manner, but just as HER2, the target antigen of Herceptin, and EGFR, the target antigen of cetuximab, are also expressed in normal tissues, the number of cancer antigens that are expressed in an extremely cancer-specific manner is thought to be limited. Therefore, although cytotoxic activity against cancer can be strengthened, side effects due to cytotoxic effects on normal tissues can become a problem.

[0007] Recently, it has been shown that ipilimumab, which enhances tumor immunity by inhibiting CTLA4, which contributes to immunosuppression in cancer, prolongs overall survival in metastatic melanoma (Non-Patent Document 12). However, because ipilimumab systemically inhibits CTLA4, while tumor immunity is enhanced, it also causes serious autoimmune disease-like side effects due to systemic immune activation, which has become a problem (Non-Patent Document 13).

[0008] On the other hand, antibody drugs for diseases other than cancer are known to exert therapeutic effects by inhibiting inflammatory cytokines in inflammatory and autoimmune diseases (Non-Patent Document 14). For example, Remicade and Humira, which target TNF, and Actemra, which targets IL-6R, exert high therapeutic effects against rheumatoid arthritis, but it is also known that systemic neutralization of these cytokines can cause side effects such as infections (Non-Patent Document 15).

[0009] Various technologies applicable to second-generation antibody drugs have been developed, and techniques for improving effector function, antigen-binding ability, pharmacokinetics, and stability, or reducing the risk of immunogenicity have been reported (Non-Patent Document 16). However, there are still few reports on technologies that enable antibody drugs to act specifically on target tissues to resolve the above-mentioned side effects. One reported technique involves linking an antibody to a masking peptide with a linker that is cleaved by a protease expressed in lesions such as cancer tissues and inflammatory tissues, thereby masking the antigen-binding site of the antibody with the masking peptide and inhibiting the antigen-binding activity of the antibody. When this linker is cleaved by the protease, the masking peptide is released, restoring the antigen-binding activity of the antibody and enabling it to bind to the antigen in the target pathological tissue (Non-Patent Documents 17, 18, Patent Document 1). [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. WO2010 / 081173 [Non-patent literature]

[0011] [Non-Patent Document 1] Monoclonal antibody successes in the clinic. Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz, Nat. Biotechnol. (2005) 23, 1073 - 1078 [Non-patent document 2] The therapeutic antibodies market to 2008. Pavlou AK, Belsey MJ., Eur. J. Pharm. Biopharm. (2005) 59 (3), 389-396 [Non-patent document 3] Monoclonal antibodies: versatile platforms for cancer immunotherapy. Weiner LM, Surana R, Wang S., Nat. Rev. Immunol. (2010) 10 (5), 317-327 [Non-patent document 4] Differential responses of human tumor cell lines to anti-p185HER2 monoclonal antibodies. Lewis GD, Figari I, Fendly B, Wong WL, Carter P, Gorman C, Shepard HM, Cancer Immunol. Immunotherapy (1993) 37, 255-263 [Non-patent document 5] ING-1, a monoclonal antibody targeting Ep-CAM in patients with advanced adenocarcinomas. de Bono JS, Tolcher AW, Forero A, Vanhove GF, Takimoto C, Bauer RJ, Hammond LA, Patnaik A, White ML, Shen S, Khazaeli MB, Rowinsky EK, LoBuglio AF, Clin. Cancer Res. (2004) 10(22), 7555–7

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Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

[0012] The present inventors considered that in the above-mentioned technique of recovering the antigen-binding activity of an antibody by dissociating a mask peptide that inhibits the antigen-binding activity of the antibody through protease cleavage, the cleavage by protease is irreversible, and therefore the antibody cleaved at the lesion site may enter the bloodstream and be distributed to normal tissues, possibly causing side effects. The present invention was made based on this idea, and one of its objects is to provide a pharmaceutical composition useful for disease treatment with reduced side effects, and an active ingredient thereof, as well as a method for screening and producing said pharmaceutical composition and said active ingredient. [Means for solving the problem]

[0013] As a result of extensive research, the present inventors have created a polypeptide that comprises an antigen-binding domain and a delivery moiety having an inhibition domain that inhibits the binding activity of the antigen-binding domain, and that has a longer half-life than the antigen-binding domain present alone. It is believed that use of this polypeptide can restore the antigen-binding activity of the antigen-binding domain in diseased tissue, allowing the antigen-binding activity to be exerted in diseased tissue. Furthermore, the difference in half-life between the polypeptide in which the antigen-binding activity of the antigen-binding domain is inhibited and the polypeptide in which the antigen-binding activity of the antigen-binding domain is restored can inhibit the systemic distribution of the activated antigen-binding domain. The present inventors have also found that this polypeptide or a pharmaceutical composition containing this polypeptide is useful for disease treatment, and that it is useful for disease treatments that involve administering this polypeptide, and that this polypeptide is useful in the manufacture of pharmaceuticals for disease treatment. Furthermore, the present inventors have completed the present invention by creating a method for screening and producing said polypeptide, a method for producing and screening for single domain antibodies whose antigen-binding activity is suppressed upon association with a specific VL, VH or VHH, and a library containing single domain antibodies whose antigen-binding activity is suppressed upon association with a specific VL, VH or VHH.

[0014] The present invention is based on these findings and specifically includes the embodiments exemplified below. (1) A polypeptide comprising an antigen-binding domain and a delivery moiety, wherein the delivery moiety has an inhibition domain that inhibits the antigen-binding activity of the antigen-binding domain, and wherein the antigen-binding domain has a shorter half-life in blood than the delivery moiety. (2) The polypeptide described in (1), wherein the molecular weight of the antigen-binding domain is smaller than the molecular weight of the transport moiety. (3) The polypeptide according to (1) or (2), wherein the molecular weight of the antigen-binding domain is 60 kDa or less. (4) The polypeptide of any one of (1) to (3), wherein the transport moiety has FcRn-binding activity, and the antigen-binding domain has no FcRn-binding activity or has weaker FcRn-binding activity than the transport moiety. (5) The polypeptide according to any one of (1) to (4), wherein the antigen-binding domain is releasable from the polypeptide, and the antigen-binding activity of the antigen-binding domain is increased by being released from the polypeptide compared to before the release. (6) The polypeptide according to any one of (1) to (5), wherein the antigen-binding activity of the antigen-binding domain is inhibited by association of the antigen-binding domain with the repression domain of the transport moiety. (7) The polypeptide according to (5), wherein the polypeptide comprises a cleavage site, and the antigen-binding domain can be released from the polypeptide by cleavage of the cleavage site. (8) The polypeptide according to (6), wherein the polypeptide comprises a cleavage site, and cleavage of the cleavage site dissolves the association between the antigen-binding domain and the repression domain of the transport moiety. (9) The polypeptide according to (7) or (8), wherein the cleavage site comprises a protease cleavage sequence. (10) The polypeptide according to (9), wherein the protease is a target tissue-specific protease. (11) The polypeptide according to (10), wherein the target tissue is cancer tissue or inflamed tissue. (12) The polypeptide according to (9), wherein the protease is at least one protease selected from matriptase, urokinase (uPA), and metalloprotease. (13) The polypeptide according to (12), wherein the protease is at least one protease selected from MT-SP1, uPA, MMP-2, MMP-9, ADAMTS5, MMP-7, and MMP-13. (14) The polypeptide according to (9), wherein the protease cleavage sequence comprises one or more sequences selected from the sequences shown in SEQ ID NOs: 12, 25, 26, 78 to 81, 83, 84, 91, 168 to 178, 193 to 195, 833 to 852, and 1062 to 1081, and the sequences listed in Table 1. (15) The polypeptide according to any one of (9) to (14), further comprising a first flexible linker attached to one end of the protease cleavage sequence. (16) The polypeptide according to (15), further comprising a second flexible linker attached to the other end of the protease cleavage sequence. (17) The polypeptide according to (15), wherein the first flexible linker is a flexible linker consisting of a glycine-serine polymer. (18) The polypeptide according to (16), wherein the second flexible linker is a flexible linker consisting of a glycine-serine polymer. (19) The polypeptide of any one of (1) to (18), wherein the antigen-binding domain comprises or is a single-domain antibody, and the inhibitory domain of the delivery moiety inhibits the antigen-binding activity of the single-domain antibody. (20) The polypeptide according to (19), 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. (21) The polypeptide according to any one of (1) to (20), 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. (22) The polypeptide according to any one of (1) to (21), 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 association with the VHH, or the antibody VH, or the antibody VL. (23) The polypeptide according to any one of (19) to (22), wherein the single-domain antibody is a VHH or a VH having antigen-binding activity as a single domain, the repression 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 repressed by associating with the antibody VL. (24) The polypeptide according to any one of (19) to (23), 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). (25) The polypeptide according to any one of (19) to (23), wherein the single domain antibody is a VHH, and the VHH contains at least one amino acid selected from the amino acids 37V, 44G, 45L, or 47W (all Kabat numbering). (26) The polypeptide according to any one of (19) to (23), 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). (27) The polypeptide according to any one of (19) to (23), 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). (28) The polypeptide according to any one of (19) to (23), wherein the single domain antibody is a VHH, and the VHH comprises at least one set 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). (29) The polypeptide according to any one of (19) to (23), 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). (30) The polypeptide according to any one of (19) to (22), wherein the single-domain antibody is a VL having antigen-binding activity as a single domain, the repression 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 repressed by associating with the antibody VH. (31) The polypeptide according to any one of (1) to (30), wherein the transport moiety has an FcRn-binding region. (32) The polypeptide according to any one of (1) to (31), wherein the transport moiety comprises an antibody constant region. (33) The polypeptide according to (32), wherein the antibody constant region of the transport moiety and the antigen-binding domain are fused with or without a linker. (34) The polypeptide according to (32), 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. (35) The polypeptide according to (32), 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. (36) The polypeptide according to (34), 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 further comprises a protease cleavage sequence, which 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. (37) The polypeptide according to (35), 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 further comprises a protease cleavage sequence, which is located within the sequence of the antigen-binding domain or closer to the antigen-binding domain than amino acid 113 (EU numbering) (113 in Kabat numbering) of the light chain antibody constant region. (38) The polypeptide according to any one of (33) to (35), 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, the antigen-binding domain is a single-domain antibody prepared from VH or VHH, and the polypeptide further comprises a protease cleavage sequence, which 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. (39) The polypeptide according to (33), wherein the N-terminus of the antibody constant region of the transport moiety is fused to the C-terminus of the antigen-binding domain with or without a linker, and further comprises a protease cleavage sequence located near the boundary between the antigen-binding domain and the antibody constant region. (40) The polypeptide according to (34), 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 further comprises a protease cleavage sequence located near the boundary between the antigen-binding domain and the antibody heavy chain constant region. (41) The polypeptide according to (35), 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 further comprises a protease cleavage sequence located near the boundary between the antigen-binding domain and the antibody light chain constant region. (42) The polypeptide according to (40), wherein the antigen-binding domain is a single-domain antibody or VHH constructed 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. (43) The polypeptide according to (41), wherein the antigen-binding domain is a single-domain antibody or VHH constructed 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 (EU numbering) (Kabat numbering 113) of the antibody light chain constant region. (44) The polypeptide according to (40), wherein the antigen-binding domain is a single-domain antibody made from a VL, and the protease cleavage sequence is located between amino acid 104 (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. (45) The polypeptide according to (41), wherein the antigen-binding domain is a single-domain antibody made 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 (EU numbering) (Kabat numbering 113) of the antibody light chain constant region. (46) The polypeptide according to any one of (32) to (45), wherein the antibody constant region of the polypeptide is an IgG antibody constant region. (47) The polypeptide according to any one of (1) to (46), wherein the polypeptide is an IgG antibody-like molecule. (48) The polypeptide according to any one of (1) to (47), 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. (49) The polypeptide according to any one of (1) to (48), wherein a second antigen-binding domain is further linked to the antigen-binding domain. (50) The polypeptide according to (49), wherein the second antigen-binding domain has an antigen-binding specificity different from that of the antigen-binding domain. (51) The polypeptide according to (49) or (50), wherein the second antigen-binding domain comprises a second single-domain antibody. (52) The polypeptide according to (51), 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. (53) The polypeptide according to any one of (49) to (52), wherein the second antigen-binding domain targets HER2 or GPC3 as a target antigen. (54) The polypeptide according to any one of (1) to (53), 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. (55) The polypeptide according to (54), wherein the additional antigen-binding domain has an antigen-binding specificity different from that of the antigen-binding domain. (56) The polypeptide according to any one of (1) to (55), wherein the antigen-binding domain is an antigen-binding domain whose target antigen is PlexinA1, IL-6R, or CD3. (57) A pharmaceutical composition comprising the polypeptide according to any one of (1) to (56). (58) A method for producing the polypeptide according to any one of (1) to (56). (59) The following steps: (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 precursor; The method according to (58), comprising: (60) The following steps: (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 near the interface between the single domain antibody and the delivery moiety; The method according to (58), comprising: (61) The following steps: (a) obtaining a single domain antibody that binds to a target antigen; (b) linking the single domain antibody obtained in step (a) to a delivery moiety via a protease cleavage sequence to form a polypeptide such that the antigen-binding activity of the single domain antibody is inhibited by the inhibition domain of the delivery moiety; The method according to (58), comprising: (62) further comprising the steps of: (d) confirming that the binding activity of the single domain antibody incorporated into the polypeptide or the precursor polypeptide to the target antigen is weakened or lost; The method according to any one of (59) to (61), comprising: (63) further comprising the steps of: (e) cleaving the protease cleavage sequence with a protease to release the single domain antibody, and confirming that the released single domain antibody binds to an antigen; The method according to any one of (59) to (62), comprising: (64) The method according to (58), wherein the polypeptide is an IgG antibody-like molecule. (65) The following steps: (a) obtaining a single domain antibody that binds to a target antigen; (b) forming an IgG antibody-like molecule precursor into which the single domain antibody obtained in step (a) has been introduced by associating the single domain antibody with a VL instead of a VH of an IgG antibody, or by associating the single domain antibody with a VH instead of a VL of an IgG antibody, so that the antigen-binding activity of the single domain antibody is suppressed; (c) introducing a protease cleavage sequence into the IgG antibody-like molecule precursor into which the single domain antibody has been introduced; The method for producing a compound according to (64), comprising: (66) The following steps: (a) obtaining a single domain antibody that binds to a target antigen; (b) forming an IgG antibody-like molecule precursor into which the single domain antibody obtained in step (a) has been introduced by associating the single domain antibody with a VL instead of a VH of an IgG antibody, or by associating the single domain antibody with a VH instead of a VL of an IgG antibody, so that the antigen-binding activity of the single domain antibody is suppressed; (c) introducing a protease cleavage sequence near the boundary between the single domain antibody and the antibody constant region in the IgG antibody-like molecule precursor; The method for producing a compound according to (64), comprising: (67) The following steps: (a) obtaining a single domain antibody that binds to a target antigen; (b) linking the single domain antibody obtained in step (a) to the heavy chain constant region or light chain constant region of an IgG antibody via a protease cleavage sequence, in place of the IgG antibody VH or VL, so that the antigen-binding activity of the single domain antibody is suppressed, thereby forming an IgG antibody-like molecule into which the single domain antibody has been introduced; The method for producing a compound according to (64), comprising: (68) further comprising the steps of: (d) confirming that the binding activity of the single domain antibody introduced into the IgG antibody-like molecule or the IgG antibody-like molecule precursor to the target antigen is weakened or lost; The method according to any one of (65) to (67), comprising: (69) further comprising the steps of: (e) cleaving the protease cleavage sequence with a protease to release the single domain antibody, and confirming that the released single domain antibody binds to the target antigen; The method according to any one of (65) to (68), comprising: (70) The following steps: (a) substituting amino acid residues in a single domain antibody that are involved in association with the antibody VH, or substituting amino acid residues in a single domain antibody that are involved in association with the antibody VL, to produce a modified single domain antibody that retains the binding activity of the single domain antibody to its target antigen; (b) forming an IgG antibody-like molecule precursor into which the modified single domain antibody prepared in step (a) has been introduced by associating the modified single domain antibody with an antibody VH or with an antibody VL so as to inhibit the antigen-binding activity of the modified single domain antibody; (c) introducing a protease cleavage sequence into the IgG antibody-like molecule precursor into which the modified single domain antibody has been introduced; The method for producing a compound according to (64), comprising: (71) The following steps: (a) substituting amino acid residues in a single domain antibody that are involved in association with the antibody VH, or substituting amino acid residues in a single domain antibody that are involved in association with the antibody VL, to produce a modified single domain antibody that retains the binding activity of the single domain antibody to its target antigen; (b) forming an IgG antibody-like molecule precursor into which the modified single domain antibody prepared in step (a) has been introduced by associating the modified single domain antibody with an antibody VH or with an antibody VL so as to inhibit the antigen-binding activity of the modified single domain antibody; (c) introducing a protease cleavage sequence near the interface between the engineered single domain antibody and the constant region of the IgG antibody-like molecule precursor; The method for producing a compound according to (64), comprising: (72) The following steps: (a) substituting amino acid residues in a single domain antibody that are involved in association with the antibody VH, or substituting amino acid residues in a single domain antibody that are involved in association with the antibody VL, to produce a modified single domain antibody that retains the binding activity of the single domain antibody to its target antigen; (b) linking the modified single domain antibody prepared in step (a) to the heavy chain constant region of an IgG antibody via a protease cleavage sequence, or linking the modified single domain antibody to the light chain constant region of an IgG antibody via a protease cleavage sequence, so as to inhibit the antigen-binding activity of the modified single domain antibody, thereby forming an IgG antibody-like molecule incorporating the modified single domain antibody; The method for producing a compound according to (64), comprising: (73) further comprising the steps of: (d) confirming that the binding activity of the modified single domain antibody introduced into the IgG antibody-like molecule or the IgG antibody-like molecule precursor to the target antigen is weakened or lost; The method according to any one of (70) to (72), comprising: (74) further comprising the steps of: (e) cleaving the protease cleavage sequence with a protease to release the engineered single domain antibody, and confirming that the released engineered single domain antibody binds to the target antigen; The method according to any one of (70) to (73), comprising: (75) A polynucleotide encoding the polypeptide according to any one of (1) to (56). (76) A vector comprising the polynucleotide according to (75). (77) A host cell comprising the polynucleotide according to (75) or the vector according to (76). (78) A method for producing the polypeptide according to any one of (1) to (56), comprising the step of culturing the host cell according to (77). (79) A method for screening for a single domain antibody whose antigen-binding activity is suppressed by associating with a specific VL, or a specific VH, or a specific VHH. (80) The screening method according to (79), which screens for a single domain antibody whose antigen-binding activity is suppressed by associating with a specific VL. (81) The following steps: (a) obtaining a single domain antibody having target antigen-binding activity; (b) associating the single domain antibody obtained in step (a) with a specific VL; (c) confirming that the binding activity of the single domain antibody associated with a specific VL in step (b) against the antigen is weakened or lost compared to before association; The screening method according to (80), comprising: (82) The following steps: (a) associating a single domain antibody with a specific VL; (b) selecting an aggregate of a VL and a single domain antibody in which the binding activity of the single domain antibody associated with a specific VL in step (a) to the antigen is absent or is below a certain level; (c) confirming that the binding activity of the single domain antibody in the aggregate selected in step (b) against the antigen when not associated with the specific VL is stronger than when associated; The screening method according to (80), comprising: (83) The screening method according to (79), wherein a single domain antibody whose antigen-binding activity is suppressed by associating with a specific VH is screened. (84) The following steps: (a) obtaining a single domain antibody having target antigen-binding activity; (b) associating the single domain antibody obtained in step (a) with a specific VH; (c) confirming that the binding activity of the single domain antibody associated with a specific VH in step (b) to the antigen is weakened or lost compared to before association; The screening method according to (83), comprising: (85) The following steps: (a) associating a single domain antibody with a specific VH; (b) selecting an aggregate of a VH and a single-domain antibody in which the binding activity of the single-domain antibody associated with a specific VH in step (a) to the antigen is absent or is below a certain level; (c) confirming that the binding activity of the single domain antibody in the aggregate selected in step (b) against the antigen when not associated with the specific VH is stronger than when associated; The screening method according to (83), comprising: (86) The screening method according to (79), wherein a single domain antibody whose antigen-binding activity is suppressed by association with a specific VHH is screened. (87) The following steps: (a) obtaining a single domain antibody having target antigen-binding activity; (b) associating the single domain antibody obtained in step (a) with a specific VHH; (c) confirming that the binding activity of the single domain antibody associated with a specific VHH in step (b) against the antigen is weakened or lost compared to before association; The screening method according to (86), comprising: (88) The following steps: (a) associating a single domain antibody with a specific VHH; (b) selecting an aggregate of a VHH and a single domain antibody in which the binding activity of the single domain antibody associated with a specific VHH in step (a) to the antigen is absent or below a certain level; (c) confirming that the binding activity of the single domain antibody in the aggregate selected in step (b) against the antigen when not associated with the specific VHH is stronger than when associated with the specific VHH; The screening method according to (86), comprising: (89) A method for producing a single domain antibody whose antigen-binding activity is suppressed by associating with a specific VL, or a specific VH, or a specific VHH. (90) The method of producing a single domain antibody according to (89), which produces a single domain antibody whose antigen-binding activity is suppressed by associating with a specific VL. (91) The following steps: (a) substituting amino acid residues in a single domain antibody that are involved in association with an antibody VL to produce a modified single domain antibody that retains the binding activity of the single domain antibody to its target antigen; The method for producing a compound according to (90), comprising: (92) Further steps of: (b) associating the engineered single domain antibody produced in step (a) with said VL; (c) confirming that the antigen-binding activity of the modified single domain antibody associated with the VL is weakened or lost compared to before association; The method for producing a compound according to (91), comprising: (93) The method of producing a single domain antibody according to (89), wherein the single domain antibody has suppressed antigen-binding activity upon association with a specific VH. (94) The following steps: (a) substituting amino acid residues in a single domain antibody that are involved in association with an antibody VH to produce a modified single domain antibody that retains the binding activity of the single domain antibody to its target antigen; The method for producing a compound according to (93), comprising: (95) further comprising the steps of: (b) associating the engineered single domain antibody produced in step (a) with said VH; (c) confirming that the antigen-binding activity of the modified single domain antibody associated with the VH is weakened or lost compared to before association; The method according to (94), comprising: (96) The method of producing a single domain antibody according to (89), the antigen-binding activity of which is suppressed by associating with a specific VHH. (97) The following steps: (a) substituting amino acid residues in a single domain antibody that are involved in association with VHH to produce a modified single domain antibody that retains the binding activity of the single domain antibody to its target antigen; The method according to (96), comprising: (98) further comprising the steps of: (b) associating the engineered single domain antibody produced in step (a) with said VHH; (c) confirming that the antigen-binding activity of the modified single domain antibody conjugated with the VHH is weakened or lost compared to before conjugation; The method for producing a compound according to (97), comprising: (99) A library comprising a plurality of fusion polypeptides in which a single-domain antibody is linked to a first association-supporting domain, wherein the single-domain antibody includes a single-domain antibody whose antigen-binding activity is suppressed or lost when associated with a specific VL, or a single-domain antibody whose antigen-binding activity is suppressed or lost when associated with a specific VH, or a single-domain antibody whose antigen-binding activity is suppressed or lost when associated with a specific VHH. (100) The library according to (99), wherein the single-domain antibody moieties of the fusion polypeptides in the library comprise single-domain antibodies or humanized antibodies thereof obtained from a camelid or a transgenic animal into which a gene capable of producing a single-domain antibody has been introduced, or single-domain antibodies or humanized antibodies thereof obtained by immunizing a camelid or a transgenic animal into which a gene capable of producing a single-domain antibody has been introduced, or single-domain antibodies or humanized antibodies thereof, or single-domain antibodies artificially produced starting from a human antibody VH or VL. (101) The library according to (99) or (100), comprising a plurality of fusion polypeptides each linking a single-domain antibody to a first association-supporting domain, wherein the single-domain antibody includes a single-domain antibody whose antigen-binding activity is suppressed or lost upon association with a specific VL. (102) The library according to (99) or (100), comprising a plurality of fusion polypeptides each linking a single-domain antibody to a first association-supporting domain, wherein the single-domain antibody includes a single-domain antibody whose antigen-binding activity is suppressed or lost upon association with a specific VH. (103) The library according to (99) or (100), comprising a plurality of fusion polypeptides linking a single-domain antibody to a first association-supporting domain, wherein the single-domain antibody includes a single-domain antibody whose antigen-binding activity is suppressed or lost upon association with a specific VHH. (104) A method for screening a fusion polypeptide comprising a single domain antibody whose antigen-binding activity is suppressed or lost when associated with a specific VL, or a single domain antibody whose antigen-binding activity is suppressed or lost when associated with a specific VH, or a single domain antibody whose antigen-binding activity is suppressed or lost when associated with a specific VHH, from the library according to (99) or (100). (105) A method for screening a fusion polypeptide comprising a single domain antibody whose antigen-binding activity is suppressed or lost when associated with a specific VL from the library according to (101). (106) The following steps: (a) in vitro displaying fusion polypeptides from the library; (b) providing an assembly partner in which a specific VL is fused to a second assembly-supporting domain; (c) associating the fusion polypeptide displayed in step (a) with the association partner prepared in step (b), and selecting a fusion polypeptide that does not bind to an antigen when the single domain antibody is associated with the VL, or that has an antigen-binding activity below a certain level; (d) selecting, from the fusion polypeptides selected in step (c), fusion polypeptides that bind to an antigen when the single domain antibody contained therein is not associated with the VL, or that have antigen-binding activity equal to or greater than a certain level; The screening method according to (105), comprising: (107) The screening method according to (106), wherein the association partner prepared in step (b) further comprises a protease cleavage sequence, and in step (d), the association partner is cleaved by protease treatment to dissolve the association between the single domain antibody and the VL. (108) The screening method described in (107), wherein the protease cleavage sequence of the association partner prepared in step (b) is located near the boundary between the specific VL and the second association support domain. (109) The screening method according to (106), wherein the fusion polypeptides of the library further comprise a protease cleavage sequence, and in step (d), the fusion polypeptides are cleaved by protease treatment to dissolve the association between the single domain antibody and the VL. (110) The screening method according to (109), wherein the protease cleavage sequence contained in the fusion polypeptide is located near the boundary between the single-domain antibody and the first association support domain. (111) The screening method according to (106), wherein in the step (d), the full-length or a portion containing a single domain antibody of the fusion polypeptide selected in the step (c) is again displayed in vitro. (112) In the step (d), the full length of the fusion polypeptide selected in the step (c) is again displayed in vitro, and a fusion polypeptide that binds to an antigen while associated with only the second association-supporting domain or has antigen-binding activity equal to or greater than a certain value is selected. (113) A method for screening a fusion polypeptide comprising a single domain antibody whose antigen-binding activity is suppressed or lost upon association with a specific VH from the library according to (102). (114) The following steps: (a) in vitro displaying fusion polypeptides from the library; (b) providing an assembly partner in which a specific VH is fused to a second assembly-supporting domain; (c) associating the fusion polypeptide displayed in step (a) with the association partner prepared in step (b), and selecting a fusion polypeptide that does not bind to an antigen when the single domain antibody and the VH are associated, or that has an antigen-binding activity below a certain level; (d) selecting, from the fusion polypeptides selected in step (c), fusion polypeptides that bind to an antigen when the VH contained in the fusion polypeptide is not associated with the single domain antibody, or that have antigen-binding activity equal to or greater than a certain level; The screening method according to (113), comprising: (115) The screening method according to (114), wherein the association partner prepared in step (b) further comprises a protease cleavage sequence, and in step (d), the association partner is cleaved by protease treatment to dissolve the association between the single domain antibody and the VH. (116) The screening method according to (115), wherein the protease cleavage sequence of the association partner prepared in step (b) is located near the boundary between the specific VH and the second association support domain. (117) The screening method according to (114), wherein the fusion polypeptides of the library further comprise a protease cleavage sequence, and in step (d), the fusion polypeptides are cleaved by protease treatment to dissolve the association between the single domain antibody and the VH. (118) The screening method according to (117), wherein the protease cleavage sequence contained in the fusion polypeptide is located near the boundary between the single-domain antibody and the first association support domain. (119) The screening method according to (114), wherein in the step (d), the full-length or a portion containing a single domain antibody of the fusion polypeptide selected in the step (c) is again displayed in vitro. (120) In the step (d), the full length of the fusion polypeptide selected in the step (c) is again displayed in vitro, and a fusion polypeptide that binds to an antigen while associated with only the second association support domain or has antigen-binding activity equal to or greater than a certain value is selected. (121) A method for screening a fusion polypeptide comprising a single domain antibody whose antigen-binding activity is suppressed or lost upon association with a specific VHH from the library according to (103). (122) The following steps: (a) in vitro displaying fusion polypeptides from the library; (b) providing an assembly partner in which a specific VHH is fused with a second assembly-supporting domain; (c) associating the fusion polypeptide displayed in step (a) with the association partner prepared in step (b), and selecting a fusion polypeptide that does not bind to an antigen when the single domain antibody is associated with the specific VHH, or that has an antigen-binding activity below a certain level; (d) selecting, from the fusion polypeptides selected in step (c), fusion polypeptides that bind to an antigen in the absence of association of the single domain antibody and the VHH contained therein, or that have antigen-binding activity equal to or greater than a certain level; The screening method according to (121), comprising: (123) The screening method described in (122), wherein the association partner prepared in step (b) further comprises a protease cleavage sequence, and in step (d), the association partner is cleaved by protease treatment to dissolve the association between the single domain antibody and the VHH. (124) The screening method described in (123), wherein the protease cleavage sequence of the association partner prepared in step (b) is located near the boundary between the specific VHH and the second association support domain. (125) The screening method described in (122), wherein the fusion polypeptide of the library further comprises a protease cleavage sequence, and in step (d), the fusion polypeptide is cleaved by protease treatment to dissolve the association between the single domain antibody and the VHH. (126) The screening method according to (125), wherein the protease cleavage sequence contained in the fusion polypeptide is located near the boundary between the single-domain antibody and the first association support domain. (127) The screening method according to (122), wherein in the step (d), the full-length or a portion containing a single domain antibody of the fusion polypeptide selected in the step (c) is again displayed in vitro. (128) In the step (d), the full length of the fusion polypeptide selected in the step (c) is again displayed in vitro, and a fusion polypeptide that binds to an antigen while associated with only the second association support domain or has antigen binding activity equal to or greater than a certain value is selected. (129) The screening method according to any one of (106) to (112), (114) to (120), and (122) to (128), wherein the step of preparing an association partner in step (b) is a step of simultaneously displaying the association partner and the fusion polypeptide. (130) The library according to any one of (99) to (103), wherein the first association support domain comprises an IgG antibody CH1 domain or an antibody light chain constant region. (131) The screening method according to any one of (106) to (112), (114) to (120), and (122) to (128), wherein the first association support domain comprises an IgG antibody CH1 domain and the second association support domain comprises an antibody light chain constant region. (132) The screening method according to any one of (106) to (112), (114) to (120), and (122) to (128), wherein the first association support domain comprises an antibody light chain constant region and the second association support domain comprises an IgG antibody CH1 domain. (133) The following steps: (a) in vitro displaying fusion polypeptides from the library; (b) providing an assembly partner in which a specific VL is fused to a second assembly-supporting domain; (c) selecting a fusion polypeptide in which a single domain antibody contained in the fusion polypeptide binds to an antigen or has an antigen-binding activity equal to or greater than a certain value; (d) associating the fusion polypeptide selected in step (c) with the association partner prepared in step (b), and selecting a fusion polypeptide that does not bind to an antigen when the single domain antibody is associated with the VL, or that has an antigen-binding activity below a certain level; The screening method according to (105), comprising: (134) The screening method according to (129), wherein in the step (d), the fusion polypeptide selected in the step (c) is again displayed in vitro. (135) The screening method according to (133), wherein in step (c), the fusion polypeptide is associated with only the second association support domain, or the antigen binding of the single domain antibody contained in the fusion polypeptide is confirmed in a state in which the fusion polypeptide is associated with only the second association support domain. (136) The following steps: (a) in vitro displaying fusion polypeptides from the library; (b) providing an assembly partner in which a specific VH is fused to a second assembly-supporting domain; (c) selecting a fusion polypeptide in which a single domain antibody contained in the fusion polypeptide binds to an antigen or has an antigen-binding activity equal to or greater than a certain value; (d) associating the fusion polypeptide selected in step (c) with the association partner prepared in step (b), and selecting a fusion polypeptide that does not bind to an antigen when the single domain antibody and the VH are associated, or that has an antigen-binding activity below a certain level; The screening method according to (113), comprising: (137) The screening method according to (136), wherein in the step (d), the fusion polypeptide selected in the step (c) is again displayed in vitro. (138) The screening method according to (136), wherein in step (c), the fusion polypeptide is associated with only the second association support domain, or the antigen binding of the single domain antibody contained in the fusion polypeptide is confirmed in a state where the fusion polypeptide is associated with only the second association support domain. (139) The following process: (a) in vitro displaying fusion polypeptides from the library; (b) providing an assembly partner in which a specific VHH is fused with a second assembly-supporting domain; (c) selecting a fusion polypeptide in which a single domain antibody contained in the fusion polypeptide binds to an antigen or has an antigen-binding activity equal to or greater than a certain value; (d) associating the fusion polypeptide selected in step (c) with the association partner prepared in step (b), and selecting a fusion polypeptide that does not bind to an antigen when the single domain antibody and the VHH are associated, or that has an antigen-binding activity below a certain level; The screening method according to (121), comprising: (140) The screening method according to (139), wherein in the step (d), the fusion polypeptide selected in the step (c) is again displayed in vitro. (141) The screening method according to (139), wherein in step (c), the fusion polypeptide is associated with only the second association support domain, or the antigen binding of the single domain antibody contained in the fusion polypeptide is confirmed in a state in which the fusion polypeptide is associated with only the second association support domain. (142) The screening method according to any one of (133) to (141), wherein the step of associating the fusion polypeptide with the association partner in step (d) is a step of simultaneously displaying the association partner and the fusion polypeptide. (143) The screening method according to any one of (133) to (142), wherein the first association support domain comprises an IgG antibody CH1 domain, and the second association support domain comprises an antibody light chain constant region. (144) The screening method according to any one of (133) to (142), wherein the first association support domain comprises an antibody light chain constant region, and the second association support domain comprises an IgG antibody CH1 domain.

[0015] The present invention can also specifically include the embodiments exemplified below. (B1) A polypeptide comprising an antigen-binding domain and a delivery moiety, wherein the delivery moiety has an inhibition domain that inhibits the antigen-binding activity of the antigen-binding domain, and the polypeptide has a protease cleavage sequence comprising one or more sequences selected from the sequences shown in SEQ ID NOs: 833 to 852, 1062 to 1081, and the sequences listed in Table 1. (B2) The polypeptide of (B1), 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. (B3) The polypeptide of (B1) or (B2), wherein the antigen-binding domain has a shorter half-life in blood than the delivery moiety. (B4) The polypeptide according to any one of (B1) to (B3), wherein the molecular weight of the antigen-binding domain is smaller than the molecular weight of the transport moiety. (B5) The polypeptide according to any one of (B1) to (B4), wherein the molecular weight of the antigen-binding domain is 60 kDa or less. (B6) The polypeptide of any one of (B1) to (B5), wherein the transport moiety has FcRn-binding activity, and the antigen-binding domain has no FcRn-binding activity or has weaker FcRn-binding activity than the transport moiety. (B7) The polypeptide of any one of (B1) to (B6), 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. (B8) The polypeptide according to any one of (B1) to (B7), wherein the antigen-binding activity of the antigen-binding domain is inhibited by association of the antigen-binding domain with the repression domain of the delivery moiety. (B9) The polypeptide according to (B7), wherein the antigen-binding domain can be released from the polypeptide upon cleavage of the protease cleavage sequence by a protease. (B10) The polypeptide according to (B8), 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. (B11) The polypeptide according to any one of (B1) to (B10), wherein the protease is a target tissue-specific protease. (B12) The polypeptide according to (B11), wherein the target tissue is a cancer tissue or an inflamed tissue, and the protease is a cancer tissue-specific protease or an inflamed tissue-specific protease. (B13) The polypeptide according to any one of (B1) to (B12), wherein the protease is at least one protease selected from matriptase, urokinase (uPA), and metalloprotease. (B14) The polypeptide according to any one of (B1) to (B12), wherein the protease is at least one protease selected from MT-SP1, uPA, MMP-2, MMP-9, ADAMTS5, MMP-7, and MMP-13. (B15) The polypeptide according to any one of (B1) to (B14), further comprising a first flexible linker attached to one end of the protease cleavage sequence. (B16) The polypeptide according to (B15), wherein the first flexible linker is a flexible linker consisting of a glycine-serine polymer. (B17) The polypeptide according to (B15) or (B16), further comprising a second flexible linker attached to the other end of the protease cleavage sequence. (B18) The polypeptide according to (B17), wherein the second flexible linker is a flexible linker consisting of a glycine-serine polymer. (B19) The polypeptide of any one of (B1) to (B18), wherein the antigen-binding domain comprises or is a single-domain antibody, and the inhibitory domain of the delivery moiety inhibits the antigen-binding activity of the single-domain antibody. (B20) The polypeptide according to (B19), 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. (B21) The polypeptide according to any one of (B1) to (B20), 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. (B22) The polypeptide according to any one of (B1) to (B21), 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 association with the VHH, or the antibody VH, or the antibody VL. (B23) The polypeptide according to any one of (B19) to (B22), wherein the single-domain antibody is a VHH or a VH having antigen-binding activity as a single domain, the repression 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 repressed by associating with the antibody VL. (B24) The polypeptide described in any one of (B19) to (B23), 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). (B25) A polypeptide described in any one of (B19) to (B23), 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). (B26) The polypeptide described in any one of (B19) to (B23), 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). (B27) The polypeptide according to any one of (B19) to (B23), 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). (B28) The polypeptide according to any one of (B19) to (B23), wherein the single domain antibody is a VHH, and the VHH comprises at least one set 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). (B29) The polypeptide according to any one of (B19) to (B23), 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). (B30) The polypeptide according to any one of (B19) to (B22), wherein the single-domain antibody is a VL having antigen-binding activity as a single domain, the repression 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 repressed by associating with the antibody VH. (B31) The polypeptide according to any one of (B1) to (B30), wherein the transport moiety has an FcRn binding region. (B32) The polypeptide according to any one of (B1) to (B31), wherein the transport moiety comprises an antibody constant region. (B33) The polypeptide according to (B32), wherein the antibody constant region of the transport moiety and the antigen-binding domain are fused with or without a linker. (B34) The polypeptide according to (B32), 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. (B35) The polypeptide according to (B32), 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. (B36) The polypeptide according to (B34), wherein the N-terminus of the antibody heavy chain constant region of the transport moiety 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. (B37) The polypeptide according to (B35), 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 (EU numbering) (113 in Kabat numbering) of the light chain antibody constant region. (B38) The polypeptide according to any one of (B33) to (B36), 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, 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. (B39) The polypeptide according to (B33), wherein the N-terminus of the antibody constant region of the transport moiety 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. (B40) The polypeptide according to (B34), 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. (B41) The polypeptide according to (B35), 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 interface between the antigen-binding domain and the antibody light chain constant region. (B42) The polypeptide according to (B40), 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. (B43) The polypeptide according to (B41), 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 (EU numbering) (Kabat numbering 113) of the antibody light chain constant region. (B44) The polypeptide according to (B40), 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 of the antigen-binding domain and amino acid 122 (EU numbering) of the antibody heavy chain constant region. (B45) The polypeptide according to (B41), 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 (EU numbering) (Kabat numbering 113) of the antibody light chain constant region. (B46) The polypeptide according to any one of (B32) to (B45), wherein the antibody constant region of the polypeptide is an IgG antibody constant region. (B47) The polypeptide according to any one of (B1) to (B46), wherein the polypeptide is an IgG antibody-like molecule. (B48) The polypeptide according to any one of (B1) to (B47), 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. (B49) The polypeptide according to any one of (B1) to (B48), wherein a second antigen-binding domain is further linked to the antigen-binding domain. (B50) The polypeptide according to (B49), wherein the second antigen-binding domain has an antigen-binding specificity different from that of the antigen-binding domain. (B51) The polypeptide according to (B49) or (B50), wherein the second antigen-binding domain comprises a second single-domain antibody. (B52) The polypeptide according to (B51), 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. (B53) The polypeptide according to any one of (B49) to (B52), wherein the second antigen-binding domain targets HER2 or GPC3 as a target antigen. (B54) The polypeptide according to any one of (B1) to (B53), 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. (B55) The polypeptide according to (B54), wherein the additional antigen-binding domain has an antigen-binding specificity different from that of the antigen-binding domain. (B56) The polypeptide according to any one of (B1) to (B55), wherein the antigen-binding domain is an antigen-binding domain whose target antigen is PlexinA1, IL-6R, or CD3. (B57) A pharmaceutical composition comprising the polypeptide according to any one of (B1) to (B56). (B58) A method for producing the polypeptide according to any one of (B1) to (B56). (B59) the following steps: (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 precursor; The method for producing a compound according to (B58), comprising: (B60) The following process: (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, near the interface between the single domain antibody and the delivery moiety, a protease cleavage sequence comprising one or more sequences selected from the sequences set forth in SEQ ID NOs: 833 to 852, 1062 to 1081, and the sequences set forth in Table 1; The method for producing a compound according to (B58), comprising: (B61) The following steps: (a) obtaining a single domain antibody that binds to a target antigen; (b) linking the single domain antibody obtained in step (a) to a delivery moiety via a protease cleavage sequence comprising one or more sequences selected from the sequences set forth in SEQ ID NOs: 833 to 852, 1062 to 1081, and the sequences set forth in Table 1, so that the antigen-binding activity of the single domain antibody is inhibited by the inhibition domain of the delivery moiety to form a polypeptide; The method for producing a compound according to (B58), comprising: (B62) further comprising the steps of: (d) confirming that the binding activity of the single domain antibody incorporated into the polypeptide or the precursor polypeptide to the target antigen is weakened or lost; The method for producing a product according to any one of (B59) to (B61), comprising: (B63) further comprising the steps of: (e) cleaving the protease cleavage sequence with a protease to release the single domain antibody, and confirming that the released single domain antibody binds to an antigen; The method for producing a product according to any one of (B59) to (B62), comprising: (B64) The method for production according to (B58), wherein the polypeptide is an IgG antibody-like molecule. (B65) The following process: (a) obtaining a single domain antibody that binds to a target antigen; (b) forming an IgG antibody-like molecule precursor into which the single domain antibody obtained in step (a) has been introduced by associating the single domain antibody with a VL instead of a VH of an IgG antibody, or by associating the single domain antibody with a VH instead of a VL of an IgG antibody, so that the antigen-binding activity of the single domain antibody is suppressed; (c) introducing into the IgG antibody-like molecule precursor into which the single domain antibody has been introduced a protease cleavage sequence(s) comprising one or more sequences selected from the sequences shown in SEQ ID NOs: 833 to 852, 1062 to 1081, and the sequences listed in Table 1; The method for producing according to (B64), comprising: (B66) The following steps: (a) obtaining a single domain antibody that binds to a target antigen; (b) forming an IgG antibody-like molecule precursor into which the single domain antibody obtained in step (a) has been introduced by associating the single domain antibody with a VL instead of a VH of an IgG antibody, or by associating the single domain antibody with a VH instead of a VL of an IgG antibody, so that the antigen-binding activity of the single domain antibody is suppressed; (c) introducing a protease cleavage sequence comprising one or more sequences selected from the sequences shown in SEQ ID NOs: 833 to 852, 1062 to 1081, and the sequences listed in Table 1, near the boundary between the single domain antibody and the antibody constant region in the IgG antibody-like molecule precursor; The method for producing according to (B64), comprising: (B67) The following process: (a) obtaining a single domain antibody that binds to a target antigen; (b) linking the single domain antibody obtained in step (a) to a heavy chain constant region or a light chain constant region of an IgG antibody via a protease cleavage sequence comprising one or more sequences selected from the sequences shown in SEQ ID NOs: 833 to 852, 1062 to 1081, and the sequences listed in Table 1, in place of an IgG antibody VH or VL, so that the antigen-binding activity of the single domain antibody is suppressed, thereby forming an IgG antibody-like molecule into which the single domain antibody has been introduced; The method for producing according to (B64), comprising: (B68) further comprising the steps of: (d) confirming that the binding activity of the single domain antibody introduced into the IgG antibody-like molecule or the IgG antibody-like molecule precursor to the target antigen is weakened or lost; The method for producing a product according to any one of (B65) to (B67), comprising: (B69) further comprising the steps of: (e) cleaving the protease cleavage sequence with a protease to release the single domain antibody, and confirming that the released single domain antibody binds to the target antigen; The method for producing a product according to any one of (B65) to (B68), comprising: (B70) The following steps: (a) substituting amino acid residues in a single domain antibody that are involved in association with the antibody VH, or substituting amino acid residues in a single domain antibody that are involved in association with the antibody VL, to produce a modified single domain antibody that retains the binding activity of the single domain antibody to its target antigen; (b) forming an IgG antibody-like molecule precursor into which the modified single domain antibody prepared in step (a) has been introduced by associating the modified single domain antibody with an antibody VH or with an antibody VL so as to inhibit the antigen-binding activity of the modified single domain antibody; (c) introducing into the IgG antibody-like molecule precursor into which the modified single-domain antibody has been introduced a protease cleavage sequence(s) comprising one or more sequences selected from the sequences shown in SEQ ID NOs: 833 to 852, 1062 to 1081, and the sequences listed in Table 1; The method for producing according to (B64), comprising: (B71) The following steps: (a) substituting amino acid residues in a single domain antibody that are involved in association with the antibody VH, or substituting amino acid residues in a single domain antibody that are involved in association with the antibody VL, to produce a modified single domain antibody that retains the binding activity of the single domain antibody to its target antigen; (b) forming an IgG antibody-like molecule precursor into which the modified single domain antibody prepared in step (a) has been introduced by associating the modified single domain antibody with an antibody VH or with an antibody VL so as to inhibit the antigen-binding activity of the modified single domain antibody; (c) introducing a protease cleavage sequence comprising one or more sequences selected from the sequences shown in SEQ ID NOs: 833 to 852, 1062 to 1081, and the sequences listed in Table 1, near the boundary between the modified single-domain antibody and the constant region of the IgG antibody-like molecule precursor; The method for producing according to (B64), comprising: (B72) The following steps: (a) substituting amino acid residues in a single domain antibody that are involved in association with the antibody VH, or substituting amino acid residues in a single domain antibody that are involved in association with the antibody VL, to produce a modified single domain antibody that retains the binding activity of the single domain antibody to its target antigen; (b) linking the modified single-domain antibody produced in step (a) to the heavy chain constant region of an IgG antibody via a protease cleavage sequence comprising one or more sequences selected from the sequences shown in SEQ ID NOs: 833 to 852, 1062 to 1081, and the sequences listed in Table 1, or linking the modified single-domain antibody to the light chain constant region of an IgG antibody via a protease cleavage sequence comprising one or more sequences selected from the sequences shown in SEQ ID NOs: 833 to 852, 1062 to 1081, and the sequences listed in Table 1, so as to inhibit the antigen-binding activity of the modified single-domain antibody, thereby forming an IgG antibody-like molecule incorporating the modified single-domain antibody; The method for producing according to (B64), comprising: (B73) further comprising the steps of: (d) confirming that the binding activity of the modified single domain antibody introduced into the IgG antibody-like molecule or the IgG antibody-like molecule precursor to the target antigen is weakened or lost; The method for producing a product according to any one of (B70) to (B72), comprising: (B74) further comprising the steps of: (e) cleaving the protease cleavage sequence with a protease to release the engineered single domain antibody, and confirming that the released engineered single domain antibody binds to the target antigen; The method for producing a product according to any one of (B70) to (B73), comprising: (B75) A polynucleotide encoding a polypeptide according to any one of (B1) to (B56). (B76) A vector comprising the polynucleotide according to (B75). (B77) A host cell comprising the polynucleotide according to (B75) or the vector according to (B76). (B78) A method for producing the polypeptide according to any one of (B1) to (B56), comprising the step of culturing the host cell according to (B77). [Brief explanation of the drawings]

[0016] [Figure 1] Figure 1 shows the concept of Probody technology. This is an antibody molecule in which a peptide that masks the antigen-binding site of the antibody is linked to the antibody via a linker that is cleaved by a protease expressed at the lesion site, thereby inhibiting the antigen-binding activity of the antibody. [Figure 2] This figure shows one possible reason why probodies may exhibit side effects. Activated probodies that accumulate in the blood may exert side effects by binding to antigens expressed in normal tissues. [Figure 3] This figure shows one possible reason why a probody may exhibit side effects. The probody is in an equilibrium state where the mask peptide linked by the linker is bound to the antigen-binding site and dissociated, and the dissociated molecule can bind to the antigen. [Figure 4] This figure shows one possible reason why a probody may exhibit side effects. Anti-drug antibodies against masked peptides (anti-masked peptide antibodies) may activate the probody by binding to the masked peptide of the probody before activation, even without cleavage by a protease. [Figure 5] Figure 1 shows the concept of a polypeptide comprising 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) The antigen-binding domain is released by cleavage at the cleavage site or other means and binds to the antigen, and the released antigen-binding domain has a short half-life. [Figure 6] FIG. 1 shows one embodiment of a method for producing a polypeptide of the present invention. 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 7] 1 is a diagram showing one embodiment of the polypeptide of the present invention. In this embodiment, 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 8] Figure 1 shows an embodiment in which a second antigen-binding domain is further linked to the antigen-binding domain of the present invention. In this embodiment, 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 9A] Figure 1 shows an example of a method for screening a library containing multiple fusion polypeptides in which a single domain antibody is linked to a first association support domain for fusion polypeptides in which the antigen-binding activity is weakened or lost upon association with a specific repression domain. (1) Figure 1 shows a library containing multiple fusion polypeptides in which a single domain antibody is linked to a first association support domain. (2) Figure 1 shows the antigen-binding activity of a single domain antibody confirmed when the fusion polypeptide is associated with an association partner. In this associated state, fusion polypeptides containing single domain antibodies that do not bind to the target antigen or whose antigen-binding activity is below a certain value are selected. (3) Figure 1 shows the antigen-binding activity of a single domain antibody in the fusion polypeptide selected in (2) after dissociating the association between the single domain antibody and the repression domain in the association partner. In this unassociated state, fusion polypeptides containing single domain antibodies that bind to the target antigen or whose antigen-binding activity is above a certain value are selected. (2') Figure 1 shows the antigen-binding activity of a single domain antibody in a fusion polypeptide confirmed. In the presence of this fusion polypeptide alone, fusion polypeptides containing single domain antibodies that bind to the target antigen or whose antigen-binding activity is above a certain value are selected. (3') A diagram showing the antigen-binding activity of a single domain antibody when the fusion polypeptide selected in (2') is associated with an association partner. Fusion polypeptides containing single domain antibodies that do not bind to the target antigen or whose antigen-binding activity is below a certain level are selected in this associated state. [Figure 9B]This figure shows a more specific example of a method for screening a library containing multiple fusion polypeptides in which a single-domain antibody is linked to a first association support domain for fusion polypeptides containing a single-domain antibody whose antigen-binding activity is weakened or lost upon association with a specific repression domain: (1) A fusion polypeptide containing a single-domain antibody and a first association support domain, and an association partner in which a protease cleavage sequence has been introduced between the repression domain and the second association support domain, are simultaneously displayed to form a Fab-like structure; (2) From the displayed Fab-like structures, those that do not bind to the antigen or whose antigen-binding activity is below a certain level are selected; (3) The association partner is cleaved with a protease, and fragments containing a single-domain antibody that binds to the antigen or whose antigen-binding activity is above a certain level are selected. [Figure 9C] This figure shows another more specific example of a method for screening a library containing multiple fusion polypeptides in which a single-domain antibody is linked to a first association support domain for fusion polypeptides containing a single-domain antibody whose antigen-binding activity is weakened or lost upon association with a specific repression domain: (1) A fusion polypeptide in which a protease cleavage sequence has been introduced between the single-domain antibody and the first association support domain, and an association partner in which a repression domain and a second association support domain are linked, are simultaneously displayed to form a Fab-like structure; (2) From the displayed Fab-like structures, those that do not bind to the antigen or whose antigen-binding activity is below a certain level are selected; (3) The fusion polypeptide is cleaved with a protease, and fragments containing a single-domain antibody that binds to the antigen or whose antigen-binding activity is above a certain level are selected. [Figure 9D]Figure 1 shows another example of a method for screening a library containing multiple fusion polypeptides in which a single-domain antibody is linked to a first association support domain for fusion polypeptides containing a single-domain antibody whose antigen-binding activity is weakened or lost upon association with a specific repression domain. (1) A fusion polypeptide containing a single-domain antibody and a first association support domain and an association partner in which a repression domain and a second association support domain are linked are simultaneously displayed to form a Fab-like structure, and fragments that do not bind to the antigen or whose antigen-binding activity is below a certain level are selected from the displayed Fab-like structure. (2) The portion of the Fab-like structure selected in (1) containing the single-domain antibody is again displayed without simultaneously expressing the repression domain, and fragments that bind to the antigen or whose antigen-binding activity is above a certain level are selected. (2') and (2'') show another embodiment in which the portion containing the single-domain antibody in (2) is again displayed without simultaneously expressing the repression domain. The order of (1) and (2) / (2') / (2'') may also be (2) / (2') / (2'') to (1); that is, a portion containing a single domain antibody is displayed without simultaneously expressing the inhibition domain, and fragments with antigen-binding activity equal to or greater than a certain level are selected. Next, a single domain antibody containing a fragment with binding activity equal to or greater than a certain level, a fusion polypeptide containing a first association supporting domain, and an association partner in which an inhibition domain and a second association supporting domain are linked are simultaneously displayed to form Fab-like structures, and among the displayed Fab-like structures, those that do not bind to the antigen or have antigen-binding activity equal to or less than a certain level are selected. [Figure 10] This figure shows the results of evaluating the binding of antibody-like molecules to human IL-6R. The antibody-like molecules were prepared by associating IL6R90-G1m, an anti-human IL-6R VHH (IL6R90) fused to the human IgG1 constant region (CH1-hinge-CH2-CH3), with various light chains. The starting point on the horizontal axis is the time when the antibody-like molecule began to interact with the antigen-immobilized sensor. [Figure 11](A) A diagram showing a model of an antibody-like molecule constructed by inserting a protease cleavage sequence near the boundary between the VHH and constant region of IL6R90-G1m. (B) A diagram showing the name of each constructed antibody heavy chain, the site where the amino acid sequence was inserted, and the inserted amino acid sequence. The insertion site is indicated by [insert]. [Figure 12-1] This figure shows the results of reducing SDS-PAGE analysis of antibody-like molecules prepared by inserting a protease cleavage sequence near the boundary between the VHH and constant region of IL6R90-G1m or IL6R90-G1m, after protease (MT-SP1) treatment, to assess the degree of cleavage. Of the two new bands that emerged upon protease treatment, the band appearing at 25 kDa or less is derived from the VHH, and the band appearing at 25-50 kDa is derived from the constant region. [Figure 12-2] This is a figure showing a continuation of Figure 12-1. [Figure 13] This figure shows the results of evaluating the binding of human IL-6R to antibody-like molecules prepared by inserting a protease cleavage sequence near the boundary between the VHH and constant region of IL6R90-G1m or IL6R90-G1m, or samples of these molecules after protease (MT-SP1) treatment. Protease- represents a sensorgram evaluating the binding of the antigen to the antibody-like molecule that has not been treated with protease, while Protease+ represents a sensorgram evaluating the binding of the antigen to the protease-treated antibody-like molecule. The horizontal axis begins 30 seconds before the antibody-like molecule begins to interact with the sensor on which the antigen is immobilized. [Figure 14] This figure shows the results of evaluating the binding to human IL-6R of antibody-like molecules prepared by associating 20A11-G1m, an anti-human IL-6R VHH (20A11) fused to the human IgG1 constant region (CH1-hinge-CH2-CH3), with various light chains. The horizontal axis starts 30 seconds before the start of the interaction between the antibody-like molecule and the antigen-immobilized sensor. [Figure 15]

[0039] Figure 1 shows the results of evaluating the binding to human IL-6R of antibody-like molecules prepared by associating 20A11hu-G1m, which was prepared by fusing 20A11hu, which was prepared by introducing mutations into amino acids at the interface with the VL of 20A11, with the constant region of human IgG1 (CH1-hinge-CH2-CH3), with various light chains. The horizontal axis starts at 60 seconds before the start of the interaction between the antibody-like molecule and the antigen-immobilized sensor. [Figure 16] Four types of antibody-like molecules were prepared by inserting a protease cleavage sequence near the boundary between 20A11hu and the constant region of 20A11-G1m or 20A11hu-G1m. These four types of antibody-like molecules were treated with protease (MT-SP1), and the extent of cleavage was assessed by reducing SDS-PAGE. Of the two new bands that emerged upon protease treatment, the band appearing at 25 kDa or less is derived from VHH, and the band appearing at 25-50 kDa is derived from the constant region. [Figure 17] This figure shows the results of evaluating the binding of antibody-like molecules prepared by inserting a protease cleavage sequence near the boundary between the VHH and constant region of 20A11-G1m or 20A11hu-G1m, or samples of these molecules after protease (MT-SP1) treatment, to human IL-6R. Protease- represents a sensorgram evaluating the binding of the antibody-like molecule to the antigen that was not treated with protease, while Protease+ represents a sensorgram evaluating the binding of the antibody-like molecule to the antigen that was treated with protease. The starting point on the horizontal axis is 60 seconds before the start of the interaction of the antibody with the sensor on which the antigen was immobilized. Samples marked "not tested" indicate that they were not measured. [Figure 18]This figure shows the results of evaluating the degree of cleavage of antibody-like molecules constructed by inserting a protease cleavage sequence near the interface between the VHH and heavy-chain constant region and containing an anti-human CD3 VHH in the heavy-chain variable region with protease (MT-SP1), followed by reducing SDS-PAGE and detection with CBB. Of the two new bands generated by protease treatment, the band at approximately 10-15 kDa is derived from the VHH, and the band at approximately 37 kDa is derived from the heavy-chain constant region. [Figure 19] This figure shows the results of evaluating the binding of human CD3ed-Fc to samples of antibody-like molecules prepared by inserting a protease cleavage sequence near the boundary between the VHH and heavy chain constant region and the protease (MT-SP1) treatment. Protease- represents a sensorgram evaluating the binding of the antigen to the antibody-like molecule that was not treated with protease, while Protease+ represents a sensorgram evaluating the binding of the antigen to the protease-treated antibody-like molecule. The horizontal axis starts 30 seconds before the start of interaction between the antibody-like molecule and the sensor on which the antigen was immobilized. The binding amount (response) before antigen binding is set to 0, and the binding amount before antibody application is set to 100. The graph begins 30 seconds before antibody application. [Figure 20]

[0049] Figure 1 shows the results of evaluating the degree of cleavage by protease (MT-SP1) treatment of antibody-like molecules prepared by inserting a protease cleavage sequence near the boundary between the light-chain variable region and the light-chain constant region of a molecule having IL6R90-G1m as a heavy chain and Vk1-39-k0MT as a light chain, or a molecule having IL6R90-G1m as a heavy chain and Vk1-39-k0MT as a light chain. The molecules were then subjected to reducing SDS-PAGE and detected with CBB. Protease treatment produced two bands derived from the light chain, indicating that the light chain was cleaved by the protease. [Figure 21]This figure shows the results of evaluating the binding of antibody-like molecules, constructed by inserting a protease cleavage sequence near the boundary between the light-chain variable region and the light-chain constant region of a molecule with IL6R90-G1m as the heavy chain and Vk1-39-k0MT as the light chain, or a molecule with IL6R90-G1m as the heavy chain and Vk1-39-k0MT as the light chain, to human IL-6R after protease (MT-SP1) treatment. Protease- represents a sensorgram evaluating the binding of the antibody-like molecule to the antigen that has not been treated with protease, while Protease+ represents a sensorgram evaluating the binding of the antibody-like molecule to the antigen that has been treated with protease. An antibody (MRA) that has been confirmed to bind to IL-6R was used as a positive control. The starting point of the horizontal axis is the time when the antibody-like molecule begins to interact with the antigen-immobilized sensor. [Figure 22] Figure 1 shows the results of SDS-PAGE evaluating protease cleavage of IgG antibody-like molecules incorporating human Plexin A1-binding VHH. The protease (+) lane represents a sample that underwent protease cleavage, while the protease (-) lane represents a negative control sample that was not subjected to protease cleavage. [Figure 23] This figure shows an Octet sensorgram of an IgG antibody-like molecule incorporating a human Plexin A1-binding VHH, which was then cleaved with protease to release the VHH. The binding of the released VHH to human Plexin A1 was assessed. Protease + represents a sample that had undergone protease cleavage, while protease - represents a sample that had not. The concentrations of the IgG antibody-like molecule used are indicated on the left side of the figure. [Figure 24] FIG. 1 shows the results of SDS-PAGE evaluating protease cleavage of bispecific VHH-VHH-containing polypeptides. [Figure 25] 1 shows luciferase activity before and after protease cleavage, with the dashed line representing the sample without protease treatment and the solid line representing the sample with protease treatment. [Figure 26]1 shows luciferase activity before and after protease cleavage, with the dashed line representing the sample without protease treatment and the solid line representing the sample with protease treatment. [Figure 27] Fig. 1 shows the results of SDS-PAGE evaluation of protease cleavage of anti-human IL-6R VHH-containing IgG antibody-like molecules. [Figure 28] FIG. 1 shows an evaluation of protease cleavage of IgG antibody-like molecules in which a protease cleavage sequence has been introduced into the light chain. [Figure 29] FIG. 1 shows an evaluation of the degree of activation of IgG-like antibody molecules in which a protease cleavage sequence has been introduced into the light chain, with or without protease treatment. [Figure 30A] FIG. 1 shows an evaluation of protease cleavage of IgG antibody-like molecules in which a protease cleavage sequence has been introduced into the heavy chain. [Figure 30B] This figure shows an evaluation of protease cleavage of an IgG antibody-like molecule in which a protease cleavage sequence was introduced into the heavy chain. Protease cleavage was performed using assay buffer (MMP Activity Assay Kit (Fluorometric - Green) (ab112146), Component C: Assay Buffer). [Figure 31] FIG. 1 shows the results of evaluating the in vivo cleavage efficiency of an antibody molecule into which a protease cleavage sequence has been inserted, when the antibody molecule was administered to mice. DETAILED DESCRIPTION OF THE INVENTION

[0017] The term "polypeptide" as used herein generally refers to peptides and proteins having a length of about four amino acids or more. The polypeptide of the present invention is generally a polypeptide consisting of an artificially designed sequence, but is not particularly limited thereto and may be, for example, a polypeptide derived from a living organism. It may also be a natural polypeptide, a synthetic polypeptide, a recombinant polypeptide, or the like. Furthermore, fragments of the above polypeptides are also included in the polypeptide of the present invention.

[0018] 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. When expressing an amino acid at a specific position, a number representing the specific position and the one-letter or three-letter code for the amino acid may be used as appropriate. For example, the amino acid 37V contained in a single-domain antibody represents Val at position 37 according to the Kabat numbering system.

[0019] 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 an amino acid by substituting an amino acid other than a natural amino acid (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 stop codon, can be suitably used. As used herein, modification includes, but is not limited to, substitution.

[0020] 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.

[0021] Herein, expressions for amino acid modifications may be appropriately expressed by listing the one-letter or three-letter codes for the amino acids 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 contained in an antibody variable region or a single-domain antibody 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 contained in an antibody constant region represents a substitution of Pro at position 238 according to the EU numbering system with Ala. That is, the number indicates the position of the amino acid as expressed in EU numbering, the one-letter or three-letter code of the amino acid written before it indicates the amino acid before substitution, and the one-letter or three-letter code of the amino acid written after it indicates the amino acid after substitution.

[0022] 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), single domain antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] As used herein, the term "complementarity determining region" or "CDR" refers to each region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops ("hypervariable loops") and / or antigen contact residues ("antigen contacts"). Typically, antibodies contain six CDRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary CDRs herein include the following: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) A combination of (a), (b), and / or (c), comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

[0027] Typically, a single domain antibody comprises three CDRs: CDR1, CDR2, and CDR3. When the single domain antibody is a VHH or a single domain antibody made from antibody VH, 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).

[0028] Where the single domain antibody is a single domain antibody made from an antibody VL, the CDRs of the single domain antibody illustratively include the following: (a) hypervariable loops occurring at amino acid residues 26-32 (CDR1), 50-52 (CDR2), and 91-96 (CDR3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigen contacts occurring at amino acid residues 27c-36 (CDR1), 46-55 (CDR2), and 89-96 (CDR3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) 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).

[0029] Unless otherwise indicated, CDR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0030] "Framework" or "FR" refers to variable domain residues other than complementarity-determining region (CDR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR sequences typically appear in the VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. In single-domain antibodies, the CDR and FR sequences typically appear in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0031] The single domain antibodies of the present invention 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).

[0032] More specifically, but not exclusively, it can be defined as a polypeptide comprising any 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.

[0033] 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 chain domain or heavy chain variable domain, followed by a heavy chain constant region (CH) containing a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain. Similarly, from the N-terminus to the C-terminus, each light chain contains a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chains of native antibodies can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0034] 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, with the exception 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 the Fc region or constant region is according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0035] 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.

[0036] 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, as long as 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).

[0037] Suitable examples of antigen-binding domains of the present invention include antigen-binding domains that can exhibit antigen-binding function in a molecule composed only of the antigen-binding domain, and antigen-binding domains that can exhibit antigen-binding function independently after being released from other peptides to which they are linked. Examples of such antigen-binding domains include, but are not limited to, single-domain antibodies, scFv, Fv, Fab, Fab', and F(ab')2.

[0038] A suitable example of an antigen-binding domain of the present invention 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, preferred examples of antigen-binding domains of the present invention include antigen-binding domains with a blood half-life of 12 hours or less, including, but not limited to, single-domain antibodies, scFv, Fab, and Fab'.

[0039] A suitable example of the antigen-binding domain of the present invention is a single-domain antibody (sdAb).

[0040] As used herein, the term "single-domain antibody" is not limited by its structure, as long as the domain alone can exhibit antigen-binding activity. Conventional antibodies, exemplified by IgG antibodies, exhibit antigen-binding activity when the variable region is formed by pairing of VH and VL, whereas single-domain antibodies are known to be able to exhibit antigen-binding activity solely through the domain structure of the single-domain antibody itself, without pairing with any other domain. Single-domain antibodies usually have a relatively low molecular weight and exist in the form of a monomer. Examples of single domain antibodies include, but are not limited to, camelid VHHs, shark VHHs, and the like. NARExamples 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, or antibody fragments comprising all or a portion of the VH domain or all or a portion of the VL domain of an antibody. Examples of single-domain antibodies, which are antibody fragments comprising all or a portion of the VH / VL domains of an antibody, include, but are not limited to, single-domain 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. In some embodiments of the present invention, a single-domain antibody has three CDRs (CDR1, CDR2, and CDR3). 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 of the present invention. A "humanized single-domain antibody" refers to a chimeric single-domain antibody comprising amino acid residues from non-human CDRs and human FRs. In one embodiment, a humanized single-domain antibody has 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 still considered an example in which substantially all of the FRs correspond to those of a human antibody. For example, when humanizing a VHH, which is one embodiment of a single-domain antibody, some of the residues in the FRs must be changed to residues that do not correspond to those of a human antibody (C. Vincke et al., The Journal of Biological Chemistry 284, 3273-3284). 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 prepared 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)).

[0041] 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 used to treat diseases caused by target tissues 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.

[0042] 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.

[0043] Although the above examples of antigens also include receptors, these receptors present in soluble form in biological fluids can also be used as antigens to which the antigen-binding domains of the present invention bind. A non-limiting example of such a soluble receptor is the protein represented by SEQ ID NO: 35, which is the soluble IL-6R described by Mullberg et al. (J. Immunol. (1994) 152(10), 4958-4968).

[0044] Examples of the antigens include membrane molecules expressed on the cell membrane and soluble molecules secreted extracellularly from cells. When the antigen-binding domain of the present invention binds to a soluble molecule secreted from cells, it is preferable that the antigen-binding domain has neutralizing activity.

[0045] There is no limitation on the solution in which a soluble molecule exists, and the soluble molecule can exist in biological fluids, i.e., all fluids filling the vessels or 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 invention can 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 gastrointestinal tract and other body cavities), such as alveolar fluid (bronchoalveolar lavage fluid), ascites, pleural effusion, pericardial fluid, cystic fluid, or aqueous humor (aqueous humor).

[0046] An epitope, meaning an antigenic determinant present in an antigen, refers to a site on the antigen to which the antigen-binding domain disclosed herein binds. Thus, for example, an epitope can be defined by its structure. Alternatively, an epitope can be defined by the binding activity of the antigen-binding domain that recognizes the epitope to the antigen. When the antigen is a peptide or polypeptide, the epitope can also be identified by the amino acid residues that make up the epitope. Furthermore, when the epitope is a glycan, the epitope can also be identified by a specific glycan structure.

[0047] A linear epitope is one in which the primary amino acid sequence comprises a recognized epitope, typically comprising at least three, and most usually at least five, e.g., about 8 to about 10, 6 to 20 amino acids in a unique sequence.

[0048] Conformational epitopes, in contrast to linear epitopes, are epitopes in which the primary sequence of amino acids comprising the epitope is not the single, defined component of the recognized epitope (e.g., an epitope in which the primary sequence of amino acids is not necessarily recognized by the antibody that defines the epitope). Conformational epitopes may encompass an increased number of amino acids relative to linear epitopes. In recognizing conformational epitopes, the antigen-binding domain recognizes the three-dimensional structure of a peptide or protein. For example, when a protein molecule folds to form a three-dimensional structure, certain amino acids and / or polypeptide backbones that form a conformational epitope are juxtaposed, allowing the antibody to recognize the epitope. Methods for determining the conformational structure of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-directed spin labeling and electromagnetic paramagnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

[0049] The structure of the antigen-binding domain that binds to the epitope is called the paratope. The epitope and paratope bind stably due to hydrogen bonds, electrostatic forces, van der Waals forces, hydrophobic bonds, etc. that act between them. The binding strength between this epitope and paratope is called affinity. The sum of the binding strengths when multiple antigens bind to multiple antigen-binding domains is called avidity. When an antibody or other substance containing multiple antigen-binding domains (i.e., a polyvalent antibody) binds to multiple epitopes, the binding strengths act synergistically, so avidity is higher than affinity.

[0050] In certain embodiments, the antigen-binding domains provided herein have an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 It has a dissociation constant (Kd) of 1 M.

[0051] Methods for confirming epitope binding by an antigen-binding domain for IL-6R or a polypeptide containing an antigen-binding domain are exemplified below; however, methods for confirming epitope binding by an antigen-binding domain for an antigen other than IL-6R or a polypeptide containing an antigen-binding domain can also be appropriately carried out in accordance with the examples below.

[0052] For example, whether an antigen-binding domain for IL-6R recognizes a linear epitope present in the IL-6R molecule can be confirmed, for example, as follows. For this purpose, a linear peptide consisting of the amino acid sequence constituting the extracellular domain of IL-6R is synthesized. This peptide can be chemically synthesized. Alternatively, it can be obtained by genetic engineering techniques using a region of IL-6R cDNA encoding the amino acid sequence corresponding to the extracellular domain. The binding activity of the linear peptide consisting of the amino acid sequence constituting the extracellular domain to the antigen-binding domain for IL-6R is then assessed. For example, the binding activity of the antigen-binding domain to the peptide can be assessed by ELISA using an immobilized linear peptide as the antigen. Alternatively, the binding activity of the antigen-binding domain to the linear peptide can be determined based on the level of inhibition by the linear peptide of the binding of the antigen-binding domain to IL-6R-expressing cells. These tests can determine the binding activity of the antigen-binding domain to the linear peptide.

[0053] Furthermore, recognition of a conformational epitope by an IL-6R antigen-binding domain can be confirmed as follows. For the above purpose, cells expressing IL-6R are prepared. Examples of such cases include when an IL-6R antigen-binding domain strongly binds to IL-6R-expressing cells upon contact with the cells, but does not substantially bind to a linear peptide consisting of the amino acid sequence constituting the extracellular domain of IL-6R to which the antigen-binding domain has been immobilized, or to a linear peptide consisting of the amino acid sequence constituting the extracellular domain of IL-6R denatured using a common denaturing agent such as guanidine. Here, "not substantially binding" refers to a binding activity that is 80% or less, typically 50% or less, preferably 30% or less, and particularly preferably 15% or less of the binding activity to human IL-6R-expressing cells.

[0054] Another method for confirming the antigen-binding activity of an antigen-binding domain is to measure the Kd value by radiolabeled antigen binding assay (RIA). In one embodiment, RIA is performed using the antigen-binding domain of interest and its antigen. For example, the solution binding affinity of the antigen-binding domain for an antigen is measured by equilibrating the antigen-binding domain with a minimum concentration of (I)-labeled antigen in the presence of increasing amounts of unlabeled antigen, and then capturing the bound antigen using a plate coated with the antigen-binding domain. (See, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)).

[0055] In another embodiment, Kd is measured by surface plasmon resonance using a BIACORE®. For example, measurements using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) are performed at 25°C using a CM5 chip with approximately 10 response units (RU) of antigen immobilized thereon. In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8, before injection at a flow rate of 5 μl / min to achieve protein binding of approximately 10 response units (RU). After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions (0.78 nM to 500 nM) of the antigen-binding domain are injected in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at a flow rate of approximately 25 μl / min at 25°C. The association rate (k) and dissociation rate (k) are calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software Version 3.2). The equilibrium dissociation constant (K) is calculated as the ratio of k / k. Furthermore, the apparent dissociation constant (K) can also be determined using equilibrium analysis. For these methods, refer to the protocols provided with BIACORE®. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999) and Methods Enzymol. 2000;323:325-40. In a surface plasmon resonance assay, the amount of protein immobilized, the amount of protein used in the reaction, the temperature, and the solution composition can be varied as desired by those skilled in the art.On-rate of 10 by the surface plasmon resonance assay described above. 6 M -1 s -1 If the on-rate exceeds , the on-rate can be determined by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandpass 16 nm) of 20 nM antigen-binding domain in PBS, pH 7.2 at 25°C in the presence of increasing concentrations of antigen, as measured in a spectrometer (e.g., a stopped-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) using a stirred cuvette).

[0056] Furthermore, the antigen-binding activity of an antigen-binding domain can also be measured by known methods for measuring intermolecular interactions, such as electrochemiluminescence.

[0057] Methods for measuring the binding activity of an IL-6R antigen-binding domain to IL-6R-expressing cells include, for example, the method described in Antibodies: A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420). Specifically, the binding activity can be assessed by ELISA or fluorescence activated cell sorting (FACS) using IL-6R-expressing cells as antigens.

[0058] In the ELISA format, the binding activity of an IL-6R antigen-binding domain to IL-6R-expressing cells is quantitatively assessed by comparing the signal levels generated by the enzymatic reaction. Specifically, a test antigen-binding domain is added to an ELISA plate on which IL-6R-expressing cells have been immobilized, and the cell-bound test antigen-binding domain is detected using an enzyme-labeled antibody that recognizes the test antigen-binding domain. Alternatively, in FACS, a dilution series of the test antigen-binding domain is prepared, and the antibody binding titer to IL-6R-expressing cells is determined, allowing the binding activity of the test antigen-binding domain to IL-6R-expressing cells to be compared.

[0059] The binding of a test antigen-binding domain to an antigen expressed on the surface of cells suspended in a buffer solution or the like can be detected using a flow cytometer. Known flow cytometers include, for example, the following: FACSCanto™ II FACSAria™ FACSArray™ FACSVantage™ SE FACSCalibur™ (both are trade names of BD Biosciences) EPICS ALTRA HyperSort Cytomics FC 500 EPICS XL-MCL ADC EPICS XL ADC Cell Lab Quanta / Cell Lab Quanta SC (both are trade names of Beckman Coulter)

[0060] For example, the following method is an example of a suitable method for measuring the antigen-binding activity of an antigen-binding domain against IL-6R. First, the test antigen-binding domain is reacted with cells expressing IL-6R and stained with an FITC-labeled secondary antibody that recognizes the test antigen-binding domain. The test antigen-binding domain is diluted with an appropriate buffer solution to prepare the desired concentration for use. For example, the antigen-binding domain can be used at any concentration between 10 μg / ml and 10 ng / ml. Next, the fluorescence intensity and cell number are measured using a FACSCalibur (BD). The amount of antigen-binding domain binding to the cells is reflected in the fluorescence intensity, i.e., the Geometric Mean value, obtained by analysis using CELL QUEST Software (BD). In other words, by obtaining the Geometric Mean value, the binding activity of the test antigen-binding domain, represented by the amount of binding of the test antigen-binding domain, can be measured.

[0061] Whether an antigen-binding domain for IL-6R shares an epitope with another antigen-binding domain can be confirmed by competition between the two for the same epitope. Competition between antigen-binding domains can be detected by cross-blocking assays, for example. For example, competitive ELISA assays are preferred cross-blocking assays.

[0062] Specifically, in a cross-blocking assay, IL-6R protein coated on the wells of a microtiter plate is preincubated in the presence or absence of a candidate competing antigen-binding domain, after which a test antigen-binding domain is added. The amount of test antigen-binding domain bound to IL-6R protein in the well is indirectly correlated with the binding ability of the candidate competing antigen-binding domain that competes for binding to the same epitope. In other words, the greater the affinity of the competing antigen-binding domain for the same epitope, the lower the binding activity of the test antigen-binding domain to wells coated with IL-6R protein.

[0063] The amount of test antigen-binding domain bound to the well via the IL-6R protein can be easily measured by labeling the antigen-binding domain in advance. For example, a biotin-labeled antigen-binding domain can be measured using an avidin-peroxidase conjugate and an appropriate substrate. Cross-blocking assays using enzyme labels such as peroxidase are particularly known as competitive ELISA assays. The antigen-binding domain can also be labeled with other detectable or measurable labeling substances. Specific examples include radiolabels and fluorescent labels.

[0064] A test antigen-binding domain binds to substantially the same epitope as a competitor antigen-binding domain or competes for binding to the same epitope as a competitor antigen-binding domain if the competitor antigen-binding domain can block the binding of the antigen-binding domain to IL-6R by at least 20%, preferably at least 20-50%, and more preferably at least 50%, compared to the binding activity obtained in a control test performed in the absence of the candidate competitor antigen-binding domain.

[0065] When the structure of the epitope to which an IL-6R antigen-binding domain binds has been identified, whether a test antigen-binding domain and a control antigen-binding domain share a common epitope can be assessed by comparing the binding activity of both antigen-binding domains toward a peptide or polypeptide in which amino acid mutations have been introduced into the peptide constituting the epitope.

[0066] For example, such binding activity can be measured by comparing the binding activity of a test antigen-binding domain and a control antigen-binding domain toward a mutated linear peptide in the above-described ELISA format. As a method other than ELISA, the binding activity toward the mutant peptide bound to a column can also be measured by passing the test antigen-binding domain and the control antigen-binding domain down the column and then quantifying the antigen-binding domain eluted in the eluate. Methods for adsorbing mutant peptides to a column, for example, as fusion peptides with GST, are known.

[0067] Furthermore, if the identified epitope is a conformational epitope, whether the test antigen-binding domain and the control antigen-binding domain share a common epitope can be assessed by the following method. First, cells expressing IL-6R and cells expressing IL-6R with an epitope mutation introduced are prepared. These cells are suspended in an appropriate buffer, such as PBS, and the test antigen-binding domain and the control antigen-binding domain are added to the cell suspension. Next, an FITC-labeled antibody that can recognize the test antigen-binding domain and the control antigen-binding domain is added to the cell suspension after washing with an appropriate buffer. The fluorescence intensity and cell count of cells stained with the labeled antibody are measured using a FACSCalibur (BD). The test antigen-binding domain and the control antigen-binding domain are adjusted to the desired concentration by diluting them with a suitable buffer. For example, they are used at a concentration between 10 μg / ml and 10 ng / ml. The amount of labeled antibody bound to the cells is reflected in the fluorescence intensity, i.e., the geometric mean value, obtained by analysis using CELL QUEST Software (BD). That is, by obtaining the Geometric Mean value, the binding activity of the test antigen-binding domain and the control antigen-binding domain, represented by the amount of bound labeled antibody, can be measured.

[0068] Furthermore, in addition to the above-mentioned ELISA and FACS, radiolabeled antigen binding assay (RIA), BIACORE (registered trademark) surface plasmon resonance assay, electrochemiluminescence, etc. can also be used to confirm competition between an antigen-binding domain and another antigen-binding domain for the same epitope.

[0069] In this method, "substantially no binding to mutant IL-6R-expressing cells" can be determined, for example, by the following method. First, the test antigen-binding domain and control antigen-binding domain bound to cells expressing mutant IL-6R are stained with a labeled antibody. The fluorescence intensity of the cells is then detected. When a FACSCalibur is used for flow cytometry to detect fluorescence, the obtained fluorescence intensity can be analyzed using CELL QUEST Software. The percentage increase in fluorescence intensity due to antigen-binding domain binding can be determined by calculating the comparative value (ΔGeo-Mean) from the Geometric Mean values ​​in the presence and absence of the polypeptide complex according to Formula 1 below.

[0070] (Formula 1) ΔGeo-Mean = Geo-Mean (in the presence of polypeptide complex) / Geo-Mean (in the absence of polypeptide complex)

[0071] The Geometric Mean comparison value (mutant IL-6R molecule ΔGeo-Mean value), which reflects the binding amount of the test antigen-binding domain to mutant IL-6R-expressing cells obtained by analysis, is compared with the ΔGeo-Mean comparison value, which reflects the binding amount of the test antigen-binding domain to IL-6R-expressing cells. In this case, it is particularly preferred that the test antigen-binding domains used to determine the ΔGeo-Mean comparison values ​​for mutant IL-6R-expressing cells and IL-6R-expressing cells are prepared at the same or substantially the same concentrations. An antigen-binding domain that has been previously confirmed to recognize an epitope in IL-6R is used as a control antigen-binding domain.

[0072] A test antigen-binding domain is deemed to "not substantially bind to mutant IL-6R-expressing cells" if the ΔGeo-Mean comparison value for the test antigen-binding domain with mutant IL-6R-expressing cells is at least 80%, preferably 50%, more preferably 30%, and particularly preferably 15% of the ΔGeo-Mean comparison value for the test antigen-binding domain with IL-6R-expressing cells. The formula for calculating the Geo-Mean value (Geometric Mean) is described in the CELL QUEST Software User's Guide (BD biosciences). When the comparison values ​​are substantially equivalent, the epitopes of the test antigen-binding domain and the control antigen-binding domain can be determined to be identical.

[0073] As used herein, the term "transport moiety" refers to a portion of a polypeptide other than the antigen-binding domain. The transport moiety of the present invention is typically a peptide or polypeptide composed of amino acids. In a specific embodiment, the transport moiety in the polypeptide is linked to the antigen-binding domain via a cleavage site. The transport moiety of the present invention 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.

[0074] The delivery moiety of the present invention 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 consist of the entire delivery moiety or a portion of the delivery moiety.

[0075] In some embodiments of the present invention, releasing the antigen-binding domain from the polypeptide increases its antigen-binding activity 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). In some embodiments of the present invention, the antigen-binding activity of an antigen-binding domain released from a 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 of the antigen-binding domain when not released from the polypeptide. In more specific embodiments of the present invention, when the antigen-binding activity of the antigen-binding domain before release is measured by a method selected from the above methods, no binding between the antigen-binding domain and the antigen is observed. In some embodiments of the present invention, cleavage of the cleavage site allows the antigen-binding domain to be released from the polypeptide, and in such embodiments, 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 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 antigen-binding activity measured using the uncleaved polypeptide. In more specific embodiments, when the antigen-binding activity of the uncleaved polypeptide is measured using a method selected from the above methods, no binding between the antigen-binding domain and the antigen is observed. In some embodiments of the present invention, the cleavage site is cleaved by a protease, and 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 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 antigen-binding activity measured using the polypeptide that has not been treated with a protease. In more specific embodiments, when the antigen-binding activity of the polypeptide that has not been treated with a protease is measured using a method selected from the above methods, no binding between the antigen-binding domain and the antigen is observed.

[0076] In the present invention, polypeptides comprising an antigen-binding domain and a delivery moiety have a longer serum half-life than the antigen-binding domain alone. To prolong the half-life of the polypeptide, in some embodiments of the present invention, 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, a delivery moiety that binds to FcRn, a delivery moiety that binds to albumin, or a delivery moiety that is PEGylated. Additionally, in some embodiments of the present invention, 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).

[0077] In the present invention, 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).

[0078] 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.

[0079] One embodiment for extending the blood half-life of a delivery moiety is to confer FcRn-binding ability to the delivery moiety. This can usually be achieved by providing an FcRn-binding domain within the delivery moiety. The FcRn-binding domain refers to a domain that has FcRn-binding ability, and any structure can be used as long as it has FcRn-binding ability. Delivery moieties containing the FcRn-binding domain can be taken up into cells via the FcRn salvage pathway and then returned to the plasma. For example, the relatively long 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 regions capable of binding to polypeptides 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 invention may also be a region that binds to such polypeptides capable of binding to FcRn.

[0080] The binding activity of the FcRn-binding region of the present invention 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), dissociation rate kd (Apparent dissociation rate), or apparent kd (Apparent dissociation rate). These can be measured by methods known to those skilled in the art. For example, Biacore (GE Healthcare), Scatchard plot, flow cytometer, etc. may be used.

[0081] 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 invention can be measured by methods known to those skilled in the art, such as using Biacore (GE Healthcare). The binding activity of the FcRn-binding region to FcRn can be assessed by passing FcRn, the FcRn-binding region, or the FcRn-binding region as an analyte through a chip on which the FcRn-binding region or a carrier moiety containing the FcRn-binding region is immobilized, or FcRn is immobilized.

[0082] 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 invention.

[0083] An example of an FcRn-binding region is, but is not limited to, an IgG antibody Fc region. When an IgG antibody Fc region is used, the type is not limited, and Fc regions such as IgG1, IgG2, IgG3, and IgG4 can be used. For example, an Fc region comprising one amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOs: 21, 22, 23, and 24 can be used.

[0084] 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.

[0085] More specifically, EU numbering in the Fc region of an IgG antibody 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:

[0086] From another perspective, the EU numbering in the IgG antibody Fc region 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 position 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 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 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:

[0087] 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.

[0088] 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).

[0089] 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.).

[0090] In some embodiments of the invention, the delivery moiety comprises an antibody Fc region. In one specific embodiment, the delivery moiety comprises the CH2 and CH3 domains of a human IgG antibody. In one specific embodiment, the delivery moiety comprises a portion of a human IgG1 antibody heavy chain extending from Cys226 or from 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.

[0091] In some embodiments of the invention, the delivery moiety comprises an antibody constant region. In more preferred embodiments, the delivery moiety comprises an IgG antibody constant region. In more preferred embodiments, the delivery moiety comprises a human IgG antibody constant region.

[0092] In some further embodiments of the present invention, the delivery 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, bound to the heavy chain constant region by covalent bonds such as disulfide bonds or non-covalent bonds such as hydrogen bonds or hydrophobic interactions.

[0093] As used herein, a "polypeptide comprising an antigen-binding domain and a transport moiety" is typically a sequence of polypeptides linked by amide bonds, or a protein comprising multiple sequences of polypeptides linked by amide bonds.

[0094] In some embodiments of the present invention, the antigen-binding domain is releasable from the polypeptide, and release of the antigen-binding domain from the polypeptide increases the antigen-binding activity. As used herein, the term "release" refers to the separation of two portions of a polypeptide from each other. Release of the antigen-binding domain from the polypeptide can be caused by the dissolution of the interaction between the antigen-binding domain and the delivery moiety. Since the antigen-binding activity of an antigen-binding domain incorporated into a polypeptide is suppressed, release of the antigen-binding domain from the polypeptide can be confirmed by measuring the antigen-binding activity of the target substance and comparing it with the antigen-binding activity of the antigen-binding domain incorporated into the polypeptide.

[0095] In some embodiments, the polypeptide contains a cleavage site, and cleavage of the cleavage site releases the antigen-binding domain from the polypeptide. The cleavage site can be cleaved, for example, by an enzyme, by reduction with a reducing agent, or by photolysis. The cleavage site may be located at any position in the polypeptide, as long as it allows the antigen-binding domain to be released and does not abolish the antigen-binding activity of the released antigen-binding domain. Furthermore, in addition to the cleavage site for releasing the antigen-binding domain, another cleavage site may be contained in the polypeptide. In one embodiment of the present invention, the cleavage site contains a protease cleavage sequence and can be cleaved by a protease.

[0096] As used herein, the term "cleaved" refers to a state in which the antigen-binding domain and the transport moiety are separated after modification of the cleavage site by a protease and / or reduction of the cysteine-cysteine ​​disulfide bond at the cleavage site and / or photoactivation. As used herein, the term "uncleaved" refers to a state in which the antigen-binding domain and the transport moiety are linked in the absence of cleavage of the cleavage site by a protease and / or reduction of the cysteine-cysteine ​​disulfide bond at the cleavage site and / or in the absence of light.

[0097] Cleavage at the cleavage site can be detected by subjecting a solution containing the cleavage site-containing polypeptide to SDS-PAGE (polyacrylamide gel electrophoresis) and measuring the molecular weight of the fragments, or by detecting the change in molecular weight before and after cleavage.

[0098] The cleavage site varies from approximately 0.001 to 1500 × 10 depending on the agent (i.e., protease, reducing agent, light). 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, reduced or photolyzed) at rates of .

[0099] Specific cleavage by a protease requires contact between the protease and the cleavage site or a molecule containing the cleavage site. In the presence of sufficient enzymatic activity, the cleavage site can be cleaved. Sufficient enzymatic activity can refer to the ability of the enzyme to contact the cleavage site and effect cleavage.

[0100] As used herein, the term "protease" refers to an enzyme, such as an endopeptidase or exopeptidase, that hydrolyzes peptide bonds, typically an endopeptidase. The protease used in the present invention is limited only by its ability to cleave the protease cleavage sequence, and its type is not particularly limited. In some embodiments, a target tissue-specific protease is used. The target tissue-specific protease may be, for example, (1) a protease that is expressed at a higher level in target tissue than in normal tissue; (2) a protease that has higher activity in target tissues than in normal tissues; (3) a protease that is expressed at a higher level in target cells than in normal cells; (4) a protease that has higher activity in target cells than in normal cells; It can refer to either of the following. In more specific embodiments, cancer tissue-specific proteases or inflamed tissue-specific proteases are used.

[0101] As used herein, the term "target tissue" refers to a tissue that contains at least one target cell. In some embodiments of the present invention, the target tissue is cancerous tissue. In some embodiments of the present invention, the target tissue is inflamed tissue.

[0102] 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.

[0103] 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

[0104] Proteases that are specifically expressed or specifically activated in several types of target tissues or that are thought to be associated with the disease state of the target tissues (target tissue-specific proteases) are known. For example, International Publication Nos. WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846 disclose proteases that are specifically expressed in cancer tissues. Furthermore, proteases thought to be associated with inflammation have been disclosed in J Inflamm (Lond). 2010; 7: 45., Nat Rev Immunol. 2006 Jul; 6(7): 541-50., Nat Rev Drug Discov. 2014 Dec; 13(12): 904-27., Respir Res. 2016 Mar 4; 17: 23., Dis Model Mech. 2014 Feb; 7(2): 193-203., and Biochim Biophys Acta. 2012 Jan; 1824(1): 133-45. In addition to proteases that are specifically expressed in target tissues, there are also proteases that are specifically activated in target tissues. For example, proteases can be expressed in an inactive form and then become active. In many tissues, substances that inhibit active proteases exist, and their activity is controlled by the activation process and the presence of inhibitors (Nat Rev Cancer. 2003 Jul;3(7):489-501.). In target tissues, active proteases can escape inhibition and become specifically activated. Active proteases can be measured using an antibody that recognizes the active form of the protease (PNAS 2013 Jan 2; 110(1): 93-98.) or a method in which a peptide recognized by the protease is fluorescently labeled, and the fluorescent light is quenched before cleavage but emits light after cleavage (Nat Rev Drug Discov. 2010 Sep;9(9):690-701. doi: 10.1038 / nrd3053.). From one perspective, the term "target tissue-specific protease" means: (i) a protease that is expressed at a higher level in the target tissue than in normal tissue; (ii) a protease that has higher activity in target tissue than in normal tissue; (iii) a protease that is expressed at a higher level in target cells than in normal cells; (iv) a protease that has higher activity in target cells than in normal cells; It can refer to either of the following.

[0105] Specific proteases include, but are not limited to, cysteine ​​proteases (including cathepsin family B, L, S, etc.), aspartyl proteases (cathepsin D, E, K, O, etc.), serine proteases (including matriptase (MT-SP1), cathepsin A and G, thrombin, plasmin, urokinase (uPA), tissue plasminogen activator (tPA), elastase, proteinase 3, thrombin, kallikrein, tryptophan, etc.), and the like. metalloproteases (including membrane-bound (MMP14-17 and MMP24-25) and secreted (MMP1-13, MMP18-23, and MMP26-28) metalloproteases (MMP1-28); 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.

[0106] From another perspective, the target tissue-specific protease can refer to a cancer tissue-specific protease or an inflamed tissue-specific protease. Examples of cancer tissue-specific proteases include proteases that are specifically expressed in cancer tissues, such as those disclosed in International Publication Nos. WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846.

[0107] The more specific the type of cancer tissue-specific protease is expressed in the cancer tissue to be treated, the more effective it is in reducing side effects. The concentration of the cancer tissue-specific protease in cancer tissue is preferably at least 5 times higher than the concentration in normal tissue, more preferably at least 10 times higher, even more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. Furthermore, the activity of the cancer tissue-specific protease in cancer tissue is preferably at least 2 times higher than the activity in normal tissue, more preferably at least 3 times higher, more preferably at least 4 times higher, more preferably at least 5 times higher, even more preferably at least 10 times higher, even more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. Furthermore, the cancer tissue-specific protease may be one that is bound to the cell membrane of cancer cells, or one that is not bound to the cell membrane and is secreted extracellularly. When the cancer tissue-specific protease is not bound to the cell membrane of cancer cells, it is preferable that the cancer tissue-specific protease is present inside or near the cancer tissue, so that cytotoxicity by immune cells is specific to cancer cells. As used herein, "near the cancer tissue" means within a range in which the cancer tissue-specific protease cleavage sequence is cleaved and the antigen-binding domain exerts its antigen-binding activity. However, it is preferable that the range be one in which normal cells are not damaged as much as possible. From another perspective, cancer tissue-specific proteases are (i) proteases that are expressed at higher levels in cancer tissues than in normal tissues; (ii) a protease that has higher activity in cancer tissue than in normal tissue; (iii) proteases that are expressed at higher levels in cancer cells than in normal cells; (iv) proteases that have higher activity in cancer cells than in normal cells; Either: The cancer tissue-specific protease may be used alone or in combination of two or more types. The number of types of cancer tissue-specific proteases can be appropriately determined by those skilled in the art, taking into consideration the type of cancer to be treated.

[0108] From the above viewpoints, among the proteases exemplified above, serine proteases and metalloproteases are preferred as cancer tissue-specific proteases, matriptase (including MT-SP1), urokinase (uPA) and metalloproteases are more preferred, and MT-SP1, uPA, MMP-2 and MMP-9 are even more preferred.

[0109] The more specific the type of inflammatory tissue-specific protease is expressed in the inflamed tissue of the treatment target, the more effective it is in reducing side effects. The concentration of the inflammatory tissue-specific protease in the inflamed tissue is preferably at least 5 times higher than the concentration in normal tissue, more preferably at least 10 times higher, even more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. Furthermore, the activity of the inflammatory tissue-specific protease in the inflamed tissue is preferably at least 2 times higher than the activity in normal tissue, more preferably at least 3 times higher, at least 4 times higher, at least 5 times higher, or at least 10 times higher, more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. Furthermore, the inflammatory tissue-specific protease may be bound to the cell membrane of an inflammatory cell, or may be secreted extracellularly without being bound to the cell membrane. When the inflammatory tissue-specific protease is not bound to the cell membrane of an inflammatory cell, it is preferable that the inflammatory tissue-specific protease is present inside or near the inflammatory tissue, so that cytotoxicity by immune cells is specific to the inflammatory cell. As used herein, "near the inflammatory tissue" means within a range in which the inflammatory tissue-specific protease cleavage sequence is cleaved and the antigen-binding domain exerts its antigen-binding activity. However, it is preferable that the range be within which normal cells are not damaged as much as possible. From another perspective, inflammatory tissue-specific proteases are (i) proteases that are expressed at higher levels in inflamed tissues than in normal tissues; (ii) proteases that have higher activity in inflamed tissue than in normal tissue; (iii) proteases that are expressed at higher levels in inflammatory cells than in normal cells; (iv) proteases that have higher activity in inflammatory cells than in normal cells; Either: The inflammatory tissue-specific protease may be used alone or in combination of two or more types. The number of types of inflammatory tissue-specific proteases can be appropriately determined by those skilled in the art, taking into consideration the condition of the target to be treated.

[0110] From the above viewpoint, among the above-mentioned proteases, metalloproteases are preferred as the inflamed tissue-specific proteases, and among the metalloproteases, ADAMTS5, MMP-2, MMP-7, MMP-9, and MMP-13 are more preferred.

[0111] A protease cleavage sequence is a particular amino acid sequence that is specifically recognized by a target tissue-specific protease when a polypeptide is hydrolyzed by the target tissue-specific protease in an aqueous solution. From the viewpoint of reducing side effects, the protease cleavage sequence is preferably an amino acid sequence that is hydrolyzed with high specificity by a target tissue-specific protease that is more specifically expressed or more specifically activated in the target tissue / cells to be treated. Specific examples of protease cleavage sequences include target sequences that are specifically hydrolyzed by the proteases specifically expressed in cancer tissues and proteases specific to inflammatory tissues, as disclosed in International Publication Nos. WO 2013 / 128194, WO 2010 / 081173, WO 2009 / 025846, etc. Artificially modified sequences, such as those obtained by introducing appropriate amino acid mutations into target sequences that are specifically hydrolyzed by known proteases, can also be used. Furthermore, protease cleavage sequences identified by methods known to those skilled in the art, such as those described in Nature Biotechnology 19, 661-667 (2001), may also be used. Furthermore, a naturally occurring protease cleavage sequence may be used. For example, a protease cleavage sequence in a protein whose molecular shape changes upon protease cleavage may be used, just as TGF-β is converted to its latent form upon protease cleavage.

[0112] Examples of protease cleavage sequences include, but are not limited to, those described in International Publication No. WO2015 / 116933, International Publication No. WO2015 / 048329, International Publication No. WO2016 / 118629, International Publication No. WO2016 / 179257, International Publication No. WO2016 / 179285, International Publication No. WO2016 / 179335, International Publication No. WO2016 / 179003, International Publication No. WO2016 / 046778, International Publication No. WO2016 / 014974, U.S. Patent Publication No. US2016 / 0289324, U.S. Patent Publication No. US2016 / 0311903, PNAS (2000) 97: 7754-7759, Biochemical Journal (2010) 426: 219-228., Beilstein J Nanotechnol. (2016) 7: 364-373. can be used. As described above, the protease cleavage sequence is preferably an amino acid sequence that is specifically hydrolyzed by a suitable target tissue-specific protease. Among the amino acid sequences that are specifically hydrolyzed by a target tissue-specific protease, a sequence containing the following amino acid sequence is preferred. LSGRSDNH (SEQ ID NO: 12, MT-SP1, cleavable by uPA) PLALAG (SEQ ID NO: 25, cleavable by MMP-2 and MMP-9) VPLSLTMG (SEQ ID NO: 26, cleavable by MMP-7) The following sequences can also be used as protease cleavage sequences: TSTSGRSANPRG (SEQ ID NO: 74, MT-SP1, cleavable by uPA) ISSGLLSGRSDNH (SEQ ID NO: 75, MT-SP1, cleavable by uPA) AVGLLAPPGGLSGRSDNH (SEQ ID NO: 76, MT-SP1, cleavable by uPA) GAGVPMSMRGGAG (SEQ ID NO: 77, cleavable by MMP-1) GAGIPVSLRSGAG ​​(SEQ ID NO: 78, cleavable by MMP-2) GPLGIAGQ (SEQ ID NO: 79, cleavable by MMP-2) GGPLGMLSQS (SEQ ID NO: 80, cleavable by MMP-2) PLGLWA (SEQ ID NO: 81, cleavable by MMP-2) GAGRPFSMIMGAG (SEQ ID NO: 82, cleavable by MMP-3) GAGVPLSLTMGAG (SEQ ID NO: 83, cleavable by MMP-7) GAGVPLSLYSGAG (SEQ ID NO: 84, cleavable by MMP-9) AANLRN (SEQ ID NO: 85, cleavable by MMP-11) AQAYVK (SEQ ID NO: 86, cleavable by MMP-11) AANYMR (SEQ ID NO: 87, cleavable by MMP-11) AAALTR (SEQ ID NO: 88, cleavable by MMP-11) AQNLMR (SEQ ID NO: 89, cleavable by MMP-11) AANYTK (SEQ ID NO: 90, cleavable by MMP-11) GAGPQGLAGQRGIVAG (SEQ ID NO: 91, cleavable by MMP-13) PRFKIIGG (SEQ ID NO: 92, pro-cleavable by urokinase) PRFRIIGG (SEQ ID NO: 93, pro-urokinase cleavable) GAGSGRSAG (SEQ ID NO: 94, cleavable by uPA) SGRSA (SEQ ID NO: 95, cleavable by uPA) GSGRSA (SEQ ID NO: 96, cleavable by uPA) SGKSA (SEQ ID NO: 97, cleavable by uPA) SGRSS (SEQ ID NO: 98, cleavable by uPA) SGRRA (SEQ ID NO: 99, cleavable by uPA) SGRNA (SEQ ID NO: 100, cleavable by uPA) SGRKA (SEQ ID NO: 101, cleavable by uPA) QRGRSA (SEQ ID NO: 102, cleavable by tPA) GAGSLLKSRMVPNFNAG (SEQ ID NO: 103, cleavable by cathepsin B) TQGAAA (SEQ ID NO: 104, cleavable by cathepsin B) GAAAAAA (SEQ ID NO: 105, cleavable by cathepsin B) GAGAAG (SEQ ID NO: 106, cleavable by cathepsin B) AAAAAG (SEQ ID NO: 107, cleavable by cathepsin B) LCGAAI (SEQ ID NO: 108, cleavable by cathepsin B) FAQALG (SEQ ID NO: 109, cleavable by cathepsin B) LLQANP (SEQ ID NO: 110, cleavable by cathepsin B) LAAANP (SEQ ID NO: 111, cleavable by cathepsin B) LYGAQF (SEQ ID NO: 112, cleavable by cathepsin B) LSQAQG (SEQ ID NO: 113, cleavable by cathepsin B) ASAASG (SEQ ID NO: 114, cleavable by cathepsin B) FLGASL (SEQ ID NO: 115, cleavable by cathepsin B) AYGATG (SEQ ID NO: 116, cleavable by cathepsin B) LAQATG (SEQ ID NO: 117, cleavable by cathepsin B) GAGSGVVIATVIVITAG (SEQ ID NO: 118, cleavable by cathepsin L) APMAEGGG (SEQ ID NO: 119, cleavable by meprin α and meprin β) EAQGDKII (SEQ ID NO: 120, cleavable by meprin α and meprin β) LAFSDAGP (SEQ ID NO: 121, cleavable by meprin α and meprin β) YVADAPK (SEQ ID NO: 122, cleavable by meprin α and meprin β) RRRRR (SEQ ID NO: 123, cleavable by furin) RRRRRR (SEQ ID NO: 124, cleavable by furin) GQSSRHRRAL (SEQ ID NO: 125, cleavable by furin) SSRHRRALD (SEQ ID NO: 126) RKSSIIIRMRDVVL (SEQ ID NO: 127, cleavable by plasminogen) SSSFDKGKYKKGDDA (SEQ ID NO: 128, cleavable by Staphylokinase) SSSFDKGKYKRGDDA (SEQ ID NO: 129, cleavable by Staphylokinase) IEGR (SEQ ID NO: 130, cleavable by Factor Xa) IDGR (SEQ ID NO: 131, cleavable by Factor Xa) GGSIDGR (SEQ ID NO: 132, cleavable by Factor Xa) GPQGIAGQ (SEQ ID NO: 133, cleavable by collagenase) GPQGLLGA (SEQ ID NO: 134, cleavable by collagenase) GIAGQ (SEQ ID NO: 135, cleavable by collagenase) GPLGIAG (SEQ ID NO: 136, cleavable by collagenase) GPEGLRVG (SEQ ID NO: 137, cleavable by collagenase) YGAGLGVV (SEQ ID NO: 138, cleavable by collagenase) AGLGVVER (SEQ ID NO: 139, cleavable by collagenase) AGLGISST (SEQ ID NO: 140, cleavable by collagenase) EPQALAMS (SEQ ID NO: 141, cleavable by collagenase) QALAMSAI (SEQ ID NO: 142, cleavable by collagenase) AAYHLVSQ (SEQ ID NO: 143, cleavable by collagenase) MDAFLESS (SEQ ID NO: 144, cleavable by collagenase) ESLPVVAV (SEQ ID NO: 145, cleavable by collagenase) SAPAVESE (SEQ ID NO: 146, cleavable by collagenase) DVAQFVLT (SEQ ID NO: 147, cleavable by collagenase) VAQFVLTE (SEQ ID NO: 148, cleavable by collagenase) AQFVLTEG (SEQ ID NO: 149, cleavable by collagenase) PVQPIGPQ (SEQ ID NO: 150, cleavable by collagenase) LVPRGS (SEQ ID NO: 151, cleavable by thrombin) TSGSGRSANARG (SEQ ID NO: 168, cleavable by uPA and MT-SP1) TSQSGRSANQRG (SEQ ID NO: 169, cleavable by uPA and MT-SP1) TSPSGRSAYPRG (SEQ ID NO: 170, cleavable by uPA and MT-SP1) TSGSGRSATPRG (SEQ ID NO: 171, cleavable by uPA and MT-SP1) TSQSGRSATPRG (SEQ ID NO: 172, cleavable by uPA and MT-SP1) TSASGRSATPRG (SEQ ID NO: 173, cleavable by uPA and MT-SP1) TSYSGRSAVPRG (SEQ ID NO: 174, cleavable by uPA and MT-SP1) TSYSGRSANFRG (SEQ ID NO: 175, cleavable by uPA and MT-SP1) TSSSGRSATPRG (SEQ ID NO: 176, cleavable by uPA and MT-SP1) TSTTGRSASPRG (SEQ ID NO: 177, cleavable by uPA and MT-SP1) TSTSGRSANPRG (SEQ ID NO: 178, cleavable by uPA and MT-SP1)

[0113] The sequences shown in Table 1 can also be used as protease cleavage sequences.

[0114] [Table 1] TIFF0007757458000002.tif252150TIFF0007757458000003.tif252150TIFF000 7757458000004.tif252150TIFF0007757458000005.tif252150TIFF00077574580 00006.tif252150TIFF0007757458000007.tif252150TIFF0007757458000008.t if252150TIFF0007757458000009.tif238150TIFF0007757458000010.tif240134

[0115] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 833) wherein X1 to X8 each represent an amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 represents R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

[0116] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 834) X1 to X8 each represent an amino acid, and X1 is an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X7 is A, X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

[0117] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 835) X1 to X8 each represent an amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, F, L, M, P, Q, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X7 is an amino acid selected from A, D, E, X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X9 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

[0118] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 836) X1 to X8 each represent an amino acid, and X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X7 is A, X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

[0119] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 837) X1 to X8 each represent an amino acid, and X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, G, H, I, K, L, M, N, Q, R, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X7 is A, X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

[0120] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 838) X1 to X8 each represent an amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from E, F, K, M, N, P, Q, R, S, and W; and X7 is A, D, E, F, G, X8 is an amino acid selected from H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

[0121] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 839) X1 to X8 each represent an amino acid, and X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X6 is A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

[0122] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 840) X1 to X8 each represent an amino acid, and X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X6 is A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, I, K, N, T and W.

[0123] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 841) X6 is A; X7 is an amino acid selected from H, I, and V; and X8 is an amino acid selected from H, V, and Y.

[0124] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 842) X6 is an amino acid selected from A and E; X7 is an amino acid selected from N and V; and X8 is an amino acid selected from H, P, V, and Y.

[0125] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 843) X1 to X9 each represent an amino acid, and X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X6 is A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and X9 is an amino acid selected from A, G, H, I, L and R.

[0126] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 844) X1 to X9 each represent an amino acid, X1 is an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is A, X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X9 is an amino acid selected from A, G, H, I, L and R.

[0127] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 845) X1 to X9 each represent an amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, F, L, M, P, Q, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X7 is an amino acid selected from A, D, E, X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X9 is an amino acid selected from A, G, H, I, L and R.

[0128] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 846) X1 to X9 each represent an amino acid, and X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X7 is A, X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X9 is an amino acid selected from A, G, H, I, L and R.

[0129] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 847) X1 to X9 each represent an amino acid, and X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, G, H, I, K, L, M, N, Q, R, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X7 is A, X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X9 is an amino acid selected from A, G, H, I, L and R.

[0130] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 848) X1 to X9 each represent an amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from E, F, K, M, N, P, Q, R, S, and W; and X7 is A, D, E, F, G, X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X9 is an amino acid selected from A, G, H, I, L and R.

[0131] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 849) X1 to X9 each represent an amino acid, and X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X6 is A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. X7 is an amino acid selected from A, D, F, G, L, M, P, Q, V and W; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and X9 is an amino acid selected from A, G, H, I, L and R.

[0132] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 850) X1 to X9 each represent an amino acid, and X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X6 is A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, I, K, N, T and W; and X9 is an amino acid selected from A, G, H, I, L and R.

[0133] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 851) X6 is A; X7 is an amino acid selected from H, I, and V; X8 is an amino acid selected from H, V, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0134] The following protease cleavage sequences may also be used: X1-X2-X3-X4-X5-X6-X7-X8-X9 (Sequence number: 852) X6 is an amino acid selected from A and E; X8 is an amino acid selected from H, P, V, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0135] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1062) X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

[0136] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1063) X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

[0137] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1064) X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, F, L, M, P, Q, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is A, D, E, F, H, I, K, L, M, N, P, Q, R, X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

[0138] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1065) X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

[0139] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1066) X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, G, H, I, K, L, M, N, Q, R, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

[0140] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1067) X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is E, F, K, M, N, P, Q, R, X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

[0141] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1068) X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, X7 is an amino acid selected from A, D, F, G, L, M, P, Q, V, and W; and X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0142] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1069) X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, I, K, N, T and W.

[0143] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1070) X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, G, I, P, Q, S, and Y; X2 is an amino acid selected from K or T; X3 is G; X4 is R; X5 is S; X6 is A; X7 is an amino acid selected from H, I, and V; and X8 is an amino acid selected from H, V, and Y.

[0144] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8 (Sequence number: 1071) X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is Y; X2 is an amino acid selected from S and T; X3 is G; X4 is R; X5 is S; X6 is an amino acid selected from A and E; X7 is an amino acid selected from N and V; and X8 is an amino acid selected from H, P, V, and Y.

[0145] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 1072) X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and X9 is an amino acid selected from A, G, H, I, L and R.

[0146] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 1073) X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and X9 is an amino acid selected from A, G, H, I, L and R.

[0147] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 1074) X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, F, L, M, P, Q, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is A, D, E, F, H, I, K, L, M, N, P, Q, R, X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and X9 is an amino acid selected from A, G, H, I, L and R.

[0148] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 1075) X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and X9 is an amino acid selected from A, G, H, I, L and R.

[0149] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 1076) X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, G, H, I, K, L, M, N, Q, R, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and X9 is an amino acid selected from A, G, H, I, L and R.

[0150] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 1077) X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is E, F, K, M, N, P, Q, R, X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and X9 is an amino acid selected from A, G, H, I, L and R.

[0151] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 1078) X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, X7 is an amino acid selected from A, D, F, G, L, M, P, Q, V, and W; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0152] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 1079) X1 to X11 each represent an amino acid, X10 is an amino acid selected from I, T, and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, I, K, N, T and W; and X9 is an amino acid selected from A, G, H, I, L and R.

[0153] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 1080) X1 is an amino acid selected from H, I, and V; X8 is an amino acid selected from H, V, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0154] The following protease cleavage sequences may also be used: X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9 (Arrangement number: 1081) X6 is an amino acid selected from A and E; X7 is an amino acid selected from N and V; X8 is an amino acid selected from H, P, V and Y; and X9 is an amino acid selected from A, G, H, I, L and R.

[0155] In addition to using the protease cleavage sequences described above, new protease cleavage sequences can be obtained through screening. For example, new protease cleavage sequences can be identified by altering the interactions between the cleavage sequence and the enzyme's active and recognition residues based on the results of crystal structure analysis of known protease cleavage sequences. New protease cleavage sequences can also be identified by modifying amino acids in known protease cleavage sequences and confirming their interaction with the protease. Another example is the discovery of sequences cleaved by proteases by displaying peptide libraries using in vitro display methods such as phage display and ribosome display, or by using peptide arrays immobilized on chips or beads to confirm their interaction with the protease. The interaction between a protease cleavage sequence and a protease can be confirmed by confirming protease cleavage in vitro or in vivo.

[0156] 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, protease activity, and cleavage rate of a molecule into which a 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.

[0157] 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.

[0158] For example, the protease cleavage sequences exemplified in Table 1 were all newly discovered by the present inventors. Polypeptides containing these protease cleavage sequences are useful as protease substrates that are hydrolyzed by the action of proteases. That is, the present invention provides protease substrates containing sequences selected from the sequences set forth in SEQ ID NOS: 833-852, 1062-1081, and the sequences set forth in Table 1. The protease substrates of the present invention can be used as libraries to select those with desired properties for incorporation into the polypeptides of the present invention. Specifically, the protease sensitivity of the polypeptides of the present invention can be evaluated in order to selectively cleave them with proteases localized in lesions. After administration to the body, the polypeptides of the present invention may potentially reach lesions through contact with various proteases. Therefore, it is desirable for the polypeptides to be sensitive to proteases localized in lesions while exhibiting as high resistance as possible to other proteases. To select a desired protease cleavage sequence depending on the purpose, the protease resistance of each protease substrate can be determined by comprehensively analyzing the sensitivity of each substrate to various proteases in advance. Based on the obtained protease resistance spectrum, a protease cleavage sequence having the required sensitivity and resistance can be found. Alternatively, 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 substrate to such external factors, a protease cleavage sequence with desired properties according to the purpose can be selected.

[0159] In one embodiment of the invention, 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 each 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.).

[0160] 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 invention, the flexible linker is a peptide linker of 1 to 7 amino acids.

[0161] 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:27)n, and (GGGS: SEQ ID NO:28)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: 28) Gly·Gly·Ser·Gly (GGSG, SEQ ID NO: 29) Gly·Ser·Gly·Gly (GSGG, SEQ ID NO: 46) Ser·Gly·Gly·Gly (SGGG, SEQ ID NO: 47) Gly·Ser·Ser·Gly (GSSG, SEQ ID NO: 48) Gly·Gly·Gly·Gly·Ser (GGGGS, SEQ ID NO: 49) Gly·Gly·Gly·Ser·Gly (GGGSG, SEQ ID NO: 33) Gly·Gly·Ser·Gly·Gly (GGSGG, SEQ ID NO: 30) Gly·Ser·Gly·Gly·Gly (GSGGG, SEQ ID NO: 32) Gly·Ser·Gly·Gly·Ser (GSGGS, SEQ ID NO: 27) Ser·Gly·Gly·Gly·Gly (SGGGG, SEQ ID NO: 51) Gly·Ser·Ser·Gly·Gly (GSSGG, SEQ ID NO: 52) Gly·Ser·Gly·Ser·Gly (GSGSG, SEQ ID NO: 31) Ser·Gly·Gly·Ser·Gly (SGGSG, SEQ ID NO: 53) Gly·Ser·Ser·Ser·Gly (GSSSG, SEQ ID NO: 34) Gly·Gly·Gly·Gly·Gly·Ser (GGGGGS, SEQ ID NO: 50) Ser·Gly·Gly·Gly·Gly·Gly·Gly (SGGGGG, SEQ ID NO: 54) Gly·Gly·Gly·Gly·Gly·Gly·Ser (GGGGGGS, SEQ ID NO: 55) Ser·Gly·Gly·Gly·Gly·Gly·Gly·Gly (SGGGGGG, SEQ ID NO: 56) (Gly·Gly·Gly·Gly·Ser (GGGGS, SEQ ID NO: 49))n (Ser·Gly·Gly·Gly·Gly (SGGGG, SEQ ID NO: 51)) Examples include:

[0162] 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.

[0163] In some embodiments of the present invention, the repression domain of the delivery moiety is associated with the antigen-binding domain. The repression domain may be a portion of the delivery moiety or the entire delivery moiety. Alternatively, 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.

[0164] 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.

[0165] 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 an antibody, ligand, receptor, substrate, or the like. Specific examples of such polypeptide regions in antibodies include heavy chain variable regions and light chain variable regions, and in some embodiments of the present invention, include antigen-binding domains and inhibitory domains. 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.

[0166] In some embodiments of the present invention, 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 between 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 that form 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 amino acid residue or an uncharged amino acid residue, modifying a negatively charged amino acid residue to a positively charged amino acid residue or an 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.

[0167] In some embodiments of the present invention, an antigen-binding domain, VHH, associates with an 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.

[0168] 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 purpose 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.

[0169] In some other embodiments of the present invention, 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.

[0170] 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.

[0171] In some embodiments of the present invention, the delivery moiety and the antigen-binding domain are fused via a linker. In more specific embodiments, the delivery moiety and the antigen-binding domain are fused via a linker that contains a cleavage site. In another specific embodiment, the delivery moiety and the antigen-binding domain are fused via a linker, and the resulting fusion protein contains a cleavage site.

[0172] In another embodiment of the present invention, 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 cleavage site. In a specific embodiment, 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 cleavage site near the fusion site. More specifically, for example, a cleavage site can be formed by substituting the LSGR sequence for the four C-terminal amino acids of the antigen-binding domain and the SDNH sequence for the four N-terminal amino acids of the delivery moiety.

[0173] In some embodiments of the present invention, the cleavage site of a polypeptide comprising a transport moiety and an antigen-binding domain comprises a protease cleavage sequence. 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.

[0174] In some embodiments of the invention, the delivery moiety comprises an antibody constant region, fused at its N-terminus 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, preferably 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 (EU numbering) (Kabat numbering 130) in the antibody light chain constant region, preferably on the antigen-binding domain side of amino acid 113 (EU numbering) (Kabat numbering 113) in the antibody light chain constant region.

[0175] In some embodiments of the invention, 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 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 transport moiety than amino acid 35b (Kabat numbering) of the single-domain antibody, preferably closer to the transport moiety than amino acid 95 (Kabat numbering) of the single-domain antibody, and more preferably closer to the transport moiety than amino acid 109 (Kabat numbering) of the single-domain antibody. 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 transport moiety than amino acid 32 (Kabat numbering) of the single-domain antibody, preferably closer to the transport moiety than amino acid 91 (Kabat numbering) of the single-domain antibody, and more preferably closer to the transport moiety than amino acid 104 (Kabat numbering) of the single-domain antibody.

[0176] In some embodiments of the present invention, the delivery 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 a more specific embodiment, the protease cleavage sequence is located closer to the single-domain antibody than amino acid 140 (EU numbering) in the antibody heavy chain constant region, preferably 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 closer to the antigen-binding domain than amino acid 130 (EU numbering) (Kabat numbering number 130) in the antibody light chain constant region, preferably closer to the antigen-binding domain than amino acid 113 (EU numbering) (Kabat numbering number 113) 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), preferably closer to amino acid 95 (Kabat numbering), and more preferably closer to 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), preferably closer to amino acid 91 (Kabat numbering), and more preferably closer to 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. The "near the interface between the antigen-binding domain and the antibody constant region" can refer to the interface between the antigen-binding domain and the antibody heavy chain constant region, or 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 VH and is linked to an antibody heavy chain constant region, the "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, preferably 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 refers to the region between amino acid 101 (Kabat numbering) for single-domain antibodies and amino acid 130 (EU numbering) (Kabat numbering number 130) for antibody light-chain constant regions, and preferably refers to the region between amino acid 109 (Kabat numbering) for single-domain antibodies and amino acid 113 (EU numbering) (Kabat numbering number 113) 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, and preferably from amino acid 104 (Kabat numbering) for single-domain antibodies.

[0177] In some embodiments of the present invention, the polypeptide is an IgG antibody-like molecule. Examples of such embodiments include, but are not limited to, embodiments in which the carrier moiety comprises an IgG antibody constant region, and a single-domain antibody antigen-binding domain replaces the VH of the IgG antibody, with the VL inhibiting antigen-binding activity; embodiments in which the carrier moiety comprises an IgG antibody constant region, and a single-domain antibody antigen-binding domain replaces the VL of the IgG antibody, with the VH inhibiting antigen-binding activity; and embodiments in which the carrier moiety comprises an IgG antibody constant region, and a single-domain antibody antigen-binding domain replaces one of the VH / VL of the IgG antibody, with another single-domain antibody that inhibits the antigen-binding activity of the antigen-binding domain replacing the other of the VH / VL of the IgG antibody.

[0178] 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.

[0179] The polypeptide may contain one or more antigen-binding domains. The repression domains that repress 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 transport moiety. Each of the multiple antigen-binding domains may be releasable from the polypeptide. There may be multiple cleavage sites for releasing the multiple antigen-binding domains, each corresponding to one of the antigen-binding domains.

[0180] 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 7, would be an embodiment that would be understood by a person skilled in the art after reading the present invention. 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 the present invention, and it is clear that this does not deviate from the scope of the present invention.

[0181] In some embodiments of the invention, 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 8, 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. 8 illustrates one embodiment in which the antigen-binding domain is further linked to a second antigen-binding domain.

[0182] 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.

[0183] The present invention also relates to a pharmaceutical composition (medicament) comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.

[0184] As used herein, "treatment" (and its grammatical derivatives, such as "treat," "treating," etc.) refers to a clinical intervention intended to alter the natural course of the individual being treated and can be performed prophylactically or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the polypeptides of the invention are used to delay the onset of disease or slow the progression of disease.

[0185] In the present invention, a pharmaceutical composition generally refers to a drug for treating or preventing a disease, or for testing or diagnosing a disease. Furthermore, in the present invention, the term "pharmaceutical composition comprising a polypeptide" can be rephrased as "a method for treating a disease, comprising administering a polypeptide to a subject to be treated," or as "use of a polypeptide in the manufacture of a medicament for treating a disease." Furthermore, the term "pharmaceutical composition comprising a polypeptide" can be rephrased as "use of a polypeptide for treating a disease."

[0186] The pharmaceutical compositions of the present invention can be formulated using methods known to those skilled in the art. For example, they can be used parenterally in the form of injections of sterile solutions or suspensions in water or other pharmaceutically acceptable liquids. For example, they can be formulated by appropriately combining them with pharmacologically acceptable carriers or vehicles, specifically, sterile water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, etc., and blending them into unit dosage forms required for generally accepted pharmaceutical practice. The amount of active ingredient in these preparations is set so that an appropriate volume within the specified range is obtained.

[0187] Sterile compositions for injection can be formulated according to standard pharmaceutical practices using a vehicle such as distilled water for injection. Examples of aqueous solutions for injection include isotonic solutions containing physiological saline, glucose, or other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). Suitable solubilizers, such as alcohol (ethanol, etc.), polyalcohols (propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (polysorbate 80™, HCO-50, etc.), can be used in combination.

[0188] Oily liquids include sesame oil and soybean oil, and may also contain benzyl benzoate and / or benzyl alcohol as a solubilizing agent. They may also contain buffers (e.g., phosphate buffer and sodium acetate buffer), soothing agents (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants. The prepared injection solution is usually filled into an appropriate ampule.

[0189] The pharmaceutical composition of the present invention is preferably administered parenterally. For example, the composition may be administered in the form of an injection, a nasal administration, a pulmonary administration, or a transdermal administration. For example, the composition may be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.

[0190] The administration method can be selected appropriately depending on the patient's age and symptoms. The dosage of a pharmaceutical composition containing a polypeptide can be set, for example, in the range of 0.0001 mg to 1,000 mg per kg of body weight per administration. Alternatively, the dosage can be set, for example, in the range of 0.001 to 100,000 mg per patient, although the present invention is not necessarily limited to these numerical values. The dosage and administration method vary depending on the patient's weight, age, symptoms, etc., but those skilled in the art can determine an appropriate dosage and administration method taking these conditions into consideration.

[0191] The present invention also relates to methods for producing polypeptides comprising a delivery moiety having a repression domain and an antigen-binding domain. One method for producing the polypeptides of the present invention involves obtaining an antigen-binding domain with antigen-binding activity, linking the antigen-binding domain to a delivery moiety to form a polypeptide precursor such that the antigen-binding activity of the antigen-binding domain is inhibited by an inhibition domain, and then inserting a cleavage site into the polypeptide precursor or modifying a portion of the polypeptide precursor into a cleavage site. It is sufficient to introduce a cleavage site into the polypeptide precursor, and the method for introducing the cleavage site may be either inserting a cleavage site or 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 modification site into the polypeptide precursor, and this does not depart from the scope of the present invention. Another method for producing the polypeptides of the present invention involves obtaining an antigen-binding domain with antigen-binding activity and linking it to a delivery moiety via a cleavage site to form a polypeptide such that the antigen-binding activity of the antigen-binding domain is inhibited by an inhibition domain. When the antigen-binding domain and delivery moiety are linked via a cleavage site, the cleavage site may be sandwiched between the antigen-binding domain and the delivery moiety, or part of the antigen-binding domain and / or part of the delivery moiety may be modified and used as part of the cleavage site.

[0192] "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).

[0193] The following method for producing the polypeptide is described for an embodiment in which a single-domain antibody is used as the antigen-binding domain and a protease cleavage sequence is used as the cleavage site.

[0194] In one embodiment of the present invention, 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 precursor; The manufacturing method includes:

[0195] In one embodiment of the present invention, 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 near the interface between the single domain antibody and the delivery moiety; The manufacturing method includes:

[0196] In one embodiment of the present invention, 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 a delivery moiety via a protease cleavage sequence to form a polypeptide such that the antigen-binding activity of the single domain antibody is inhibited by the inhibition domain of the delivery moiety; The manufacturing method includes:

[0197] In certain embodiments, the method for producing a polypeptide comprising an antigen binding domain and a delivery moiety having a repression domain further comprises the steps of: (d) confirming that the binding activity of the single domain antibody incorporated into the polypeptide or the precursor polypeptide to the target antigen is weakened or lost; In the present invention, "weakened binding activity" means that the binding activity to the target antigen is reduced compared to before linkage, and the degree of reduction does not matter.

[0198] In certain embodiments, the method for producing a polypeptide comprising an antigen binding domain and a delivery moiety having a repression domain further comprises the steps of: (e) cleaving the protease cleavage sequence with a protease to release the single domain antibody, and confirming that the released single domain antibody binds to an antigen; The manufacturing method includes:

[0199] In one embodiment of the present invention, a method for producing a polypeptide that comprises a delivery moiety having a repression domain and an antigen-binding domain and is an IgG antibody-like molecule comprises the steps of: (a) obtaining a single domain antibody that binds to a target antigen; (b) forming an IgG antibody-like molecule precursor into which the single domain antibody obtained in step (a) has been introduced by associating the single domain antibody with a VL instead of a VH of an IgG antibody, or by associating the single domain antibody with a VH instead of a VL of an IgG antibody, so that the antigen-binding activity of the single domain antibody is suppressed; (c) introducing a protease cleavage sequence into the IgG antibody-like molecule precursor into which the single domain antibody has been introduced; The manufacturing method includes:

[0200] In one embodiment of the present invention, a method for producing a polypeptide that comprises a delivery moiety having a repression domain and an antigen-binding domain and is an IgG antibody-like molecule comprises the steps of: (a) obtaining a single domain antibody that binds to a target antigen; (b) forming an IgG antibody-like molecule precursor into which the single domain antibody obtained in step (a) has been introduced by associating the single domain antibody with a VL instead of a VH of an IgG antibody, or by associating the single domain antibody with a VH instead of a VL of an IgG antibody, so that the antigen-binding activity of the single domain antibody is suppressed; (c) introducing a protease cleavage sequence near the boundary between the single domain antibody and the antibody constant region in the IgG antibody-like molecule precursor; The manufacturing method includes:

[0201] In one embodiment of the present invention, a method for producing a polypeptide that comprises a delivery moiety having a repression domain and an antigen-binding domain and is an IgG antibody-like molecule 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 heavy chain constant region or light chain constant region of an IgG antibody via a protease cleavage sequence, in place of the IgG antibody VH or VL, so that the antigen-binding activity of the single domain antibody is suppressed, thereby forming an IgG antibody-like molecule into which the single domain antibody has been introduced; The manufacturing method includes:

[0202] In certain embodiments, a method for producing a polypeptide comprising a delivery moiety having a repression domain and an antigen binding domain, wherein the polypeptide is an IgG antibody-like molecule, further comprises the steps of: (d) confirming that the binding activity of the single domain antibody introduced into the IgG antibody-like molecule or the IgG antibody-like molecule precursor to the target antigen is weakened or lost; In the present invention, "weakened binding activity" means that the binding activity to a target antigen is reduced compared to before association or linkage, and the degree of reduction does not matter.

[0203] In certain embodiments, a method for producing a polypeptide comprising a delivery moiety having a repression domain and an antigen binding domain, wherein the polypeptide is an IgG antibody-like molecule, further comprises the steps of: (e) cleaving the protease cleavage sequence with a protease to release the single domain antibody, and confirming that the released single domain antibody binds to the target antigen; The manufacturing method includes:

[0204] When VH / VL / VHH is used as the repression domain, one method for suppressing the antigen-binding activity of a single-domain antibody with the repression domain of the carrier moiety is to associate the single-domain antibody with VH / VL / VHH. VH / VL / VHH that suppress the antigen-binding activity of a prepared single-domain antibody can be screened by associating a known VH / VL / VHH with the single-domain antibody and comparing the antigen-binding activity of the single-domain antibody before and after association. Alternatively, as another method for inhibiting the antigen-binding activity of a single-domain antibody with a specific VH / VL / VHH, amino acid residues in the single-domain antibody that are involved in association with VH / VL / VHH can be substituted to promote association, or a single-domain antibody can be used in which those amino acid residues are originally amino acids that can promote association. This makes it possible to prepare a single-domain antibody / inhibitory domain pair in which the difference in antigen-binding activity before and after association is at a desired level.

[0205] In one embodiment of the present invention, a method for producing an IgG antibody-like molecule polypeptide comprising a delivery moiety having a repression domain and an antigen-binding domain comprises the steps of: (a) substituting amino acid residues in a single domain antibody that are involved in association with the antibody VH, or substituting amino acid residues in a single domain antibody that are involved in association with the antibody VL, to produce a modified single domain antibody that retains the binding activity of the single domain antibody to its target antigen; (b) forming an IgG antibody-like molecule precursor into which the modified single domain antibody prepared in step (a) has been introduced by associating the modified single domain antibody with an antibody VH or with an antibody VL so as to suppress the antigen-binding activity of the modified single domain antibody; (c) introducing a protease cleavage sequence into the IgG antibody-like molecule precursor into which the modified single domain antibody has been introduced; The manufacturing method includes:

[0206] In one embodiment of the present invention, a method for producing a polypeptide that comprises a delivery moiety having a repression domain and an antigen-binding domain and is an IgG antibody-like molecule comprises the steps of: (a) substituting amino acid residues in a single domain antibody that are involved in association with the antibody VH, or substituting amino acid residues in a single domain antibody that are involved in association with the antibody VL, to produce a modified single domain antibody that retains the binding activity of the single domain antibody to its target antigen; (b) forming an IgG antibody-like molecule precursor into which the modified single domain antibody prepared in step (a) has been introduced by associating the modified single domain antibody with an antibody VH or with an antibody VL so as to suppress the antigen-binding activity of the modified single domain antibody; (c) introducing a protease cleavage sequence near the interface between the engineered single domain antibody and the constant region of the IgG antibody-like molecule precursor; The manufacturing method includes:

[0207] In one embodiment of the present invention, a method for producing a polypeptide that comprises a delivery moiety having a repression domain and an antigen-binding domain and is an IgG antibody-like molecule comprises the steps of: (a) substituting amino acid residues in a single domain antibody that are involved in association with the antibody VH, or substituting amino acid residues in a single domain antibody that are involved in association with the antibody VL, to produce a modified single domain antibody that retains the binding activity of the single domain antibody to its target antigen; (b) linking the modified single-domain antibody prepared in step (a) to the heavy chain constant region of an IgG antibody via a protease cleavage sequence, or linking the modified single-domain antibody to the light chain constant region of an IgG antibody via a protease cleavage sequence, so as to suppress the antigen-binding activity of the modified single-domain antibody, thereby forming an IgG antibody-like molecule incorporating the modified single-domain antibody; The manufacturing method includes:

[0208] In certain embodiments, a method for producing a polypeptide comprising a delivery moiety having a repression domain and an antigen binding domain, wherein the polypeptide is an IgG antibody-like molecule, further comprises the steps of: (d) confirming that the binding activity of the modified single domain antibody introduced into the IgG antibody-like molecule or the IgG antibody-like molecule precursor to the target antigen is weakened or lost; In the present invention, "weakened binding activity" means that the binding activity to a target antigen is reduced compared to before association or linkage, and the degree of reduction does not matter.

[0209] In certain embodiments, a method for producing a polypeptide comprising a delivery moiety having a repression domain and an antigen binding domain, wherein the polypeptide is an IgG antibody-like molecule, further comprises the steps of: (e) cleaving the protease cleavage sequence with a protease to release the engineered single domain antibody, and confirming that the released engineered single domain antibody binds to the target antigen; The manufacturing method includes:

[0210] The present invention also relates to polynucleotides that encode polypeptides that include a delivery moiety having a repression domain and an antigen binding domain.

[0211] The polynucleotide of the present invention is typically carried (inserted) into an appropriate vector and introduced into a host cell. The vector is not particularly limited as long as it stably retains the inserted nucleic acid. For example, if Escherichia coli is used as the host, a cloning vector such as the pBluescript vector (Stratagene) is preferred, although various commercially available vectors can also be used. When using a vector for the purpose of producing the polypeptide of the present invention, an expression vector is particularly useful. The expression vector is not particularly limited as long as it expresses a polypeptide in a test tube, in Escherichia coli, in cultured cells, or in an individual organism. For example, the pBEST vector (Promega) is preferred for in vitro expression, the pET vector (Invitrogen) is preferred for Escherichia coli, the pME18S-FL3 vector (GenBank Accession No. AB009864) is preferred for cultured cells, and the pME18S vector (Mol Cell Biol. 8:466-472 (1988)) is preferred for individual organisms. The DNA of the present invention can be inserted into a vector by conventional methods, for example, by ligase reaction using a restriction enzyme site (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Sections 11.4-11.11).

[0212] The host cell is not particularly limited, and various host cells can be used depending on the purpose. Examples of cells for expressing a polypeptide include bacterial cells (e.g., Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, Bacillus subtilis), fungal cells (e.g., yeast, Aspergillus), insect cells (e.g., Drosophila S2, Spodoptera SF9), animal cells (e.g., CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells), and plant cells. Vectors can be introduced into host cells by known methods, such as calcium phosphate precipitation, electroporation (Current protocols in Molecular Biology, edited by Ausubel et al. (1987) Published by John Wiley & Sons, Sections 9.1-9.9), lipofectamine (GIBCO-BRL), and microinjection.

[0213] To allow secretion of a polypeptide expressed in a host cell into the lumen of the endoplasmic reticulum, into the periplasmic space, or into the extracellular environment, appropriate secretion signals can be incorporated into the polypeptide of interest. These signals can be endogenous to the polypeptide of interest or heterologous signals.

[0214] In the above production method, when the polypeptide of the present invention is secreted into the medium, the medium is recovered, whereas when the polypeptide of the present invention is produced intracellularly, the cells are first lysed and then the polypeptide is recovered.

[0215] Polypeptides of the invention can be recovered and purified from recombinant cell cultures using known methods, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography.

[0216] Furthermore, the antigen-binding domain used in some embodiments of the present invention includes a single-domain antibody, and in these embodiments, the antigen-binding activity of the single-domain antibody is suppressed by associating with a specific VL, a specific VH, or a specific VHH. The present invention also relates to methods for screening such single-domain antibodies.

[0217] VL / VH / VHH with known sequences, such as those registered in the IMGT or Kabat database, can be used as VL / VH / VHH that inhibit the antigen-binding activity of a single-domain antibody. New VL / VH / VHH sequences identified from human antibody libraries, etc., can also be used. Proteins can be prepared by combining these sequences, and the binding activity can be measured using the above-described method to select VL / VH / VHH that inhibit the binding activity of a single-domain antibody.

[0218] In some embodiments of the present invention, VL / VH / VHH that inhibit the antigen-binding activity of a single-domain antibody can have a human antibody germline sequence. For example, when VL is used as an inhibition domain, VL having a kappa chain framework sequence or a lamda chain framework sequence can be used. VL having a modified framework sequence, such as a framework sequence that combines a kappa chain framework sequence and a lamda chain framework sequence, can also be used.

[0219] In one embodiment of the present invention, the method comprises the steps of: (a) obtaining a single domain antibody having target antigen-binding activity; (b) associating the single domain antibody obtained in step (a) with a specific VL; (c) confirming that the binding activity of the single domain antibody associated with a specific VL in step (b) to the antigen is weakened or lost; The present invention provides a method for screening for single domain antibodies whose antigen-binding activity is suppressed upon association with a specific VL, comprising: (a) a single domain antibody having a reduced binding activity to a target antigen compared to before association;

[0220] In one embodiment of the present invention, the method comprises the steps of: (a) obtaining a single domain antibody having target antigen-binding activity; (b) associating the single domain antibody obtained in step (a) with a specific VH; (c) confirming that the binding activity of the single domain antibody associated with a specific VH in step (b) to the antigen is weakened or lost; The present invention provides a method for screening for single domain antibodies whose antigen-binding activity is suppressed upon association with a specific VH, comprising: (a) a VH comprising: (i) a VH ...b) a VH comprising: (c) a VH comprising: (d) a VH comprising: (i) a VH comprising: (c) a VH comprising: (a) a VH comprising: (b) a VH comprising: (

[0221] In one embodiment of the present invention, the method comprises the steps of: (a) obtaining a single domain antibody having target antigen-binding activity; (b) associating the single domain antibody obtained in step (a) with a specific VHH; (c) confirming that the binding activity of the single domain antibody associated with the specific VHH in step (b) to the antigen is weakened or lost; The present invention provides a method for screening for single domain antibodies whose antigen-binding activity is suppressed upon association with a specific VHH, comprising: (a) a VHH comprising: (i) a VHH comprising: (a) a VHH;

[0222] An example of a method for associating a single-domain antibody with a specific VL / VH / VHH is to design a molecule in which the sequence of a single-domain antibody is substituted for the sequence of one of the VH and VL in a complete antibody, antibody or antibody fragment containing both VH and VL, such as Fab, Fab', or (Fab)2, and then express a polypeptide having that sequence.

[0223] Furthermore, the present invention relates to screening for single domain antibodies whose antigen-binding activity is suppressed by associating with a specific VL, or a specific VH, or a specific VHH, as well as to methods for producing single domain antibodies whose antigen-binding activity is suppressed by promoting association with a specific VL, or a specific VH, or a specific VHH.

[0224] In one embodiment of the present invention, the method comprises the steps of: (a) substituting amino acid residues in a single domain antibody that are involved in association with an antibody VL to produce a modified single domain antibody that retains the binding activity of the single domain antibody to its target antigen; The present invention provides a method for producing a single domain antibody whose antigen-binding activity is suppressed by associating with a specific VL, comprising:

[0225] In certain embodiments, further comprising the steps of: (b) associating the engineered single domain antibody produced in step (a) with a specific VL; (c) confirming that the antigen-binding activity of the engineered single domain antibody associated with the VL is reduced or lost; The present invention provides a method for producing a single domain antibody whose antigen-binding activity is suppressed upon association with a specific VL, comprising: (a) a single domain antibody having a reduced binding activity to a target antigen compared to before association;

[0226] In one embodiment of the present invention, the method comprises the steps of: (a) substituting amino acid residues in a single domain antibody that are involved in association with an antibody VH to produce a modified single domain antibody that retains the binding activity of the single domain antibody to its target antigen; The present invention provides a method for producing a single domain antibody whose antigen-binding activity is suppressed by associating with a specific VH, comprising:

[0227] In certain embodiments, further comprising the steps of: (b) associating the engineered single domain antibody produced in step (a) with a specific VH; (c) confirming that the antigen-binding activity of the engineered single domain antibody associated with the VH is weakened or lost; The present invention provides a method for producing a single domain antibody whose antigen-binding activity is suppressed upon association with a specific VH, comprising: (a) a single domain antibody having a reduced binding activity to a target antigen compared to before association;

[0228] In one embodiment of the present invention, the method comprises the steps of: (a) substituting amino acid residues in a single domain antibody that are involved in association with VHH to produce a modified single domain antibody that retains the binding activity of the single domain antibody to its target antigen; The present invention provides a method for producing a single domain antibody whose antigen-binding activity is suppressed by associating with a specific VHH, comprising:

[0229] In certain embodiments, further comprising the steps of: (b) associating the engineered single domain antibody produced in step (a) with a specific VHH; (c) confirming that the antigen-binding activity of the modified single domain antibody associated with the VHH is weakened or lost; The present invention provides a method for producing a single domain antibody whose antigen-binding activity is suppressed upon association with a specific VHH, comprising: (a) a single domain antibody having a reduced binding activity to a target antigen compared to before association;

[0230] The step of associating a single-domain antibody with a specific VL / VH / VHH is carried out by designing an antibody or antibody fragment containing both VH and VL, such as a complete antibody, Fab, Fab', or (Fab)2, in which the sequence of the single-domain antibody is substituted for the sequence of one of the VH and VL, and expressing a polypeptide having that sequence.

[0231] According to one embodiment of the present invention, single domain antibodies whose antigen-binding activity is suppressed or lost upon association with a specific VL / VH / VHH of the present invention can be obtained from a library containing multiple fusion polypeptides in which a single domain antibody is linked to a first association-supporting domain.

[0232] As an embodiment of the "library" herein, a library can be provided that can efficiently obtain single domain antibodies whose antigen-binding activity is suppressed or lost upon association with a specific VL / VH / VHH.

[0233] As used herein, the term "library" refers to a set of multiple fusion polypeptides each having a different sequence, or a set of nucleic acids or polynucleotides encoding these fusion polypeptides. The multiple fusion polypeptides contained in the library do not have a single sequence, but are fusion polypeptides with different sequences from each other.

[0234] As used herein, the term "different in sequence" in the description of multiple fusion polypeptides with different sequences means that the sequences of the individual fusion polypeptides in the library are different from each other. More preferably, it means that the sequences of the single domain antibody moieties in the individual fusion polypeptides in the library are different. In other words, the number of different sequences in the library reflects the number of independent clones with different sequences in the library, and is sometimes referred to as the "library size." In a typical phage display library, 10 6 From 10 12 By applying known techniques such as ribosome display, the library size can be increased to 1014 However, the actual number of phage particles ...

Claims

1. a polypeptide comprising an antigen-binding domain and a delivery moiety, wherein the delivery moiety has a repression domain that inhibits the antigen-binding activity of the antigen-binding domain by associating with the antigen-binding domain; the antigen-binding domain comprises or is a single-domain antibody; the repression domain of the delivery moiety is a VHH, antibody VH, or antibody VL; the polypeptide has a cleavage site comprising a protease cleavage sequence comprising one or more sequences selected from the sequences shown in SEQ ID NOs: 833 to 852, 854 to 951, and 1062 to 1081; the antigen-binding domain and the delivery moiety are fused via the cleavage site or via a linker comprising the cleavage site; and cleavage of the protease cleavage sequence by a protease dissolves the association between the antigen-binding domain and the repression domain of the delivery moiety and / or allows the antigen-binding domain to be released from the polypeptide.

2. The polypeptide of claim 1, wherein the protease cleavage sequence comprises a sequence selected from SEQ ID NOs: 854-951.

3. 3. The polypeptide of claim 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 when the protease cleavage sequence is not cleaved.

4. 4. The polypeptide of claim 1, wherein the antigen-binding domain has a shorter serum half-life than the delivery moiety.

5. The polypeptide of claim 4, wherein the molecular weight of the antigen-binding domain is 60 kDa or less.

6. The polypeptide of claim 4 or claim 5, wherein the transport moiety has FcRn binding activity, and the antigen-binding domain has no FcRn binding activity or has weaker FcRn binding activity than the transport moiety.

7. 7. The polypeptide according to claim 1 , 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.

8. The polypeptide according to any one of claims 1 to 7, wherein the protease is a cancer tissue-specific protease or an inflamed tissue-specific protease.

9. The polypeptide of claim 1 , wherein the transport moiety comprises an antibody constant region.

10. The polypeptide described in claim 9, wherein the antibody constant region comprises an antibody light chain constant region (CL) and at least CH1 of an antibody heavy chain constant region.

11. 11. The polypeptide of claim 9 or claim 10, wherein the N-terminus of the antibody constant region of the transport moiety and the C-terminus of the antigen-binding domain are fused with or without a linker.

12. The polypeptide of any one of claims 9 to 11, wherein the protease cleavage sequence is located near the boundary between the antigen-binding domain and the antibody constant region.

13. 13. The polypeptide of any one of claims 9 to 12, wherein the antibody constant region of the polypeptide is an IgG antibody constant region.

14. The polypeptide of any one of claims 1 to 13, wherein the polypeptide is an IgG antibody-like molecule.

15. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 14.

16. A method for producing a polypeptide described in any one of claims 1 to 14, comprising the step of culturing a host cell containing a polynucleotide encoding a polypeptide described in any one of claims 1 to 14, or a vector containing said polynucleotide.

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