Polypeptide containing antigen-binding domain and transporter portion

Abstract

The present invention provides a polypeptide comprising an antigen-binding domain and a transport portion having an inhibitory domain that suppresses the antigen-binding activity of the antigen-binding domain, a method for producing the polypeptide and a screening method, and a pharmaceutical composition comprising the polypeptide. [Solution] A polypeptide is provided, wherein the polypeptide comprises an antigen-binding domain, a transport portion, and a cleavage site, the transport portion having an inhibitory domain that suppresses the antigen-binding activity of the antigen-binding domain, the antigen-binding domain having a shorter half-life in the blood than the transport portion, the antigen-binding domain comprising or being a monodomain antibody, the inhibitory domain of the transport portion being VHH, antibody VH, or antibody VL, and the monodomain antibody having its antigen-binding activity suppressed by the VHH, antibody VH, or antibody VL.

Description

[Technical Field]

[0001] The present invention relates to a polypeptide comprising an antigen-binding domain and a transport portion having an inhibitory domain that suppresses the antigen-binding activity of the antigen-binding domain, and having a longer half-life than an antigen-binding domain existing alone; a method for producing and screening the polypeptide; a pharmaceutical composition containing the polypeptide; a method for producing and screening a monodomain antibody whose antigen-binding activity is suppressed by association with a specific VL / VH / VHH; and a library of fusion polypeptides containing a monodomain antibody whose antigen-binding activity is suppressed by association with a specific VL / VH / VHH. [Background technology]

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

[0003] To date, antibody drugs such as rituxan for the CD20 antigen, cetuximab for the EGFR antigen, and herceptin for the HER2 antigen have been approved as cancer treatments (Non-Patent Literature 3). These antibody molecules bind to antigens expressed on cancer cells and exert cytotoxic activity against cancer cells through ADCC activity, etc. It is known that this cytotoxic activity by ADCC activity, etc. depends on the number of antigens expressed on the target cells of the therapeutic antibody (Non-Patent Literature 4), so a high expression level of the target antigen is preferable from the viewpoint 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 tissue, it will exert cytotoxic activity such as ADCC activity against normal cells, which becomes a major problem in terms of side effects. For this reason, it is preferable that the antigen targeted by therapeutic antibodies as cancer treatments is specifically expressed on cancer cells. For example, antibody molecules against the EpCAM antigen, known as a cancer antigen, were considered promising as cancer treatments. However, the EpCAM antigen is also known to be expressed in the pancreas, and in fact, clinical trials have reported that administering anti-EpCAM antibodies can cause pancreatitis as a side effect due to cytotoxic activity against the pancreas (Non-Patent Literature 5).

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

[0005] Antibody molecules that exhibit such potent cytotoxic activity can exert cytotoxic activity even against cancer cells that do not express the antigen in large quantities, but they also exert cytotoxic activity against normal tissues that do not express the antigen in large quantities, just as they do against cancer cells. In fact, compared to cetuximab, a natural human IgG1 against the EGFR antigen, EGFR-BiTE, a bispecific antibody against CD3 and EGFR, can exert potent cytotoxic activity against cancer cells and exert an antitumor effect by recruiting T cells to cancer cells. On the other hand, since EGFR is also expressed in normal tissues, serious side effects have been observed when EGFR-BiTE is administered to cynomolgus monkeys (Non-Patent Literature 10). Furthermore, bivatuzumab mertansine, an ADC in which mertansine is conjugated to an antibody against CD44v6, which is highly expressed in cancer cells, has been shown to cause serious skin toxicity and hepatotoxicity in clinical practice because CD44v6 is also expressed in normal tissues (Non-Patent Literature 11).

[0006] When using antibodies that can exert potent cytotoxic activity even against cancer cells with low antigen expression, the target antigen needs to be expressed in an extremely cancer-specific manner. However, 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, while it is possible to enhance cytotoxic activity against cancer, side effects due to cytotoxicity in normal tissues may become a problem.

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

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

[0009] Various technologies have been developed that can be applied to second-generation antibody drugs, and technologies that improve effector function, antigen binding ability, pharmacokinetics, and stability, or reduce immunogenicity risk have been reported (Non-Patent Literature 16). However, there are still few reports on technologies that enable antibody drugs to act specifically on target tissues in order to solve the above-mentioned side effects. One reported technology involves linking a masking peptide and an antibody with a linker that is cleaved by a protease expressed in lesion sites such as cancerous or inflammatory tissue. This masks the antigen-binding site of the antibody with the masking peptide, inhibiting the antigen-binding activity of the antibody. When this linker is cleaved by the protease, the mask peptide is released, restoring the antigen-binding activity of the antibody and enabling it to bind to the antigen in the target diseased tissue (Non-Patent Literature 17, 18, Patent Literature 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

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Non - Patent Document 16

[0012] The inventors of the present invention considered that in the technique described above, which involves dissociating a mask peptide that inhibits the antigen-binding activity of an antibody by protease cleavage and thereby restoring the antigen-binding activity of the antibody, the cleavage by the protease is irreversible, and therefore, antibodies cleaved at the lesion site may enter the bloodstream and be distributed to normal tissue, potentially causing side effects. This invention is based on the above idea, and one of its objectives is to provide a pharmaceutical composition useful for treating diseases with reduced side effects, and its active ingredient. Another objective is to provide a screening method and manufacturing method for the pharmaceutical composition and its active ingredient. [Means for solving the problem]

[0013] The inventors, through diligent research, have created a polypeptide comprising an antigen-binding domain and a transport portion having an inhibitory domain that suppresses the binding activity of the antigen-binding domain, and having a longer half-life than an antigen-binding domain existing alone. It is believed that by using this polypeptide, the antigen-binding activity of the antigen-binding domain can be restored in diseased tissue, and thus the antigen-binding activity can be exerted in diseased tissue. Furthermore, the difference in half-life between the polypeptide with suppressed antigen-binding activity and the polypeptide with restored antigen-binding activity can suppress the systemic distribution of the activated antigen-binding domain. The inventors have also found that this polypeptide or a pharmaceutical composition containing this polypeptide is useful for treating diseases, as well as useful in disease treatment involving the administration of this polypeptide, and useful in the manufacture of pharmaceuticals for disease treatment. Furthermore, the inventors have completed the present invention by creating a screening method and a manufacturing method for the polypeptide, a manufacturing method and a screening method for a single-domain antibody whose antigen-binding activity is suppressed by association with a specific VL, VH, or VHH, and a library containing a single-domain antibody whose antigen-binding activity is suppressed by association with a specific VL, VH, or VHH.

[0014] The present invention is based on these findings and specifically includes the embodiments described below as illustrative. (1) A polypeptide comprising an antigen-binding domain and a transport portion, wherein the transport portion has an inhibitory domain that suppresses the antigen-binding activity of the antigen-binding domain, and the antigen-binding domain has a blood half-life shorter than that of the transport portion. (2) The polypeptide according to (1), wherein the molecular weight of the antigen-binding domain is smaller than the molecular weight of the transport portion. (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 according to any one of (1) to (3), wherein the transport portion has FcRn binding activity, and the antigen-binding domain does not have FcRn binding activity or has weaker FcRn binding activity than the transport portion. (5) The polypeptide according to any one of (1) to (4), wherein the antigen-binding domain is detachable from the polypeptide, and the antigen-binding activity of the antigen-binding domain increases upon detachment from the polypeptide. (6) The polypeptide according to any one of (1) to (5), wherein the antigen-binding activity of the antigen-binding domain is suppressed by association between the antigen-binding domain and the inhibitory domain of the transport portion. (7) The polypeptide according to (5), wherein the polypeptide includes a cleavage site, and the antigen-binding domain becomes detachable from the polypeptide when the cleavage site is cleaved. (8) The polypeptide according to (6), wherein the polypeptide includes a cleavage site, and the association between the antigen-binding domain and the repressive domain of the transport portion is resolved by cleavage of the cleavage site. (9) The polypeptide according to (7) or (8), wherein the cleavage site comprises a protease cleavage sequence. (10) The polypeptide described in (9), wherein the protease is a target tissue-specific protease. (11) The polypeptide according to (10), wherein the target tissue is cancerous tissue or inflammatory tissue. (12) The polypeptide according to (9), wherein the protease is at least one protease selected from matryptase, urokinase (uPA), and metalloprotease. (13) The polypeptide according to (12), wherein the protease is at least one protease selected from MT-SP1, uPA, MMP2, MMP9, ADAMTS5, MMP7, and MMP13. (14) The polypeptide according to (9), wherein the protease cleavage sequence includes a sequence selected from SEQ ID NOs: 12, 25, 34, 35, 70-73, 75, 76, 91, 178, 193-195. (15) The polypeptide according to any one of (9) to (14), wherein a first movable linker is further added to one end of the protease cleavage sequence. (16) The polypeptide according to (15), wherein a second movable linker is further added to the other end of the protease cleavage sequence. (17) The polypeptide according to (15), wherein the first movable linker is a movable linker made of a glycine-serine polymer. (18) The polypeptide according to (16), wherein the second movable linker is a movable linker made of a glycine-serine polymer. (19) The polypeptide according to any one of (1) to (18), wherein the antigen-binding domain comprises or is a single-domain antibody, and the inhibitory domain of the transport portion inhibits the antigen-binding activity of the single-domain antibody. (20) The polypeptide according to (19), wherein the single-domain antibody is VHH, or VH having antigen-binding activity in a single domain, or VL having antigen-binding activity in a single domain. (21) The polypeptide according to any one of (1) to (20), wherein the antigen-binding domain comprises a single-domain antibody, the repression domain of the transport portion is VHH, or antibody VH, or antibody VL, and the antigen-binding activity of the single-domain antibody is suppressed by the VHH, or antibody VH, or antibody VL. (22) The polypeptide according to any one of (1) to (21), wherein the antigen-binding domain comprises a single-domain antibody, the repression domain of the transport portion is VHH, or antibody VH, or antibody VL, and the antigen-binding activity of the single-domain antibody is suppressed by association with the VHH, or antibody VH, or antibody VL. (23) The polypeptide according to any one of (19) to (22), wherein the monodomain antibody is VHH or VH having antigen-binding activity in a monodomain, the repression domain of the transport portion is antibody VL, and the antigen-binding activity of the VHH or VH having antigen-binding activity in a monodomain is suppressed by association with the antibody VL. (24) The polypeptide according to any one of (19) to (23), wherein the monodomain antibody is VHH, and the VHH is substituted 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 monodomain antibody is 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 monodomain antibody is VHH, and the VHH comprises 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 monodomain antibody is VHH, and the VHH is substituted with amino acids at at least one set 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 monodomain antibody is VHH, and the VHH comprises at least one set of amino acids selected from 37V / 44G, 37V / 45L, 37V / 47W, 44G / 45L, 44G / 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 monodomain antibody is VHH, and the VHH comprises 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 monodomain antibody is a VL having antigen-binding activity in a single domain, the repression domain of the transport portion is an antibody VH, and the antigen-binding activity of the monodomain antibody VL is suppressed by association with the antibody VH. (31) The transport portion has an FcRn binding region, wherein the polypeptide is according to any one of (1) to (30). (32) The polypeptide according to any one of (1) to (31), wherein the transport portion includes an antibody constant region. (33) The polypeptide according to (32), wherein the antibody constant region of the transport portion and the antigen-binding domain are fused via or without a linker. (34) The polypeptide according to (32), wherein the transport portion includes an antibody heavy chain constant region, and the antibody heavy chain constant region and the antigen-binding domain are fused via or without a linker. (35) The polypeptide according to (32), wherein the transport portion includes an antibody light chain constant region, and the antibody light chain constant region and the antigen-binding domain are fused via or without a linker. (36) The polypeptide according to (34), wherein the N-terminus of the constant region of the antibody heavy chain of the transport portion and the C-terminus of the antigen-binding domain are fused via or without a linker, and further has a protease cleavage sequence, the protease cleavage sequence being located in the sequence of the antigen-binding domain or on the antigen-binding domain side of amino acid 122 (EU numbering) of the constant region of the heavy antibody. (37) The polypeptide according to (35), wherein the N-terminus of the constant region of the antibody light chain of the transport portion and the C-terminus of the antigen-binding domain are fused via or without a linker, and further has a protease cleavage sequence, the protease cleavage sequence being located in the sequence of the antigen-binding domain or on the antigen-binding domain side of amino acid 113 (EU numbering) (Kabat numbering 113) of the constant region of the light chain antibody. (38) The polypeptide according to any one of (33) to (35), wherein the N-terminus of the antibody constant region of the transport portion and the C-terminus of the antigen-binding domain are fused via or without a linker, the antigen-binding domain is a single-domain antibody or VHH made from VH, and the polypeptide further has a protease cleavage sequence, the protease cleavage sequence is located in the sequence of the antibody constant region or on the antibody constant region side of 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 portion and the C-terminus of the antigen-binding domain are fused via or without a linker, and further has a protease cleavage sequence, the protease cleavage sequence being 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 constant region of the antibody heavy chain of the transport portion and the C-terminus of the antigen-binding domain are fused via or without a linker, and further has a protease cleavage sequence, the protease cleavage sequence being located near the boundary between the antigen-binding domain and the constant region of the antibody heavy chain. (41) The polypeptide according to (35), wherein the N-terminus of the constant region of the antibody light chain of the transport portion and the C-terminus of the antigen-binding domain are fused via or without a linker, and further has a protease cleavage sequence, the protease cleavage sequence being located near the boundary between the antigen-binding domain and the constant region of the antibody light chain. (42) The polypeptide according to (40), wherein the antigen-binding domain is a single-domain antibody or VHH prepared from VH, and the protease cleavage sequence is located between the 109th amino acid (Kabat numbering) of the single-domain antibody of the antigen-binding domain and the 122nd amino acid (EU numbering) of the constant region of the antibody heavy chain. (43) The polypeptide according to (41), 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 constant region of the antibody light chain. (44) The polypeptide according to (40), wherein the antigen-binding domain is a single-domain antibody produced from VL, and the protease cleavage sequence is located between the 104th amino acid (Kabat numbering) of the single-domain antibody of the antigen-binding domain and the 122nd amino acid (EU numbering) of the constant region of the antibody heavy chain. (45) The polypeptide according to (41), wherein the antigen-binding domain is a single-domain antibody produced from 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 constant region of the antibody light chain. (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) A polypeptide according to any one of (1) to (47), wherein, when the antigen-binding domain is not released, no binding between the antigen-binding domain and the antigen is observed when measured using the BLI (Bio-Layer Interferometry) method (Octet). (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 different antigen-binding specificity from the antigen-binding domain. (51) The polypeptide according to (49) or (50), wherein the second antigen-binding domain comprises a second monodomain antibody. (52) The polypeptide according to (51), wherein the antigen-binding domain is a monodomain antibody, the second antigen-binding domain is a second monodomain antibody, the antigen-binding domain and the second antigen-binding domain are releaseable from the polypeptide, and in the release state of the antigen-binding domain and the second antigen-binding domain, the monodomain antibody and the second monodomain antibody form a bispecific antigen-binding molecule. (53) The second antigen-binding domain is a polypeptide according to any one of (49) to (52), with HER2 or GPC3 as the target antigen. (54) The polypeptide according to any one of (1) to (53), wherein the polypeptide further has another antigen-binding domain in addition to the antigen-binding domain, and the antigen-binding activity is suppressed by the linkage of the other antigen-binding domain to the transport portion of the polypeptide. (55) The polypeptide according to (54), wherein the other antigen-binding domain has different antigen-binding specificity from 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 that targets PlexinA1, IL6R, or CD3 as the target antigen. A pharmaceutical composition comprising the polypeptide described in any of (57)(1) to (56). (58) A method for producing the polypeptide described in any of (1) to (56). (59) The following steps: (a) A step to obtain a single-domain antibody that binds to a target antigen; (b) A step of forming a polypeptide precursor by linking the single-domain antibody obtained in step (a) with the transport portion such that the antigen-binding activity of the single-domain antibody is suppressed by the inhibitory domain of the transport portion; (c) A step of introducing a protease cleavage sequence into the polypeptide precursor; The manufacturing method described in (58), including (60) Steps below: (a) A step to obtain a single-domain antibody that binds to a target antigen; (b) A step of forming a polypeptide precursor by linking the single-domain antibody obtained in step (a) with the transport portion such that the antigen-binding activity of the single-domain antibody is suppressed by the inhibitory domain of the transport portion; (c) A step of introducing a protease cleavage sequence near the boundary between the monodomain antibody and the transport portion; The manufacturing method described in (58), including (61) The following steps: (a) A step to obtain a single-domain antibody that binds to a target antigen; (b) A step of forming a polypeptide by linking the single-domain antibody obtained in step (a) with the transport portion via a protease cleavage sequence such that the antigen-binding activity of the single-domain antibody is suppressed by the inhibitory domain of the transport portion; The manufacturing method described in (58), including (62) The following steps: (d) A step of confirming that the binding activity of the monodomain antibody incorporated in the polypeptide or polypeptide precursor to the target antigen is weakened or lost; A manufacturing method according to any one of (59) to (61), including the above. (63) The following steps: (e) A step of releasing the monodomain antibody by cleaving the protease cleavage sequence with a protease, and confirming that the released monodomain antibody binds to the antigen; A manufacturing method according to any one of (59) to (62), including the above. (64) The method for producing the polypeptide described in (58), wherein the polypeptide is an IgG antibody-like molecule. (65) The following steps: (a) A step to obtain a single-domain antibody that binds to a target antigen; (b) A step of forming an IgG antibody-like molecular precursor into which the single-domain antibody has been introduced, by associating the single-domain antibody with the VL of the IgG antibody in place of the VH, or by associating the single-domain antibody with the VH of the IgG antibody in place of the VL, so as to suppress the antigen-binding activity of the single-domain antibody obtained in step (a); (c) A step of introducing a protease cleavage sequence into the IgG antibody-like molecular precursor into which the monodomain antibody has been introduced; The manufacturing method described in (64), including (66) The following steps: (a) A step to obtain a single-domain antibody that binds to a target antigen; (b) A step of forming an IgG antibody-like molecular precursor into which the single-domain antibody has been introduced, by associating the single-domain antibody with the VL of the IgG antibody in place of the VH, or by associating the single-domain antibody with the VH of the IgG antibody in place of the VL, so as to suppress the antigen-binding activity of the single-domain antibody obtained in step (a); (c) A step of introducing a protease cleavage sequence near the boundary between the monodomain antibody and the antibody constant region in the IgG antibody-like molecular precursor; The manufacturing method described in (64), including (67) The following steps: (a) A step to obtain a single-domain antibody that binds to a target antigen; (b) A step of forming an IgG antibody-like molecule into which the monodomain antibody obtained in step (a) is introduced, by linking the monodomain antibody 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 as to suppress the antigen-binding activity of the monodomain antibody; The manufacturing method described in (64), including (68) The following steps: (d) A step of confirming that the binding activity of the monodomain antibody introduced into the IgG antibody-like molecule or the IgG antibody-like molecule precursor to the target antigen is weakened or lost; A manufacturing method according to any one of (65) to (67), including the above. (69) The following steps: (e) A step of releasing the monodomain antibody by cleaving the protease cleavage sequence with a protease, and confirming that the released monodomain antibody binds to the target antigen; A manufacturing method according to any one of (65) to (68), including the above. (70) Steps below: (a) A step of producing a modified single-domain antibody in which an amino acid residue involved in association with antibody VH in a single-domain antibody is substituted, or an amino acid residue involved in association with antibody VL in a single-domain antibody is substituted, thereby maintaining the binding activity of the single-domain antibody to the target antigen; (b) A step of forming an IgG antibody-like molecular precursor into which the modified single-domain antibody has been introduced, by associating the modified single-domain antibody with antibody VL or with antibody VH in such a way as to suppress the antigen-binding activity of the modified single-domain antibody prepared in step (a); (c) A step of introducing a protease cleavage sequence into an IgG antibody-like molecular precursor into which the modified single-domain antibody has been introduced; The manufacturing method described in (64), including (71) The following steps: (a) A step of producing a modified single-domain antibody in which an amino acid residue involved in association with antibody VH in a single-domain antibody is substituted, or an amino acid residue involved in association with antibody VL in a single-domain antibody is substituted, thereby maintaining the binding activity of the single-domain antibody to the target antigen; (b) A step of forming an IgG antibody-like molecular precursor into which the modified single-domain antibody has been introduced, by associating the modified single-domain antibody with antibody VL or with antibody VH in such a way as to suppress the antigen-binding activity of the modified single-domain antibody prepared in step (a); (c) A step of introducing a protease cleavage sequence near the boundary between the modified single-domain antibody and the constant region of the IgG antibody-like molecular precursor; The manufacturing method described in (64), including (72) The following steps: (a) A step of producing a modified single-domain antibody in which an amino acid residue involved in association with antibody VH in a single-domain antibody is substituted, or an amino acid residue involved in association with antibody VL in a single-domain antibody is substituted, thereby maintaining the binding activity of the single-domain antibody to the target antigen; (b) A step of forming an IgG antibody-like molecule into which the modified single-domain antibody has been introduced, by 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 by linking the modified single-domain antibody to the light chain constant region of an IgG antibody via a protease cleavage sequence, in order to suppress the antigen-binding activity of the modified single-domain antibody; The manufacturing method described in (64), including (73) The following steps: (d) A step of 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; A manufacturing method according to any one of (70) to (72), including the above. (74) The following steps: (e) A step of releasing the modified single-domain antibody by cleaving the protease cleavage sequence with a protease, and confirming that the released modified single-domain antibody binds to the target antigen; A manufacturing method according to any one of (70) to (73), including the above. A polynucleotide encoding a polypeptide as described in any of (75)(1) to (56). A vector containing the polynucleotides described in (76)(75). A host cell containing the polynucleotide described in (77)(75) or the vector described in (76). A method for producing a polypeptide according to any one of (1) to (56), comprising the step of culturing the host cells described in (78) or (77). (79) A method for screening single-domain antibodies whose antigen-binding activity is suppressed by association with a specific VL, or by association with a specific VH, or by association with a specific VHH. (80) The screening method described in (79) for screening single-domain antibodies whose antigen-binding activity is suppressed by association with a specific VL. (81) The following steps: (a) A step to obtain a monodomain antibody having target antigen-binding activity; (b) A step of associating the single-domain antibody obtained in step (a) with a specific VL; (c) A step to confirm 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 the association; The screening method described in (80), including the following. (82) The following steps: (a) A step of associating a single-domain antibody with a specific VL; (b) A step of selecting an aggregate of a VL and a single-domain antibody in which the single-domain antibody associated with a specific VL in step (a) has no binding activity to the antigen or is below a certain value; (c) A step to confirm that the binding activity of the single-domain antibody in the aggregate selected in step (b) to the antigen is stronger when it is not associated with the specific VL compared to when it is associated; The screening method described in (80), including the following. (83) The screening method described in (79) for screening single-domain antibodies whose antigen-binding activity is suppressed by association with a specific VH. (84) The following steps: (a) A step to obtain a monodomain antibody having target antigen-binding activity; (b) A step of associating the single-domain antibody obtained in step (a) with a specific VH; (c) A step to confirm that the binding activity of the monodomain antibody associated with a specific VH in step (b) against the antigen is weakened or lost compared to before the association; The screening method described in (83), including the following. (85) The following steps: (a) A step of associating a single-domain antibody with a specific VH; (b) A step of selecting an aggregate of a VH and a single-domain antibody in which the single-domain antibody associated with a specific VH in step (a) has no binding activity to the antigen or is below a certain value; (c) A step to confirm that the binding activity of the single-domain antibody in the aggregate selected in step (b) to the antigen is stronger when it is not associated with the specific VH compared to when it is associated; The screening method described in (83), including the following. (86) The screening method described in (79) for screening single-domain antibodies whose antigen-binding activity is suppressed by association with a specific VHH. (87) The following steps: (a) A step to obtain a monodomain antibody having target antigen-binding activity; (b) A step of associating the single-domain antibody obtained in step (a) with a specific VHH; (c) A step to confirm that the binding activity of the monodomain antibody associated with a specific VHH in step (b) against the antigen is weakened or lost compared to before the association; The screening method described in (86), including the following: (88) The following steps: (a) A step of associating a single-domain antibody with a specific VHH; (b) A step of selecting an aggregate of VHH and a single-domain antibody in which the single-domain antibody associated with a specific VHH in step (a) has no binding activity to the antigen or is below a certain value; (c) A step to confirm that the binding activity of the single-domain antibody in the aggregate selected in step (b) against the antigen is stronger when it is not associated with the specific VHH compared to when it is associated; The screening method described in (86), including the following: (89) A method for producing a single-domain antibody whose antigen-binding activity is suppressed by association with a specific VL, or by association with a specific VH, or by association with a specific VHH. (90) A method for producing a monodomain antibody whose antigen-binding activity is suppressed by association with a specific VL, as described in (89). (91) The following steps: (a) A step of producing a modified single-domain antibody in which an amino acid residue involved in association with the antibody VL is substituted in the single-domain antibody, thereby maintaining the binding activity of the single-domain antibody against the target antigen; The manufacturing method described in (90), including (92) The following steps: (b) A step of associating the modified single-domain antibody prepared in step (a) with the VL; (c) A step to confirm that the antigen-binding activity of the modified single-domain antibody associated with the VL is weakened or lost compared to before association; The manufacturing method described in (91), including (93) The method for producing a monodomain antibody whose antigen-binding activity is suppressed by association with a specific VH, as described in (89). (94) The following steps: (a) A step of producing a modified single-domain antibody in which an amino acid residue involved in the association of an IgG antibody-like molecule with VH is substituted in the single-domain antibody, thereby maintaining the binding activity of the single-domain antibody against the target antigen; The manufacturing method described in (93), including (95) The following steps: (b) A step of associating the modified single-domain antibody prepared in step (a) with the VH; (c) A step to confirm that the antigen-binding activity of the modified single-domain antibody associated with the VH is weakened or lost compared to before the association; The manufacturing method described in (94), including (96) The method for producing a monodomain antibody whose antigen-binding activity is suppressed by association with a specific VHH, as described in (89). (97) The following steps: (a) A step of producing a modified single-domain antibody in which an amino acid residue involved in association with VHH is substituted in the single-domain antibody, thereby maintaining the binding activity of the single-domain antibody to the target antigen; The manufacturing method described in (96), including (98) The following steps: (b) A step of associating the modified single-domain antibody prepared in step (a) with the VHH; (c) A step to confirm that the antigen-binding activity of the modified single-domain antibody associated with the VHH is weakened or lost compared to before the association; The manufacturing method described in (97), including (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 comprises a single-domain antibody whose antigen-binding activity is suppressed or lost upon association with a specific VL, or a single-domain antibody whose antigen-binding activity is suppressed or lost upon association with a specific VH, or a single-domain antibody whose antigen-binding activity is suppressed or lost upon association with a specific VHH. (100) The library according to (99), wherein the single-domain antibody portion of the fusion polypeptide in the library includes a single-domain antibody or a humanized antibody thereof obtained from a camelid or a genetically modified animal into which a gene capable of producing a single-domain antibody has been introduced, or a single-domain antibody or a humanized antibody thereof obtained by immunizing a camelid or a genetically modified animal into which a gene capable of producing a single-domain antibody has been introduced, or a single-domain antibody artificially created starting from a human antibody VH or VL. (101) A library comprising a plurality of fusion polypeptides in which a monodomain antibody is linked to a first association-supporting domain, wherein the monodomain antibody includes a monodomain antibody whose antigen-binding activity is suppressed or lost upon association with a specific VL, as described in (99) or (100). (102) A library comprising a plurality of fusion polypeptides in which a monodomain antibody is linked to a first association-supporting domain, wherein the monodomain antibody includes a monodomain antibody whose antigen-binding activity is suppressed or lost upon association with a specific VH, as described in (99) or (100). (103) A library comprising a plurality of fusion polypeptides in which a monodomain antibody is linked to a first association-supporting domain, wherein the monodomain antibody includes a monodomain antibody whose antigen-binding activity is suppressed or lost upon association with a specific VHH, as described in (99) or (100). A method for screening fusion polypeptides from the libraries described in (104)(99) or (100) for monodomain antibodies whose antigen-binding activity is suppressed or lost upon association with a specific VL, or monodomain antibodies whose antigen-binding activity is suppressed or lost upon association with a specific VH, or monodomain antibodies whose antigen-binding activity is suppressed or lost upon association with a specific VHH. A method for screening fusion polypeptides containing monodomain antibodies whose antigen-binding activity is suppressed or lost upon association with a specific VL from the libraries described in (105)(101). (106) The following steps: (a) A step of displaying the fused polypeptide of the library in vitro; (b) The process of preparing a meeting partner by fusing a specific VL with a second meeting support domain; (c) A step of 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 the antigen when the single-domain antibody and the VL are associated, or whose antigen-binding activity is below a certain value; (d) A step of selecting a fusion polypeptide from the fusion polypeptide selected in step (c) that binds to the antigen without the single-domain antibody contained therein associating with the VL, or that has an antigen-binding activity of a certain value or higher; The screening method described in (105), including the following. (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 resolve the VL association with the single-domain antibody. (108) The screening method according to (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 polypeptide of the library further comprises a protease cleavage sequence, and in step (d), the fusion polypeptide is cleaved by protease treatment to resolve the association between the monodomain 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-supporting domain. (111) The screening method according to (106), wherein in step (d), the full-length or single-domain antibody portion of the fusion polypeptide selected in step (c) is again displayed in vitro. (112) The screening method according to (106), wherein in step (d), the entire length of the fusion polypeptide selected in step (c) is again displayed in vitro, and a fusion polypeptide is selected that binds to the antigen while associated only with the second association supporting domain, or that has an antigen-binding activity of a certain value or higher. A method for screening fusion polypeptides containing single-domain antibodies whose antigen-binding activity is suppressed or lost upon association with a specific VH from the libraries described in (113)(102). (114) The following steps: (a) A step of displaying the fused polypeptide of the library in vitro; (b) The process of preparing a meeting partner by fusing a specific VH with a second meeting support domain; (c) A step of 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 the antigen when the single-domain antibody and the VH are associated, or whose antigen-binding activity is below a certain value; (d) A step of selecting a fusion polypeptide from the fusion polypeptides selected in step (c) that binds to the antigen without the single-domain antibody contained therein associating with the VH, or that has an antigen-binding activity of a certain value or higher; The screening method described in (113), including the following. (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 resolve 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-supporting domain. (117) The screening method according to (114), 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 resolve the association between the monodomain 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-supporting domain. (119) The screening method according to (114), wherein in step (d), the full-length or single-domain antibody portion of the fusion polypeptide selected in step (c) is again displayed in vitro. (120) The screening method according to (114), wherein in step (d), the entire length of the fusion polypeptide selected in step (c) is again displayed in vitro, and a fusion polypeptide is selected that binds to the antigen while associated only with the second association supporting domain, or that has an antigen-binding activity of a certain value or higher. A method for screening fusion polypeptides containing single-domain antibodies whose antigen-binding activity is suppressed or lost upon association with a specific VHH, from the libraries described in (121) and (103). (122) The following steps: (a) A step of displaying the fused polypeptide of the library in vitro; (b) The process of preparing a meeting partner by fusing a specific VHH with a second meeting support domain; (c) A step of 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 the antigen when the single-domain antibody and the specific VHH are associated, or whose antigen-binding activity is below a certain value; (d) A step of selecting a fusion polypeptide from the fusion polypeptide selected in step (c) that binds to the antigen without the single-domain antibody contained therein associating with the VHH, or that has an antigen-binding activity of a certain value or higher; The screening method described in (121), including the following. (123) The screening method according to (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 resolve the association between the single-domain antibody and the VHH. (124) The screening method according to (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-supporting domain. (125) The screening method according to (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 resolve the association between the monodomain 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-supporting domain. (127) The screening method according to (122), wherein in step (d), the full-length or single-domain antibody portion of the fusion polypeptide selected in step (c) is again displayed in vitro. (128) The screening method according to (122), wherein in step (d), the entire length of the fusion polypeptide selected in step (c) is again displayed in vitro, and a fusion polypeptide is selected that binds to the antigen while associated only with the second association supporting domain, or that has an antigen-binding activity of a certain value or higher. (129) The screening method according to any one of (106) to (112), (114) to (120), or (122) to (128), wherein the step of preparing an association partner in step (b) is a step of displaying the association partner and the fusion polypeptide simultaneously. (130) A library according to any one of (99) to (103), wherein the first association-supporting 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), or (122) to (128), wherein the first association-supporting domain comprises an IgG antibody CH1 domain and the second association-supporting domain comprises an antibody light chain constant region. (132) The screening method according to any one of (106) to (112), (114) to (120), or (122) to (128), wherein the first association-supporting domain comprises an antibody light chain constant region and the second association-supporting domain comprises an IgG antibody CH1 domain. (133) The following steps: (a) A step of displaying the fused polypeptide of the library in vitro; (b) The process of preparing a meeting partner by fusing a specific VL with a second meeting support domain; (c) A step of selecting a fusion polypeptide in which a single-domain antibody contained in the fusion polypeptide binds to an antigen, or in which the antigen-binding activity is above a certain value; (d) A step of 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 the antigen when the single-domain antibody and the VL are associated, or whose antigen-binding activity is below a certain value; The screening method described in (105), including the following. (134) The screening method according to (129), wherein in step (d), the fusion polypeptide selected in step (c) is displayed again in vitro. (135) The screening method according to (133), wherein in step (c), the fusion polypeptide is associated only with the second association-supporting domain, or antigen binding of a single-domain antibody contained in the fusion polypeptide is confirmed while the fusion polypeptide is associated only with the second association-supporting domain. (136) The following steps: (a) A step of displaying the fused polypeptide of the library in vitro; (b) The process of preparing a meeting partner by fusing a specific VH with a second meeting support domain; (c) A step of selecting a fusion polypeptide in which a single-domain antibody contained in the fusion polypeptide binds to an antigen, or in which the antigen-binding activity is above a certain value; (d) A step of 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 the antigen when the single-domain antibody and the VH are associated, or whose antigen-binding activity is below a certain value; The screening method described in (113), including the following. (137) The screening method according to (136), wherein in step (d), the fusion polypeptide selected in step (c) is displayed again in vitro. (138) The screening method according to (136), wherein in step (c), the fusion polypeptide is associated only with the second association-supporting domain, or antigen binding of a single-domain antibody contained in the fusion polypeptide is confirmed while the fusion polypeptide is associated only with the second association-supporting domain. (139) The following steps: (a) A step of displaying the fused polypeptide of the library in vitro; (b) The process of preparing a meeting partner by fusing a specific VHH with a second meeting support domain; (c) A step of selecting a fusion polypeptide in which a single-domain antibody contained in the fusion polypeptide binds to an antigen, or in which the antigen-binding activity is above a certain value; (d) A step of 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 the antigen when the single-domain antibody and the VHH are associated, or whose antigen-binding activity is below a certain value; The screening method described in (121), including the following. (140) The screening method according to (139), wherein in step (d), the fusion polypeptide selected in step (c) is displayed again in vitro. (141) The screening method according to (139), wherein in step (c), the fusion polypeptide is associated only with the second association-supporting domain, or antigen binding of a single-domain antibody contained in the fusion polypeptide is confirmed while the fusion polypeptide is associated only with the second association-supporting 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 displaying the association partner and the fusion polypeptide simultaneously. (143) The screening method according to any one of (133) to (142), wherein the first association-supporting domain comprises an IgG antibody CH1 domain and the second association-supporting domain comprises an antibody light chain constant region. (144) The screening method according to any one of (133) to (142), wherein the first association-supporting domain comprises an antibody light chain constant region, and the second association-supporting domain comprises an IgG antibody CH1 domain. [Brief explanation of the drawing]

[0015] [Figure 1]This diagram illustrates the concept of Probody technology. It is an antibody molecule in which the antigen-binding activity of the antibody is inhibited by linking a peptide that masks the antigen-binding site of the antibody to the antibody via a linker that is cleaved by a protease expressed at the lesion site. [Figure 2] This diagram illustrates one possible cause of side effects from probody. Activated probody accumulated in the bloodstream can bind to antigens expressed in normal tissues, potentially causing adverse effects. [Figure 3] This diagram illustrates one possible cause of side effects in Probody. Probody exists in an equilibrium state where the mask peptide linked by the linker is either bound to the antigen-binding site or dissociated. The dissociated molecule can then bind to the antigen. [Figure 4] This diagram illustrates one possible cause of side effects from Probody. Antidrug antibodies against mask peptides (anti-mask peptide antibodies) can potentially activate Probody even without protease cleavage by binding to the mask peptide of Probody before it is activated. [Figure 5] This diagram illustrates the concept of a polypeptide containing an antigen-binding domain and a transporter. (A) A polypeptide with the antigen-binding domain and transporter linked has a long half-life and does not bind to the antigen. (B) The antigen-binding domain is released by cleavage at a cleavage site, etc., and binds to the antigen. The released antigen-binding domain then has a short half-life. [Figure 6] This figure shows one embodiment of the method for producing the polypeptide of the present invention. In this embodiment, the target polypeptide is an IgG antibody-like molecule. (A) A monodomain antibody that binds to the target antigen is obtained. (B) The monodomain antibody is associated with the VL of the IgG antibody instead of the VH such that the antigen-binding activity of the monodomain antibody is suppressed. (C) A protease cleavage sequence is introduced into the IgG antibody-like molecule precursor into which the monodomain antibody has been introduced. [Figure 7]This figure shows one embodiment of the polypeptide of the present invention. In this embodiment, the polypeptide is an IgG antibody-like molecule, and antigen-binding domains are provided in the regions corresponding to the two variable regions of an IgG antibody. The two antigen-binding domains may have similar antigen-binding specificity or different antigen-binding specificity. [Figure 8] This figure 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) This figure shows the polypeptide in its unreleased state. The antigen-binding activity of the antigen-binding domain is suppressed. (B) This figure shows the release of the bispecific antigen-binding molecule formed by the antigen-binding domain and the second antigen-binding domain. (C) This figure shows an example of a bispecific antigen-binding molecule after release, for example, a bispecific antigen-binding molecule for T cell surface antigen and cancer cell surface antigen. [Figure 9A]This figure illustrates an example of a method for screening fusion polypeptides containing single-domain antibodies whose antigen-binding activity is attenuated or lost upon association with a specific inhibitory domain, from a library containing multiple fusion polypeptides in which single-domain antibodies are linked to a first association-supporting domain. (1) This figure shows a library containing multiple fusion polypeptides in which single-domain antibodies are linked to a first association-supporting domain. (2) This figure shows the confirmation of the antigen-binding activity of the single-domain antibody when the fusion polypeptide and its association partner are associated. 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) This figure shows the confirmation of the antigen-binding activity of the single-domain antibody when the association between the single-domain antibody in the fusion polypeptide selected in (2) and the inhibitory domain in the association partner is resolved. 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') This figure shows the confirmation of the antigen-binding activity of the single-domain antibody in the fusion polypeptide. In the standalone state of this fusion polypeptide, fusion polypeptides containing single-domain antibodies that bind to the target antigen or whose antigen-binding activity is above a certain value are selected. This figure shows the process of confirming the antigen-binding activity of a single-domain antibody in the state where the fusion polypeptide selected in (3') and (2') is associated with its association partner. In this associated state, a fusion polypeptide containing a single-domain antibody that does not bind to the target antigen or whose antigen-binding activity is below a certain value is selected. [Figure 9B]This figure shows a more specific example of a method for screening fusion polypeptides containing monodomain antibodies whose antigen-binding activity is attenuated or lost upon association with a specific repressor domain, from a library containing multiple fusion polypeptides in which a monodomain antibody is linked to a first association-supporting domain. (1) A fusion polypeptide containing a monodomain antibody and a first association-supporting domain is simultaneously displayed with an association partner in which a protease cleavage sequence is introduced between the repressor domain and the second association-supporting domain to form a Fab-like structure; (2) From the displayed Fab-like structure, those that do not bind to the antigen or have antigen-binding activity below a certain value are selected; (3) The association partner is cleaved by a protease, and a fragment containing a monodomain antibody that binds to the antigen or has antigen-binding activity above a certain value is selected. [Figure 9C] This figure shows another, more specific example of a method for screening fusion polypeptides containing single-domain antibodies whose antigen-binding activity is attenuated or lost upon association with a specific repressor domain, from a library containing multiple fusion polypeptides in which a single-domain antibody is linked to a first association-supporting domain. (1) A fusion polypeptide in which a protease cleavage sequence is introduced between a single-domain antibody and the first association-supporting domain is simultaneously displayed with an association partner in which the repressor domain and the second association-supporting domain are linked, forming a Fab-like structure; (2) From the displayed Fab-like structure, those that do not bind to the antigen or have antigen-binding activity below a certain value are selected; (3) The fusion polypeptide is cleaved by a protease and fragments containing single-domain antibodies that bind to the antigen or have antigen-binding activity above a certain value are selected. [Figure 9D]This figure shows another example of a method for screening a library containing multiple fusion polypeptides, each containing a monodomain antibody linked to a first association support domain, for fusion polypeptides containing monodomain antibodies whose antigen-binding activity is reduced or lost upon association with a specific repressor domain. (1) A fusion polypeptide containing a monodomain antibody and a first association support domain is simultaneously displayed with an association partner linked to a repressor domain and a second association support domain to form a Fab-like structure, and from the displayed Fab-like structure, a portion that does not bind to the antigen or has antigen-binding activity below a certain value is selected; (2) The portion containing the monodomain antibody in the Fab-like structure selected in (1) is redisplayed without simultaneously expressing the repressor domain, and a fragment that binds to the antigen or has antigen-binding activity above a certain value is selected. (2') and (2'') are figures showing another embodiment of (2), in which the portion containing the monodomain antibody is redisplayed without simultaneously expressing the repressor domain. Note that the order of (1) and (2) / (2') / (2'') can also be (2) / (2') / (2'') to (1), that is, the portion containing the monodomain antibody is displayed without simultaneously expressing the repressive domain, and a fragment with antigen binding activity above a certain value is selected. Next, the monodomain antibody containing the fragment with binding activity above a certain value, the fusion polypeptide containing the first association support domain, and the association partner in which the repressive domain and the second association support domain are linked are displayed simultaneously to form a Fab-like structure, and from the displayed Fab-like structure, those that do not bind to the antigen or have antigen binding activity below a certain value are selected. [Figure 10] This figure shows the results of evaluating the binding of antibody-like molecules to human IL6R, which were created by associating IL6R90-G1m (IL6R90-G1m, which is formed by fusing anti-human IL6R VHH (IL6R90) to the constant region (CH1-hinge-CH2-CH3) of human IgG1) with various light chains. The horizontal axis represents the start time when the antigen immobilized on the sensor and the antibody-like molecule began to act. [Figure 11](A) This figure shows a model of an antibody-like molecule created by inserting a protease cleavage sequence near the boundary between the VHH and constant region of IL6R90-G1m. (B) This figure shows the name of each antibody heavy chain created, 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 evaluating the degree of cleavage by reduced SDS-PAGE after protease (MT-SP1) treatment of antibody-like molecules created by inserting a protease cleavage sequence near the boundary between the VHH and constant region of IL6R90-G1m or IL6R90-G1m. Of the two new bands created by protease treatment, the band that appeared below 25 kDa is derived from the VHH, and the band that appeared at the 25-50 kDa position is derived from the constant region. [Figure 12-2] This figure is a continuation of Figure 12-1. [Figure 13] This figure shows the results of evaluating the binding of antibody-like molecules created by inserting a protease cleavage sequence near the boundary between the VHH and constant region of IL6R90-G1m or IL6R90-G1m, or samples after protease (MT-SP1) treatment of these molecules, to human IL6R. "Protease-" represents the sensorgram evaluating the binding of the protease-untreated antibody-like molecule to the antigen, while "Protease+" represents the sensorgram evaluating the binding of the protease-treated antibody-like molecule to the antigen. The horizontal axis starts 30 seconds before the start of action between the antigen-immobilized sensor and the antibody-like molecule. [Figure 14] This figure shows the results of evaluating the binding of antibody-like molecules to human IL6R, which were created by fusing anti-human IL6R VHH(20A11) to the constant region (CH1-hinge-CH2-CH3) of human IgG1 (20A11-G1m) and associating it with various light chains. The horizontal axis starts 30 seconds before the start of action between the antigen-immobilized sensor and the antibody-like molecule. [Figure 15]This figure shows the results of evaluating the binding of antibody-like molecules to human IL6R, which were created by fusing 20A11hu (created by introducing mutations into the amino acids present at the interface between 20A11-G1m or 20A11 with the VL) to the constant region (CH1-hinge-CH2-CH3) of human IgG1, and then associating these 20A11hu-G1m molecules with various light chains. The horizontal axis starts 60 seconds before the start of action between the antigen-immobilized sensor and the antibody-like molecule. [Figure 16] This figure shows the results of evaluating the degree of cleavage of four antibody-like molecules, prepared by inserting a protease cleavage sequence near the boundary between 20A11hu and the constant region of 20A11-G1m or 20A11hu-G1m, after treatment with protease (MT-SP1), using reduced SDS-PAGE. Of the two new bands generated by the protease treatment, the band that appeared below 25 kDa is derived from VHH, and the band that appeared at the 25-50 kDa position is derived from the constant region. [Figure 17] This figure shows the results of evaluating the binding of antibody-like molecules created by inserting a protease cleavage sequence near the boundary between the VHH and constant region of 20A11-G1m or 20A11hu-G1m, or samples after protease (MT-SP1) treatment of these molecules, to human IL6R. "Protease-" represents the sensorgram evaluating the binding of untreated antibody-like molecules to the antigen, while "Protease+" represents the sensorgram evaluating the binding of protease-treated antibody-like molecules to the antigen. The horizontal axis starts 60 seconds before the start of action between the antigen-immobilized sensor and the antibody. Samples marked "not tested" indicate that they were not measured. [Figure 18]This figure shows the results of evaluating the degree of cleavage by electrophoresis using reduced SDS-PAGE and detection by CBB after treating an antibody-like molecule prepared by inserting a protease cleavage sequence near the boundary between the VHH and the heavy chain constant region, which had anti-human CD3 VHH in the heavy chain variable region. Of the two new bands generated by the protease treatment, the band that appeared around 10-15 kDa is the band derived from VHH, and the band that appeared around 37 kDa is the band derived from the heavy chain constant region. [Figure 19] This figure shows the results of evaluating the binding of an antibody-like molecule, created by having anti-human CD3 VHH in the heavy chain variable region and inserting a protease cleavage sequence near the boundary between VHH and the heavy chain constant region, to human CD3ed-Fc after treatment with protease (MT-SP1). "Protease-" represents the sensorgram evaluating the binding of the protease-untreated antibody-like molecule to the antigen, while "Protease+" represents the sensorgram evaluating the binding of the protease-treated antibody-like molecule to the antigen. The horizontal axis starts 30 seconds before the start of action between the antigen-immobilized sensor and the antibody-like molecule. The binding amount before antigen binding (response) is set to 0, and the binding amount before antibody action is set to 100. The data is displayed from 30 seconds before antibody action. [Figure 20] This figure shows the results of evaluating the degree of cleavage of antibody-like molecules prepared by inserting a protease cleavage sequence near the boundary between the variable and constant regions of the light chain in a molecule with IL6R90-G1m as the heavy chain and Vk1-39-k0MT as the light chain, or in a molecule with IL6R90-G1m as the heavy chain and Vk1-39-k0MT as the light chain. These molecules were treated with protease (MT-SP1), then electrophoresed using reduced SDS-PAGE, and detected by CBB. Two bands originating from the light chain were generated after protease treatment, 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, prepared by inserting a protease cleavage sequence near the boundary between the variable and constant regions of the light chain 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 IL6R after treatment with protease (MT-SP1). "Protease-" represents the sensorgram evaluating the binding of the protease-untreated antibody-like molecule to the antigen, while "Protease+" represents the sensorgram evaluating the binding of the protease-treated antibody-like molecule to the antigen. An antibody (MRA) confirmed to bind to IL6R was used as a positive control. The starting point on the horizontal axis is when the antigen-immobilized sensor and the antibody-like molecule began to act. [Figure 22] This figure shows the SDS-PAGE results evaluating the protease cleavage of an IgG antibody-like molecule incorporating human Plexin A1-binding VHH. The Protease(+) lane represents the sample that underwent protease cleavage treatment, while the Protease(-) lane represents the negative control sample that did not undergo protease cleavage treatment. [Figure 23] This figure shows Octet sensorgrams evaluating the binding of human Plexin A1 to an IgG antibody-like molecule incorporating human Plexin A1-binding VHH, after VHH was released by protease cleavage. "Protease +" indicates samples treated with protease cleavage, while "protease -" indicates samples that were not treated with protease cleavage. The concentrations of the IgG antibody-like molecules used are indicated on the left side of the figure. [Figure 24] This figure shows the SDS-PAGE results evaluating the protease cleavage of bispecific VHH-VHH-containing polypeptides. [Figure 25] This figure shows the luciferase activity before and after protease cleavage. The dashed line represents the sample without protease treatment, and the solid line represents the sample with protease treatment. [Figure 26]This figure shows the luciferase activity before and after protease cleavage. The dashed line represents the sample without protease treatment, and the solid line represents the sample with protease treatment. [Figure 27] This figure shows the evaluation of protease cleavage of an anti-human IL6R VHH-containing IgG antibody-like molecule using SDS-PAGE. [Figure 28] This figure shows the evaluation of protease cleavage of an IgG antibody-like molecule in which a protease cleavage sequence has been introduced into the light chain. [Figure 29] This figure shows the evaluation of the degree of activation of an IgG-like antibody molecule in which a protease cleavage sequence has been introduced into the light chain, with and without protease treatment. [Figure 30A] This figure shows the 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 the 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 an assay buffer (MMP Activity Assay Kit (Fluorometric - Green) (ab112146), Component C: Assay Buffer). [Modes for carrying out the invention]

[0016] In the present invention, polypeptides typically refer to peptides and proteins having a length of about 4 amino acids or more. While polypeptides in the present invention are typically polypeptides consisting of artificially designed sequences, they are not particularly limited and may, for example, be polypeptides of biological origin. They may also be natural polypeptides, synthetic polypeptides, recombinant polypeptides, etc. Furthermore, fragments of the above-mentioned polypeptides are also included in the polypeptides of the present invention.

[0017] In this specification, amino acids are represented by single-letter codes, three-letter codes, or both, for example, Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, and Val / V. When representing an amino acid at a specific position, a representation that includes both a number indicating the specific position and the single-letter or three-letter code of the amino acid may be used as appropriate. For example, the amino acid 37V, which is contained in a single-domain antibody, represents Val, which is located at position 37 in Kabat numbering.

[0018] For modifying amino acids in the amino acid sequence of polypeptides, known methods such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR may be used as appropriate. Furthermore, several known methods for modifying amino acids by substituting them with non-natural amino acids may also be employed (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249, Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing tRNA in which a non-natural amino acid is bound to a complementary amber suppressor tRNA of the UAG codon (amber codon), one of the stop codons, is also suitably used. In this specification, substitution is mentioned as a modification, but is not limited to this.

[0019] In this specification, the term "and / or" used to describe amino acid modification sites includes any combination of "and" and "or" as appropriate. Specifically, for example, "amino acids 37, 45, and / or 47 are substituted" includes the following variations of amino acid modification: (a) No. 37, (b) No. 45, (c) No. 47, (d) No. 37 and No. 45, (e) No. 37 and No. 47, (f) No. 45 and No. 47, (g) No. 37, No. 45 and No. 47.

[0020] In this specification, expressions that include a number representing a specific position followed by a one-letter or three-letter code of the amino acid before and after the modification may be used as appropriate to represent amino acid modifications. For example, the modification F37V or Phe37Val, used when making amino acid substitutions in the antibody variable region or single-domain antibody, represents the substitution of Phe at position 37, as represented by Kabat numbering, to Val. That is, the number represents the position of the amino acid as represented by Kabat numbering, the one-letter or three-letter code of the amino acid listed before it represents the amino acid before substitution, and the one-letter or three-letter code of the amino acid listed after it represents the amino acid after substitution. Similarly, the modification P238A or Pro238Ala, used when making amino acid substitutions in the Fc region included in the antibody constant region, represents the substitution of Pro at position 238, as represented by EU numbering, to Ala. In other words, the numbers represent the position of the amino acid as expressed in EU numbering, the one-letter or three-letter code of the amino acid listed before it represents the amino acid before substitution, and the one-letter or three-letter code of the amino acid listed after it represents the amino acid after substitution.

[0021] In this specification, the term “antibody” is used in its broadest sense and is not limited to any antibody that exhibits the desired antigen-binding activity, but encompasses a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-domain antibodies, and antibody fragments.

[0022] An "antibody fragment" refers to a molecule other than the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments are not limited to these, but include Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0023] The terms "full-length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a native antibody, or having a heavy chain containing an Fc region as defined herein.

[0024] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of an antibody (VH and VL, respectively) typically have a similar structure, with each domain containing four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0025] As used herein, the terms “complementarity-determining region” or “CDR” refer to the regions of the variable domain of an antibody that are hypervariable in sequence and / or form structurally defined loops (“hypervariable loops”) and / or antigen contact residues (“antigen contacts”). Typically, an antibody contains six CDRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Illustrative CDRs as used herein include: (a) Hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) Antigen contact occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and, (d) A combination of (a), (b), and / or (c), including 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). Unless otherwise indicated, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein in accordance with Kabat et al.

[0026] The "framework" or "FR" refers to variable domain residues other than complementarity-determining region (CDR) residues. The variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the sequences of the CDR and FR usually appear in the VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0027] In this specification, the term “constant region” or “constant domain” refers to the portion of an antibody other than the variable region. For example, an IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons, composed of two identical disulfide-bonded light chains and two identical heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by a heavy chain constant region (CH) containing the CH1 domain, hinge region, CH2 domain, and CH3 domain. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chains of native antibodies may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

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

[0029] The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0030] In this specification, the term "antigen-binding domain" is limited solely to domains that bind to the target antigen. Any domain structure may be used as the antigen-binding domain, as long as it binds to the target antigen. Examples of such domains, though not limited to these, include, for example, the heavy chain variable region (VH) and light chain variable region (VL) of antibodies, single-domain antibodies (sdAb), a module called the A domain of about 35 amino acids contained in Avimer, a cell membrane protein present in living organisms (International Publication WO2004 / 044011, WO2005 / 040229), Adnectin containing the 10Fn3 domain, a protein-binding domain in fibronectin, a glycoprotein expressed on the cell membrane (International Publication WO2002 / 032925), Affibody (International Publication WO1995 / 001937), which uses an IgG-binding domain as a scaffold to form a bundle of three helices consisting of 58 amino acids of Protein A, and ankyrin repeats, which have a structure in which a turn containing 33 amino acid residues and two antiparallel helical and loop subunits are repeatedly stacked. Examples include DARPins (Designed Ankyrin Repeat proteins) (International Publication WO2002 / 020565), which are regions exposed on the molecular surface of repeat (AR); Anticalin, etc. (International Publication WO2003 / 029462), which are four loop regions in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) that support one side of a barrel structure twisted towards the center by eight highly conserved antiparallel strands; and recessed regions of parallel sheet structures within a horseshoe-shaped structure in which leucine-rich repeat (LRR) modules, which do not possess the structure of immunoglobulins, are repeatedly stacked, as part of the acquired immune system of jawless fish such as lampreys and hagfish (International Publication WO2008 / 016854).

[0031] Suitable examples of the antigen-binding domain of the present invention include an antigen-binding domain that can exhibit antigen-binding function in a molecule composed solely of the antigen-binding domain, and an antigen-binding domain that can exhibit antigen-binding function independently after being released from other linked peptides. Examples of such antigen-binding domains, though not limited to these, include single-domain antibodies, scFv, Fv, Fab, Fab', F(ab')2, and the like.

[0032] One preferred example of the 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, but not limited to them, include single-domain antibodies, scFv, Fab, and Fab'. Antigen-binding domains with a molecular weight of 60 kDa or less are usually highly likely to be cleared by the kidney when they exist in the blood as monomers (see J Biol Chem. 1988 Oct 15;263(29):15064-70). From another perspective, one preferred example of the antigen-binding domain of the present invention is an antigen-binding domain with a blood half-life of 12 hours or less. Examples of such antigen-binding domains, but not limited to them, include single-domain antibodies, scFv, Fab, Fab', etc.

[0033] One preferred example of the antigen-binding domain of the present invention is a single-domain antibody (sdAb).

[0034] In this specification, the term "monodomain antibody" is used without regard to its structure, as long as the domain alone can exhibit antigen-binding activity. While conventional antibodies, such as IgG antibodies, exhibit antigen-binding activity when a variable region is formed by the pairing of VH and VL domains, monodomain antibodies are known to exhibit antigen-binding activity solely through their own domain structure, without pairing with other domains. Monodomain antibodies usually have a relatively low molecular weight and exist in monomeric form. Examples of single-domain antibodies, though not limited to them, include, for example, VHH antibodies for camelid animals and V antibodies for sharks. NARExamples include antigen-binding molecules that congenitally lack a light chain, or antibody fragments that contain all or part of the VH domain or all or part of the VL domain of an antibody. Examples of monodomain antibodies that are antibody fragments containing all or part of the VH / VL domain of an antibody include, but are not limited to, monodomain antibodies artificially produced starting from human antibody VH or human antibody VL, as described in, for example, U.S. Patent No. 6,248,516B1. In some embodiments of the present invention, one monodomain antibody has three CDRs (CDR1, CDR2, and CDR3). Monodomain antibodies can be obtained from animals capable of producing monodomain antibodies, or by immunizing animals capable of producing monodomain antibodies. Examples of animals capable of producing monodomain antibodies include, but are not limited to, camelids and transgenic animals into which a gene capable of producing monodomain antibodies has been introduced. Camelids include camels, llamas, alpacas, dromedaries, and guanacos. Examples of transgenic animals into which a gene capable of producing monodomain antibodies has been introduced include, but are not limited to, the transgenic animals described in International Publication WO2015 / 143414 and U.S. Patent Publication US2011 / 0123527A1. Humanized monodomain antibodies can also be obtained by using a human germline sequence or a similar sequence as the framework sequence of a monodomain antibody obtained from an animal. Humanized monodomain antibodies (e.g., humanized VHH) are also one embodiment of the monodomain antibody of the present invention. Furthermore, single-domain antibodies can be obtained from polypeptide libraries containing single-domain antibodies by methods such as ELISA and panning. Examples of polypeptide libraries containing single-domain antibodies include, but are not limited to, naive antibody libraries obtained from various animals or humans (e.g., Methods in Molecular Biology 2012 911 (65-78), Biochimica et Biophysica Acta - Proteins and Proteomics 2006 1764:8 (1307-1319)), antibody libraries obtained by immunizing various animals (e.g., Journal of Applied Microbiology 2014 117:2 (528-536)), or synthetic antibody libraries created from antibody genes of various animals or humans (e.g., Journal of Biomolecular Screening 2016 21:1 (35-43), Journal of Biological Chemistry 2016 291:24 (12641-12657), AIDS 2016 30:11). (1691-1701) is one example.

[0035] In this specification, “antigen” is limited only to including an epitope to which an antigen-binding domain binds. Preferred examples of antigens, but are not limited to, include, for example, animal or human peptides, polypeptides, and proteins. Preferred examples of antigens used to treat diseases caused by target tissues, but are not limited to, include, for example, molecules expressed on the surface of target cells (e.g., cancer cells, inflammatory cells), molecules expressed on the surface of other cells in tissues containing target cells, molecules expressed on the surface of cells that have an immunological role to target cells and tissues containing target cells, and macromolecules present in the stroma of tissues containing target cells.

[0036] Antigens include the following molecules: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, Adresin, 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-Stimulating 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, β-NGF, BOK, Bombecin, 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, PD1, PDL1, 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, Cytokine-related antigen, DAN, DCC, DCR3, DC-SIGN, Complement-accelerating factor (Decay accelerating)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-activating 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, Fractal 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-Alpha 1, GFR-Alpha 2, GFR-Alpha 3, GITR, Glucagon, Glut4, Glycoprotein IIb / IIIa (GPIIb / IIIa), GM-CSF, gp130, gp72, GRO, Growth Hormone Releasing Factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFGPEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2 IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-21, IL-23, IL-27, interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like proliferation 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 duct inhibitor, Mug, MuSK, NAIP, NAP, NCAD, NC adherin, NCA 90, NCAM, NCAM, Neprilysin, Neurotrophin-3, -4, or -6, Neuroturin, 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, RANTES, relaxin A chain, relaxin B chain, renin, polynuclear respiratory 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, Thymus 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 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-β LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand) CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand, Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand, CD70), TNFSF8 (CD30 ligand, CD153), TNFSF9 (4-1BB ligand, CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TLR (Toll-likereceptor)1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen 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 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.

[0037] While the above examples of antigens include receptors, these receptors can also be used as antigens to which the antigen-binding domain of the present invention binds when they exist in a soluble form in biological fluids. One non-limiting embodiment of such a soluble receptor is the protein represented by SEQ ID NO: 35, which is a soluble IL-6R as described by Mullberg et al. (J. Immunol. (1994) 152 (10), 4958-4968).

[0038] The above examples of antigens include membrane-bound molecules expressed on cell membranes and soluble molecules secreted from cells into the extracellular space. When the antigen-binding domain of the present invention binds to a soluble molecule secreted from a cell, it is preferable that the antigen-binding domain has neutralizing activity.

[0039] The soluble molecule is not limited to the solution in which it exists; it can be present in any biological fluid, i.e., any fluid that fills the spaces between blood vessels or tissues and cells within a living organism. In one non-limiting embodiment, the soluble molecule to which the antigen-binding domain of the present invention binds can be present in extracellular fluid. Extracellular fluid refers to the general term for components of bone and cartilage such as plasma, interstitial fluid, lymph, dense connective tissue, cerebrospinal fluid, cerebrospinal fluid, puncture fluid, or synovial fluid, as well as cellular permeable fluids such as alveolar fluid (bronchial alveolar lavage fluid), ascites, pleural fluid, pericardial fluid, cystic fluid, or aqueous humor (aqueous humor) (fluids in various glandular lumens resulting from the active transport and secretory activity of cells, and fluids in the gastrointestinal tract and other body cavities).

[0040] An epitope, meaning an antigenic determinant present in an antigen, refers to a site on the antigen to which an antigen-binding domain, as disclosed herein, binds. Therefore, 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 it. If the antigen is a peptide or polypeptide, the epitope can also be identified by the amino acid residues that constitute it. Furthermore, if the epitope is a glycan, it can also be identified by a specific glycan structure.

[0041] A linear epitope is an epitope that contains an epitope whose amino acid primary sequence has been recognized. A linear epitope typically contains at least three, and most commonly at least five, amino acids, e.g., about eight to about ten, or six to twenty, in a specific sequence.

[0042] A stereoepitope, in contrast to a linear epitope, is an epitope in which the primary amino acid sequence containing the epitope is not a single defining component of the recognized epitope (for example, an epitope whose primary amino acid sequence is not necessarily recognized by the antibody defining the epitope). A stereoepitope may contain a larger number of amino acids than a linear epitope. In relation to the recognition of a stereoepitope, the antigen-binding domain recognizes the three-dimensional structure of the peptide or protein. For example, if a protein molecule folds to form a three-dimensional structure, certain amino acids and / or polypeptide backbone that form the stereoepitope are parallel, allowing the antibody to recognize the epitope. Methods for determining the three-dimensional structure of an epitope include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-specific spin labeling and electromagnetic paramagnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, Morris (ed.).

[0043] The structure of an antigen-binding domain that binds to an epitope is called a paratope. The epitope and paratope bind stably due to hydrogen bonds, electrostatic forces, van der Waals forces, hydrophobic bonds, etc., acting between them. This binding force between the epitope and paratope is called affinity. The sum of the binding forces when multiple antigens and multiple antigen-binding domains bind is called avidity. When antibodies containing multiple antigen-binding domains (i.e., polyvalent antibodies) bind to multiple epitopes, the binding forces (affinity) work synergistically, resulting in avidity that is higher than affinity.

[0044] In certain embodiments, the antigen-binding domains provided herein are ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (for example, 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 It has a dissociation constant (Kd) of M.

[0045] The following are examples of methods for confirming the binding of an antigen-binding domain to an epitope with an antigen-binding domain or a polypeptide containing an antigen-binding domain to an epitope with IL-6R. However, methods for confirming the binding of an antigen-binding domain to an epitope with an antigen-binding domain or a polypeptide containing an antigen-binding domain to an antigen other than IL-6R can also be carried out as appropriate in accordance with the following examples.

[0046] For example, the recognition of a linear epitope present in the IL-6R molecule by the antigen-binding domain for IL-6R can be confirmed, for instance, as follows: A linear peptide consisting of the amino acid sequence constituting the extracellular domain of IL-6R is synthesized for the above purpose. This peptide can be synthesized chemically, or obtained by genetic engineering using the region of the IL-6R cDNA that codes for the amino acid sequence corresponding to the extracellular domain. Next, the binding activity of the linear peptide, consisting of the amino acid sequence constituting the extracellular domain, and the antigen-binding domain for IL-6R is evaluated. For example, the binding activity of the antigen-binding domain to the immobilized linear peptide can be evaluated by ELISA using the immobilized linear peptide as the antigen. Alternatively, the binding activity to the linear peptide can be determined based on the level of inhibition by the linear peptide in the binding of the antigen-binding domain to IL-6R-expressing cells. Through these tests, the binding activity of the antigen-binding domain to the linear peptide can be determined.

[0047] Furthermore, the recognition of the stereoepitope by the antigen-binding domain for IL-6R can be confirmed as follows. For the above purpose, cells expressing IL-6R are prepared. When the antigen-binding domain for IL-6R comes into contact with IL-6R-expressing cells, it binds strongly to the cells, while the antigen-binding domain does not substantially bind to a linear peptide consisting of the amino acid sequence constituting the extracellular domain of immobilized IL-6R, or to a linear peptide consisting of the amino acid sequence constituting the extracellular domain of IL-6R that has been denatured using a common denaturing agent such as guanidine. Here, substantially non-binding means a binding activity of 80% or less, usually 50% or less, preferably 30% or less, and particularly preferably 15% or less of the binding activity to human IL-6R-expressing cells.

[0048] Another method for confirming the antigen-binding activity of an antigen-binding domain is to measure the Kd value using, for example, a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using the antigen-binding domain of interest and its antigen. For example, the solution-bound affinity of an antigen-binding domain to an antigen is measured by equilibrating the antigen-binding domain with a minimum concentration of (125I)-labeled antigen in the presence of an increasing dose series of unlabeled antigens, and then capturing the bound antigen with a plate coated with the antigen-binding domain. (See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999)).

[0049] In another embodiment, Kd is measured by surface plasmon resonance using BIACORE®. For example, the measurement method using BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25°C using a CM5 chip immobilized with approximately 10 response units (RUs) of antigen. 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 being injected at a flow rate of 5 μl / min to achieve binding of approximately 10 response units (RUs) of protein. After antigen injection, 1M ethanolamine is injected to block unreacted groups. For kinetics measurement, two-fold serial dilutions (0.78 nM to 500 nM) of the antigen-binding domain in PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20®) surfactant are injected at 25°C and a flow rate of approximately 25 μl / min. The binding rate (kon) and dissociation rate (koff) are calculated by simultaneously fitting binding and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® evaluation software version 3.2). The equilibrium dissociation constant (Kd) is calculated as the koff / kon ratio. Furthermore, the apparent dissociation constant (Kd) can also be determined using equilibrium analysis. Refer to the protocols included with BIACORE® for these methods. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999) and Methods Enzymol. 2000;323:325-40. Furthermore, in surface plasmon resonance assays, 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.When the on-rate exceeds 10 6 M -1 s -1 , the on-rate can be determined by using fluorescence quenching techniques that measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, bandpass 16 nm) at 25 °C of 20 nM antigen-binding domain in PBS, pH 7.2 in the presence of increasing concentrations of antigen, measured in a spectrometer (e.g., a stopped-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO (trademark) spectrophotometer (ThermoSpectronic) using a stirred cuvette).

[0050] Furthermore, the antigen-binding activity of the antigen-binding domain can also be measured by known methods for measuring intermolecular interactions, such as electrochemiluminescence methods.

[0051] Examples of methods for measuring the binding activity of the antigen-binding domain to IL-6R-expressing cells against IL-6R include, for example, the methods described in Antibodies A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420). That is, it can be evaluated by ELISA using IL-6R-expressing cells as an antigen or based on the principle of FACS (fluorescence activated cell sorting).

[0052] In the ELISA format, the binding activity of the antigen-binding domain to IL-6R-expressing cells is quantitatively evaluated by comparing the signal levels generated by the enzymatic reaction. Specifically, the test polypeptide aggregate is added to an ELISA plate immobilized with IL-6R-expressing cells, and the test antigen-binding domain bound to the cells is detected using an enzyme-labeled antibody that recognizes the test antigen-binding domain. Alternatively, in FACS, the binding activity of the test antigen-binding domain to IL-6R-expressing cells can be compared by creating a dilution series of the test antigen-binding domain and determining the antibody binding titer against IL-6R-expressing cells.

[0053] The binding of the target antigen-binding domain to an antigen expressed on the cell surface suspended in a buffer solution can be detected by a flow cytometer. Examples of known flow cytometers include the following: FACSCanto™ II FACSAria™ FACSArray™ FACSVantage™ SE FACSCalibur™ (both are product 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 product names of Beckman Coulter)

[0054] For example, the following method is a suitable method for measuring the antigen-binding activity of an antigen-binding domain to IL-6R. First, cells expressing IL-6R are stained with a FITC-labeled secondary antibody that recognizes the test antigen-binding domain. The test antigen-binding domain is then diluted with a suitable buffer to prepare it to the desired concentration. For example, it can be used at a concentration between 10 μg / ml and 10 ng / ml. Next, the fluorescence intensity and cell count are measured using FACSCalibur (BD). The amount of antigen-binding domain 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, expressed by the amount of binding, can be measured.

[0055] The sharing of an epitope between an antigen-binding domain for IL-6R and another antigen-binding domain can be confirmed by competition between the two domains for the same epitope. Competition between antigen-binding domains can be detected by cross-blocking assays, for example. A competitive ELISA assay is a preferred cross-blocking assay.

[0056] Specifically, in a cross-blocking assay, IL-6R protein coated on wells of a microtiter plate is pre-incubated in the presence or absence of candidate competitive antigen-binding domains, after which the test antigen-binding domain is added. The amount of test antigen-binding domain bound to the IL-6R protein in the well is indirectly correlated with the binding ability of candidate competitive antigen-binding domains that compete for binding to the same epitope. In other words, the greater the affinity of the competitive antigen-binding domain for the same epitope, the lower the binding activity of the test antigen-binding domain to the well coated with IL-6R protein.

[0057] The amount of antigen-binding domain bound to a well via the IL-6R protein can be easily measured by pre-labeling the antigen-binding domain. For example, biotin-labeled antigen-binding domains can be measured using an avidin peroxidase conjugate and an appropriate substrate. Cross-blocking assays utilizing enzymatic labeling such as peroxidase are specifically called competitive ELISA assays. Antigen-binding domains can be labeled with other detectable or measurable labeling substances. Specifically, radiolabeling and fluorescent labeling are well known.

[0058] If, compared to the binding activity obtained in a control test performed in the absence of a candidate competing antigen-binding domain aggregate, a competing 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%, then the test antigen-binding domain is either substantially binding to the same epitope as the competing antigen-binding domain, or is an antigen-binding domain that competes for binding to the same epitope.

[0059] If the structure of the epitope to which the antigen-binding domain for IL-6R binds has been identified, the sharing of the epitope between the test antigen-binding domain and the control antigen-binding domain can be evaluated by comparing the binding activity of the two antigen-binding domains to a peptide or polypeptide into which amino acid mutations have been introduced into the peptide constituting the epitope.

[0060] One method for measuring such binding activity is to compare the binding activity of the test antigen-binding domain and the control antigen-binding domain to a linear peptide into which a mutation has been introduced in the aforementioned ELISA format. Alternatively, the binding activity to the mutated peptide bound to a column can be measured by quantitatively determining the antigen-binding domain eluted into the eluate after the test antigen-binding domain and the control antigen-binding domain have been passed down the column. Methods for adsorbing the mutated peptide onto a column as a fusion peptide with, for example, GST, are well known.

[0061] Furthermore, if the identified epitope is a stereoepitope, the sharing of the epitope between the test antigen-binding domain and the control antigen-binding domain can be evaluated by the following method. First, cells expressing IL-6R and cells expressing IL-6R with a mutation introduced into the epitope are prepared. The test antigen-binding domain and the control antigen-binding domain are added to the cell suspension, in which these cells are suspended in a suitable buffer such as PBS. Next, a FITC-labeled antibody capable of recognizing the test antigen-binding domain and the control antigen-binding domain is added to the cell suspension, which has been washed with a buffer as appropriate. The fluorescence intensity and cell count of the cells stained with the labeled antibody are measured using FACSCalibur (BD). The concentrations of the test antigen-binding domain and the control antigen-binding domain are adjusted to the desired concentration by appropriately diluting them with a suitable buffer. For example, concentrations between 10 μg / ml and 10 ng / ml are used. 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). In other words, by obtaining the Geometric Mean value, the binding activity of the test antigen-binding domain and the control antigen-binding domain, which is represented by the amount of labeled antibody bound, can be measured.

[0062] Furthermore, to confirm competition between antigen-binding domains for the same epitope as other antigen-binding domains, methods other than ELISA and FACS, such as radiolabeled antigen binding assay (RIA), BIACORE® surface plasmon resonance assay, and electrochemiluminescence, can also be used.

[0063] In this method, for example, "substantially ineffective binding to mutant IL-6R-expressing cells" can be determined by the following method. First, the test antigen-binding domain and the control antigen-binding domain bound to cells expressing mutant IL-6R are stained with a labeled antibody. Next, the fluorescence intensity of the cells is detected. When FACSCalibur is used as flow cytometry for fluorescence detection, the obtained fluorescence intensity can be analyzed using CELL QUEST Software. By calculating this comparison value (ΔGeo-Mean) from the Geometric Mean values ​​in the presence and absence of polypeptide aggregates based on Equation 1 below, the percentage increase in fluorescence intensity due to antigen-binding domain binding can be determined.

[0064] (Formula 1) ΔGeo-Mean = Geo-Mean (in the presence of polypeptide aggregates) / Geo-Mean (in the absence of polypeptide aggregates)

[0065] The Geometric Mean comparison value (ΔGeo-Mean value of the mutant IL-6R molecule), which reflects the amount of binding of the test antigen-binding domain to mutant IL-6R-expressing cells obtained by the analysis, is compared with the ΔGeo-Mean comparison value, which reflects the amount of binding of the test antigen-binding domain to IL-6R-expressing cells. In this case, it is particularly preferable that the concentrations of the test antigen-binding domain used when determining the ΔGeo-Mean comparison value for mutant IL-6R-expressing cells and IL-6R-expressing cells are prepared at the same or substantially the same concentration. An antigen-binding domain that has been confirmed to recognize an epitope in IL-6R in advance is used as the control antigen-binding domain.

[0066] If the ΔGeo-Mean comparison value of the test antigen-binding domain to mutant IL-6R-expressing cells is less than at least 80%, preferably 50%, more preferably 30%, and particularly preferably 15% of the ΔGeo-Mean comparison value of the test antigen-binding domain to IL-6R-expressing cells, it is considered that the domain "substantially does not bind to mutant 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). If the comparison values ​​are substantially equivalent, the epitopes of the test antigen-binding domain and the control antigen-binding domain can be considered identical.

[0067] In this specification, the term "transport portion" refers to the portion of a polypeptide other than the antigen-binding domain. The transport portion of the present invention is typically a peptide or polypeptide composed of amino acids, and in one specific embodiment, the transport portion in the polypeptide is linked to the antigen-binding domain via a cleavage site. The transport portion of the present invention may be a series of peptides or polypeptides linked by amide bonds, or a complex in which multiple peptides or polypeptides are formed by covalent bonds such as disulfide bonds or non-covalent bonds such as hydrogen bonds or hydrophobic interactions.

[0068] The transport portion of the present invention has an inhibitory domain that suppresses the antigen-binding activity of the antigen-binding domain. In this specification, the term "inhibitory domain" is limited solely to domains that suppress the antigen-binding activity of the antigen-binding domain. The inhibitory domain can be any domain structure that suppresses the antigen-binding activity of the antigen-binding domain. Examples of such inhibitory domains, but not limited to them, include, for example, the heavy chain variable region (VH) of an antibody, the light chain variable region (VL) of an antibody, a pre-B cell receptor, and a single-domain antibody. The inhibitory domain may consist of the entire transport portion or a portion of the transport portion.

[0069] In some embodiments of the present invention, the antigen-binding activity of the antigen-binding domain increases upon its release from the polypeptide. In other words, when the antigen-binding domain is not released from the polypeptide, its antigen-binding activity is suppressed by the inhibitory domain. Methods to confirm that the antigen-binding activity of the antigen-binding domain is suppressed by the inhibitory domain include FACS (fluorescence activated cell sorting), ELISA (enzyme-linked immunosorbent assay), ECL (electrogenerated chemiluminescence), SPR (Surface Plasmon Resonance) (Biacore), and BLI (Bio-Layer Interferometry) (Octet). In some embodiments of the present invention, the antigen-binding activity when the antigen-binding domain is released from the polypeptide is 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 90, 1000, 2000, or 3000 times greater than the antigen-binding activity when the antigen-binding domain is not released from the polypeptide. In some more specific embodiments of the present invention, when the antigen-binding activity of the antigen-binding domain is measured by one of the methods 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 antigen-binding domain becomes detachable from the polypeptide when the cleavage site is cleaved. Therefore, in such embodiments, the comparison of antigen-binding activity can be performed 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, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or 3000 times greater than the antigen-binding activity measured using the uncleaved polypeptide. In some more specific embodiments, when the antigen-binding activity of the uncleaved polypeptide is measured using one of the methods selected from the above, 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, so the comparison of antigen-binding activity in such embodiments can be performed by comparing the antigen-binding activity of the polypeptide before and after protease treatment. That is, the antigen-binding activity measured using a protease-treated polypeptide is 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or 3000 times higher than the antigen-binding activity measured using an untreated polypeptide.

[0070] In the present invention, a polypeptide comprising an antigen-binding domain and a transporter has a longer blood half-life compared to an antigen-binding domain existing alone. To extend the half-life of the polypeptide, in some embodiments of the present invention, the transporter is designed to have a longer blood half-life. Examples of embodiments that extend the blood half-life of the transporter include, but are not limited to, a large molecular weight of the transporter, or the transporter having FcRn-binding properties, or the transporter having albumin-binding properties, or the transporter being PEGylated. Furthermore, in some embodiments of the present invention, the transporter has a longer blood half-life than the antigen-binding domain (in other words, the antigen-binding domain has a shorter blood half-life than the transporter).

[0071] In the present invention, when comparing the half-life of the antigen-binding domain alone with that of the polypeptide, or the half-life of the antigen-binding domain with that of the transporter, it is preferable to compare them using the half-life in humans. If it is difficult to measure the half-life in humans, the half-life in humans can be predicted based on the half-life in mice (e.g., normal mice, human antigen-expressing transgenic mice, human FcRn-expressing transgenic mice, etc.) or monkeys (e.g., cynomolgus monkeys, etc.).

[0072] One embodiment for extending the blood half-life of the transport portion is to increase the molecular weight of the transport portion. Another embodiment for making the blood half-life of the transport portion longer than that of the antigen-binding domain is to make the molecular weight of the transport portion larger than that of the antigen-binding domain.

[0073] One embodiment for extending the blood half-life of the transport portion is to give the transport portion FcRn binding ability. To give the transport portion FcRn binding ability, one can usually provide an FcRn binding region within the transport portion. An FcRn binding region is a region that has binding ability to FcRn, and any structure can be used as long as it has binding ability to FcRn. The transport region, including the FcRn binding domain, can be taken up into cells via the FcRn salvage pathway and then returned to the plasma. For example, the relatively long retention time (slow disappearance) of IgG molecules in plasma is due to the function of FcRn, which is known as a salvage receptor for IgG molecules. IgG molecules taken up into endosomes by pinocytosis bind to FcRn expressed in endosomes under acidic conditions. IgG molecules that do not bind to FcRn proceed to lysosomes and are degraded there, but IgG molecules that do bind to FcRn migrate to the cell surface and dissociate from FcRn under neutral conditions in plasma, returning to the plasma. The FcRn binding region is preferably a region that directly binds to FcRn. A preferred example of an FcRn binding region is the Fc region of an antibody. However, regions that can bind to polypeptides that have the ability to bind to FcRn, such as albumin and IgG, can indirectly bind to FcRn via albumin or IgG, so the FcRn binding region in the present invention may be a region that binds to such polypeptides that have the ability to bind to FcRn.

[0074] The binding activity of the FcRn binding domain in the present invention to FcRn, particularly human FcRn, can be measured by methods known to those skilled in the art, as described in the section on binding activity, and the conditions can be appropriately determined by those skilled in the art. The binding activity to human FcRn can be evaluated as KD (Dissociation constant), apparent KD (Apparent dissociation constant), dissociation rate (kd), or apparent dissociation rate (kd). These can be measured by methods known to those skilled in the art. For example, Biacore (GE Healthcare), scatchard plots, flow cytometers, etc., can be used.

[0075] The conditions for measuring the binding activity of the FcRn binding region to FcRn can be appropriately selected by those skilled in the art and are not particularly limited. For example, it can be measured under conditions of MES buffer and 37°C, as described in WO2009 / 125825. Furthermore, the measurement of the binding activity of the FcRn binding region of the present invention to FcRn can be performed by methods known to those skilled in the art, for example, by using Biacore (GE Healthcare). The binding activity between the FcRn binding region and FcRn can be evaluated by flowing FcRn, the FcRn binding region, or the transport portion containing the FcRn binding region as an analyte to the FcRn binding region, the transport portion containing the FcRn binding region, or a tip with immobilized FcRn, respectively.

[0076] The binding affinity between the FcRn binding region and FcRn may be evaluated at any pH between pH 4.0 and pH 6.5 as the pH used for measurement conditions. Preferably, a pH between pH 5.8 and pH 6.0, which is close to the pH in early endosomes in vivo, is used to determine the binding affinity between the FcRn binding region and human FcRn. The binding affinity between the FcRn binding region and FcRn may be evaluated at any temperature between 10°C and 50°C as the temperature used for measurement conditions. Preferably, a temperature between 15°C and 40°C is used to determine the binding affinity between the FcRn binding region and human FcRn. More preferably, any temperature between 20°C and 35°C, such as any one of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35°C, is also used to determine the binding affinity between the FcRn binding region and FcRn. The temperature of 25°C is a non-limiting example of an embodiment of the present invention.

[0077] One example of an FcRn binding region, though not limited to this, is the Fc region of an IgG antibody. When using the Fc region of an IgG antibody, the type is not limited, and it is possible to use Fc regions of IgG1, IgG2, IgG3, IgG4, etc. For example, it is possible to use an Fc region containing one sequence selected from the amino acid sequences shown in SEQ ID NOs: 21, 22, 23, and 24.

[0078] Furthermore, not only the Fc region of natural IgG antibodies, but also modified Fc regions with one or more amino acid substitutions can be used, as long as they retain FcRn binding ability. For example, EU numbering in the IgG antibody Fc region: 237th, 238th, 239th, 248th, 250th, 252nd, 254th, 255th, 256th, 257th, 258th, 265th, 270th, 286th, 289th, 297th, 298th, 303rd, 305th, 307th, 308th, 309th, 311th, 312th, 314th It is possible to use a modified Fc region containing an amino acid sequence in which at least one amino acid selected from positions 315, 317, 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 is substituted with another amino acid.

[0079] More specifically, EU numbering in the Fc region of IgG antibodies Amino acid substitution where Gly at position 237 is replaced with Met. An amino acid substitution where the 238th Pro is replaced with Ala. An amino acid substitution where Ser at position 239 is replaced with Lys. An amino acid substitution where Lys at position 248 is replaced with Ile. Amino acid substitutions that replace the 250th Thr with Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr. Amino acid substitutions that replace Met at position 252 with Phe, Trp, or Tyr. An amino acid substitution where Ser at position 254 is replaced with Thr. Amino acid substitution where Arg at position 255 is replaced with Glu, Amino acid substitutions that replace the 256th Thr with Asp, Glu, or Gln. Amino acid substitutions that replace the 257th Pro with Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val. An amino acid substitution in which Glu at position 258 is replaced with His. An amino acid substitution in which Asp at position 265 is replaced with Ala. An amino acid substitution where Asp at position 270 is replaced with Phe. Amino acid substitutions that replace the 286th Asn with Ala or Glu. An amino acid substitution where the 289th Thr is replaced with His, An amino acid substitution in which the 297th Asn is replaced with Ala. An amino acid substitution in which Ser at position 298 is replaced with Gly. An amino acid substitution in which the 303rd Val is replaced with Ala. An amino acid substitution in which the 305th Val is replaced with Ala. Amino acid substitutions that replace the 307th Thr with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr. Amino acid substitutions that replace the 308th Val with Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr. Amino acid substitutions that replace the 309th Leu or Val with Ala, Asp, Glu, Pro, or Arg. Amino acid substitutions that replace the 311th Gln with Ala, His, or Ile. Amino acid substitutions that replace Asp at position 312 with Ala or His. Amino acid substitutions that replace the 314th Leu with Lys or Arg. Amino acid substitutions that replace the 315th Asn with Ala or His, An amino acid substitution in which Lys at position 317 is replaced with Ala. Amino acid substitution where Asn at position 325 is replaced with Gly. Amino acid substitution where Ile at position 332 is replaced with Val. An amino acid substitution where Lys at position 334 is replaced with Leu. An amino acid substitution where the 360th Lys is replaced with His. An amino acid substitution in which Asp at position 376 is replaced with Ala. An amino acid substitution where Glu at position 380 is replaced with Ala. An amino acid substitution in which Glu at position 382 is replaced with Ala. Amino acid substitutions that replace the 384th Asn or Ser with Ala, Amino acid substitutions that replace the 385th Gly with Asp or His, Amino acid substitution where Gln at position 386 is replaced with Pro. An amino acid substitution where Pro at position 387 is replaced with Glu. Amino acid substitutions that replace the 389th Asn with Ala or Ser. An amino acid substitution in which Ser at position 424 is replaced with Ala. Amino acid substitutions that replace the 428th Met with Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr. An amino acid substitution where His at position 433 is replaced with Lys. Amino acid substitutions that replace the 434th Asn with Ala, Phe, His, Ser, Trp, or Tyr, and Amino acid substitutions that replace Tyr or Phe at position 436 with His It is possible to use a modified Fc region that includes at least one amino acid substitution selected from the above.

[0080] From another perspective, EU numbering in the IgG antibody Fc region. Met at amino acid position 237, Ala at amino acid position 238, Lys at the 239th amino acid, Ile at the 248th amino acid, Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr at the 250th amino acid, Phe, Trp, or Tyr at the 252nd amino acid, Thr at amino acid 254, Glu at the 255th amino acid, Asp, Glu, or Gln at the 256th amino acid, Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val at amino acid position 257 His at amino acid position 258, Ala at the 265th amino acid, Phe at amino acid 270, Ala or Glu at the 286th amino acid, His at the 289th amino acid, Ala at the 297th amino acid, Gly at the 298th amino acid, Ala at amino acid position 303, Ala at amino acid position 305, Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr at amino acid position 307, Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr at amino acid position 308, Ala, Asp, Glu, Pro, or Arg at amino acid position 309, Ala, His, or Ile at the 311th amino acid, Ala or His at the 312th amino acid, Lys or Arg at the 314th amino acid, Ala or His at the 315th amino acid, Ala at amino acid position 317, Gly at amino acid position 325, Val at amino acid position 332, Leu at amino acid position 334, His at amino acid position 360, Ala at amino acid position 376, Ala at amino acid position 380, Ala at amino acid position 382, Ala at amino acid position 384, Asp or His at the 385th amino acid, Pro at amino acid 386, Glu at amino acid position 387, Ala or Ser at the 389th amino acid, Ala at amino acid position 424, Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr at amino acid position 428, Lys at amino acid position 433, Ala, Phe, His, Ser, Trp, or Tyr at amino acid position 434, and His at amino acid position 436 It is possible to use an Fc region containing at least one amino acid selected from the following.

[0081] Providing the transport portion with FcRn binding ability does not mean that the antigen-binding domain does not have FcRn binding ability. As an embodiment to make the blood half-life of the transport portion longer than that of the antigen-binding domain, it is not only necessary for the antigen-binding domain to not have FcRn binding ability, but even if the antigen-binding domain has FcRn binding ability, it is sufficient for the transport portion to have weaker FcRn binding ability.

[0082] Another embodiment of extending the blood half-life of the transport portion involves binding the transport portion to albumin. Albumin is not excreted by the kidneys and has FcRn-binding properties, resulting in a long blood half-life of 17-19 days (J Clin Invest. 1953 Aug; 32(8): 746-768). Therefore, the protein bound to albumin becomes bulkier and can indirectly bind to FcRn, which has been reported to increase its blood half-life (Antibodies 2015, 4(3), 141-156).

[0083] Furthermore, one embodiment of extending the blood half-life of the transport portion is to PEGylate the transport portion. It is thought that PEGylates the protein, increasing its bulk and simultaneously suppressing its degradation by proteases in the blood, thereby extending the protein's blood half-life (J Pharm Sci. 2008 Oct;97(10):4167-83.).

[0084] In some embodiments of the present invention, the transport portion includes an antibody Fc region. In one specific embodiment, the transport portion includes the CH2 and CH3 domains of a human IgG antibody. In another specific embodiment, the transport portion includes a portion extending from Cys226 or Pro230 of the human IgG1 antibody heavy chain to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region may or may not be present.

[0085] In some embodiments of the present invention, the transport portion includes an antibody constant region. In a more preferred embodiment, the transport portion includes an IgG antibody constant region. In a more preferred embodiment, the transport portion includes a human IgG antibody constant region.

[0086] In some further embodiments of the present invention, the transport portion includes a region having a structure substantially similar to that of the antibody heavy chain constant region, and a region having a structure substantially similar to that of the antibody light chain, which is linked to the said region by covalent bonds such as disulfide bonds or non-covalent bonds such as hydrogen bonds or hydrophobic interactions.

[0087] In this specification, "polypeptide comprising an antigen-binding domain and a transport portion" is typically a series of polypeptides linked by amide bonds, or a protein comprising multiple series of polypeptides linked by amide bonds.

[0088] In some embodiments of the present invention, the antigen-binding domain is detachable from the polypeptide, and the release of the antigen-binding domain from the polypeptide increases its antigen-binding activity. In this specification, the term "detachment" refers to the separation of two parts of the polypeptide from each other. The release of the antigen-binding domain from the polypeptide may result from the elimination of the interaction between the antigen-binding domain and the transporter. Since the antigen-binding activity of the antigen-binding domain incorporated into the polypeptide is suppressed, confirmation that the antigen-binding domain has been detached from the polypeptide can be confirmed by measuring the antigen-binding activity of the object and comparing it with the antigen-binding activity of the antigen-binding domain in its incorporated state within the polypeptide.

[0089] In some embodiments, the polypeptide includes a cleavage site, and cleavage of the cleavage site releases an antigen-binding domain from the polypeptide. The cleavage site can be cleaved, for example, by an enzyme, reduced by a reducing agent, or photodegraded. The cleavage site can be located at any position in the polypeptide as long as it can release the antigen-binding domain and does not cause the antigen-binding activity of the released antigen-binding domain to be lost. In addition, other cleavage sites may be included in the polypeptide besides the cleavage site for releasing the antigen-binding domain. In one embodiment of the present invention, the cleavage site includes a protease cleavage sequence and can be cleaved by a protease.

[0090] In this specification, the term "cleaved" refers to a state in which the antigen-binding domain and transporter 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. In this specification, the term "uncleaved" refers to a state in which the antigen-binding domain and transporter 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.

[0091] The cleavage site can be detected by subjecting a solution containing the polypeptide with the cleavage site to SDS-PAGE (polyacrylamide gel electrophoresis) and measuring the molecular weight of the fragment, or by detecting the change in molecular weight before and after cleavage.

[0092] The cleavage sites are approximately 0.001 to 1500 × 10⁻¹⁰ by chemicals (i.e., proteases, reducing agents, and 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 × 10 4 M -1 S -1 It can be specifically modified (cleaved, reduced, or photodegraded) at a certain rate.

[0093] Specific cleavage by proteases involves 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 be defined as the enzyme's ability to contact the cleavage site and bring about cleavage.

[0094] In this specification, the term "protease" refers to an enzyme such as an endopeptidase or exopeptidase that hydrolyzes peptide bonds, usually an endopeptidase. The proteases used in the present invention are limited only by their ability to cleave protease cleavage sequences, and their type is not particularly limited. In some embodiments, target tissue-specific proteases are used. Target tissue-specific proteases are, for example, (1) Proteases that are expressed at higher levels in target tissue than in normal tissue, (2) Proteases that have higher activity in target tissue than in normal tissue, (3) Proteases expressed at higher levels in target cells than in normal cells, (4) Proteases that have higher activity in target cells than in normal cells, It can refer to any of the following: In a more specific embodiment, a cancer tissue-specific protease or an inflammatory tissue-specific protease is used.

[0095] In this specification, the term “target tissue” means tissue containing at least one target cell. In some embodiments of the present invention, the target tissue is cancerous tissue. In some embodiments of the present invention, the target tissue is inflammatory tissue.

[0096] The term "cancer tissue" means tissue containing at least one cancer cell. Therefore, it refers to all cell types that contribute to the formation of a tumor mass, including cancer cells and endothelial cells, such as cancer tissue containing cancer cells and blood vessels. In this specification, "tumor" means a foci of tumor tissue. The term "tumor" is generally used to mean either a benign or malignant neoplasm.

[0097] In this specification, "inflammatory tissue" includes, for example, the following: Joints in rheumatoid arthritis and osteoarthritis • Lungs (alveoli) in bronchial asthma and COPD • Digestive organs in inflammatory bowel disease, Crohn's disease, and ulcerative colitis • Fibrotic tissue in fibrosis of the liver, kidneys, and lungs • Tissues that are being rejected in organ transplants • Blood vessels and heart (myocardium) in arteriosclerosis and heart failure • Visceral fat in metabolic syndrome • Skin tissue in atopic dermatitis and other skin inflammations • Spinal nerves in herniated discs and chronic lower back pain

[0098] In some types of target tissues, proteases that are specifically expressed or specifically activated, or proteases that are thought to be associated with the disease state of the target tissue (target tissue-specific proteases), are known. For example, proteases that are specifically expressed in cancer tissue are disclosed in international publications WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846, among others. Furthermore, proteases thought to be associated with inflammation have been disclosed in J Inflamm (Lond). 2010; 7: 45., Nat Rev Immunol. 2006 Jul;6(7):541-50., Nat Rev Drug Discov. 2014 Dec;13(12):904-27., Respir Res. 2016 Mar 4;17:23., Dis Model Mech. 2014 Feb;7(2):193-203., and Biochim Biophys Acta. 2012 Jan;1824(1):133-45. In addition to proteases that are specifically expressed in target tissues, there are also proteases that are specifically activated in target tissues. For example, proteases may be expressed in an inactive form and then become active, and in many tissues, substances that inhibit the active protease exist, and the activity is controlled by the activation process and the presence of inhibitors (Nat Rev Cancer. 2003 Jul;3(7):489-501.). In target tissues, the active protease may escape inhibition and be specifically activated. Active proteases can be measured using methods that employ antibodies that recognize active proteases (PNAS 2013 Jan 2; 110(1): 93-98.) or by fluorescently labeling the peptide recognized by the protease, quenching it before cleavage, and then emitting light after cleavage (Nat Rev Drug Discov. 2010 Sep;9(9):690-701. doi: 10.1038 / nrd3053.). From one perspective, the term "target tissue-specific protease" is, (i) Proteases expressed at higher levels in target tissue than in normal tissue, (ii) Proteases that have higher activity in target tissue than in normal tissue, (iii) Proteases expressed at higher levels in target cells than in normal cells, (iv) Proteases that have higher activity in target cells than in normal cells, It can refer to any of the following:

[0099] While not meant to be interpreted restrictively, specific proteases include cysteine ​​proteases (including cathepsin family B, L, S, etc.), aspartyl proteases (cathepsin D, E, K, O, etc.), serine proteases (matryptase (including MT-SP1), cathepsin A and G), thrombin, plasmin, urokinase (uPA), tissue plasminogen activator (tPA), elastase, proteinase 3, thrombin, kallikrein, trip Metalloproteinases (including tase and chymase), metalloproteinases (including both membrane-bound (MMP14-17 and MMP24-25) and secreted (MMP1-13, MMP18-23 and MMP26-28) metalloproteinases (MMP1-28), protease A disintegrin and metalloproteinase (ADAM), metalloproteinases with A disintegrin or thrombospongin motifs (ADAMTS), meprin (meprin α(meprin alpha, meprin beta, CD10 (CALLA), prostate-specific antigen (PSA), regmine, TMPRSS3, TMPRSS4, neutrophil elastase (HNE), beta-secretase (BACE), fibroblast-activating protein alpha (FAP), granzyme B, guanidinobenzoate (GB), hepsin, neprilysin, NS3 / 4A, HCV-NS3 / 4, calpain, ADAMDEC1, renin, cathepsin C, cathepsin V / L2, cathepsin X / Z / P, Kurjipain, Otsubine 2, Kallikrein-related peptidases (KLKs (KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14)), Bone morphogenetic protein 1 (BMP-1), Activated protein C, Blood coagulation-related proteases (Factor VIIa, Factor IXa, Factor Xa, Factor XIa, Factor XIIa), HtrA1, lactoferrin, malapsin, PACE4, DESC1, dipeptidyl peptidase 4 (DPP-4), TMPRSS2, cathepsin F, cathepsin H, cathepsin L2, cathepsin O, cathepsin S, granzyme A, gepsin calpain 2, glutamate carboxypeptidase 2, AMSH-LikeExamples include proteases, AMSH, gamma secretase, A anti-plasmin cleavage enzyme (APCE), Decysin 1, N-Acetylated Alpha-Linked Acidic Dipeptidase-Like 1 (NAALADL1), and furin.

[0100] From another perspective, target tissue-specific proteases can refer to cancer tissue-specific proteases or inflammatory tissue-specific proteases. Examples of cancer tissue-specific proteases include those disclosed in international publications WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846, which are specifically expressed in cancer tissue.

[0101] The type of cancer tissue-specific protease that exhibits high specificity in the target cancer tissue yields a greater reduction in side effects. It is preferable that the concentration of the cancer tissue-specific protease in cancer tissue is at least five times higher than that in normal tissue, more preferably at least ten times higher, even more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. Furthermore, it is preferable that the activity of the cancer tissue-specific protease in cancer tissue is at least twice as high as that in normal tissue, more preferably at least three times higher, four times higher, five times higher, ten times higher, even more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. Furthermore, cancer tissue-specific proteases may be bound to the cell membrane of cancer cells, or they may not be bound to the cell membrane and may be secreted extracellularly. If cancer tissue-specific proteases are not bound to the cell membrane of cancer cells, it is preferable that the cancer tissue-specific protease is located inside or near the cancer tissue in order for the cytotoxicity by immune cells to be specific to cancer cells. In this specification, "near the cancer tissue" means the range in which the cancer tissue-specific protease cleavage sequence is cleaved and the antigen-binding domain exhibits antigen-binding activity. However, it is preferable that this range does not damage normal cells as much as possible. From another perspective, cancer tissue-specific proteases are, (i) Proteases expressed at higher levels in cancer tissue than in normal tissue, (ii) Proteases that have higher activity in cancer tissue than in normal tissue, (iii) Proteases expressed at higher levels in cancer cells than in normal cells, (iv) Proteases that have higher activity in cancer cells than in normal cells, It is one of the following: Cancer tissue-specific proteases may be used individually or in combination of two or more. The number of types of cancer tissue-specific proteases can be appropriately determined by a person skilled in the art, taking into consideration the type of cancer being treated.

[0102] From the above viewpoint, among the proteases exemplified above, serine proteases and metalloproteases are preferred as cancer tissue-specific proteases, matryptase (including MT-SP1), urokinase (uPA), and metalloproteases are more preferred, and MT-SP1, uPA, MMP2, and MMP9 are even more preferred.

[0103] The type of inflammation-specific protease that exhibits high specificity in the inflammatory tissue being treated is preferable to achieve a reduction in side effects. It is preferable that the concentration of the inflammation-specific protease in inflammatory tissue is at least five times higher than that in normal tissue, more preferably at least ten times higher, even more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. Furthermore, it is preferable that the activity of the inflammation-specific protease in inflammatory tissue is at least twice as high as that in normal tissue, more preferably at least three times, four times, five times, ten times, even more preferably at least 100 times higher, particularly preferably at least 500 times higher, and most preferably at least 1000 times higher. Furthermore, the inflammation tissue-specific protease may be bound to the cell membrane of inflammatory cells, or it may not be bound to the cell membrane and may be secreted extracellularly. If the inflammation tissue-specific protease is not bound to the cell membrane of inflammatory cells, it is preferable that the inflammation tissue-specific protease is located inside or near the inflammatory tissue in order for the cytotoxicity by immune cells to be specific to inflammatory cells. In this specification, "near the inflammatory tissue" means the range in which the inflammation tissue-specific protease cleavage sequence is cleaved and the antigen-binding domain exerts antigen-binding activity. However, it is preferable that this range does not damage normal cells as much as possible. From another perspective, inflammation-specific proteases are, (i) Proteases expressed at higher levels in inflammatory tissue than in normal tissue, (ii) Proteases that have higher activity in inflammatory tissue than in normal tissue, (iii) Proteases expressed at higher levels in inflammatory cells than in normal cells, (iv) Proteases that have higher activity in inflammatory cells than in normal cells, It is one of the following: Inflammatory tissue-specific proteases may be used individually or in combination of two or more types. The number of types of inflammatory tissue-specific proteases can be appropriately determined by those skilled in the art, taking into consideration the disease condition being treated.

[0104] From the above perspective, among the proteases exemplified above, metalloproteases are preferred as inflammatory tissue-specific proteases, and among metalloproteases, ADAMTS5, MMP2, MMP7, MMP9, and MMP13 are more preferred.

[0105] A protease cleavage sequence is a specific amino acid sequence that is specifically recognized by a target tissue-specific protease when a polypeptide is hydrolyzed by that protease in an aqueous solution. From the standpoint of reducing side effects, the protease cleavage sequence is preferably an amino acid sequence that is hydrolyzed with high specificity by a target tissue-specific protease that is more specifically expressed in or more specifically activated in the target tissue / cells being treated. Specific examples of protease cleavage sequences include, for example, target sequences that are specifically hydrolyzed by proteases specifically expressed in cancer tissue, inflammatory tissue-specific proteases, etc., as disclosed in International Publications WO2013 / 128194, WO2010 / 081173, WO2009 / 025846, etc. Artificially modified sequences, such as those obtained by introducing appropriate amino acid mutations into target sequences specifically hydrolyzed by known proteases, can also be used. Furthermore, protease cleavage sequences identified by methods known to those skilled in the art, as described in Nature Biotechnology 19, 661 - 667 (2001), may also be used. Furthermore, naturally occurring protease cleavage sequences may also be used. For example, just as TGFβ changes to its latent form upon protease cleavage, sequences in proteins that undergo protease cleavage and whose molecular shape changes upon such cleavage can also be used.

[0106] Examples of protease cleavage sequences, but not limited to these, include International Publications WO2015 / 116933, WO2015 / 048329, WO2016 / 118629, WO2016 / 179257, WO2016 / 179285, WO2016 / 179335, WO2016 / 179003, WO2016 / 046778, WO2016 / 014974, U.S. Patent Publication US2016 / 0289324, U.S. Patent Publication US2016 / 0311903, PNAS (2000) 97: 7754-7759, Biochemical Journal (2010) 426: The sequences shown in pp. 219-228 and Beilstein J Nanotechnol. (2016) 7: 364-373 can be used. As described above, the protease cleavage sequence is more preferably an amino acid sequence that is specifically hydrolyzed by a suitable target tissue-specific protease. Among amino acid sequences that are specifically hydrolyzed by a target tissue-specific protease, sequences containing the following amino acid sequences are preferred. LSGRSDNH (Sequence ID: 12, MT-SP1, can be cut by uPA) PLALAG (Sequence ID: 25, can be cut using MMP2 and MMP9) VPLSLTMG (Sequence ID: 26, can be disconnected by MMP7) The following sequences can also be used as protease cleavage sequences. TSTSGRSANPRG (Sequence ID: 74, MT-SP1, can be cut by uPA) ISSGLLSGRSDNH (Sequence number: 75, MT-SP1, can be cut by uPA) AVGLLAPPGGLSGRSDNH (Sequence number: 76, MT-SP1, can be cut by uPA) GAGVPMSMRGGAG (Sequence ID: 77, can be disconnected by MMP1) GAGIPVSLRSGAG ​​(Sequence ID: 78, can be disconnected by MMP2) GPLGIAGQ (Sequence ID: 79, can be disconnected via MMP2) GGPLGMLSQS (SEQ ID: 80, cleavable by MMP2) PLGLWA (SEQ ID: 81, cleavable by MMP2) GAGRPFSMIMGAG (SEQ ID: 82, cleavable by MMP3) GAGVPLSLTMGAG (SEQ ID: 83, cleavable by MMP7) GAGVPLSLYSGAG (SEQ ID: 84, cleavable by MMP9) AANLRN (SEQ ID: 85, cleavable by MMP11) AQAYVK (SEQ ID: 86, cleavable by MMP11) AANYMR (SEQ ID: 87, cleavable by MMP11) AAALTR (SEQ ID: 88, cleavable by MMP11) AQNLMR (SEQ ID: 89, cleavable by MMP11) AANYTK (SEQ ID: 90, cleavable by MMP11) GAGPQGLAGQRGIVAG (SEQ ID: 91, cleavable by MMP13) PRFKIIGG (SEQ ID: 92, cleavable by pro - urokinase) PRFRIIGG (SEQ ID: 93, cleavable by pro - urokinase) GAGSGRSAG (SEQ ID: 94, cleavable by uPA) SGRSA (SEQ ID: 95, cleavable by uPA) GSGRSA (SEQ ID: 96, cleavable by uPA) SGKSA (SEQ ID: 97, cleavable by uPA) SGRSS (SEQ ID: 98, cleavable by uPA) SGRRA (SEQ ID: 99, cleavable by uPA) SGRNA (SEQ ID: 100, cleavable by uPA) SGRKA (SEQ ID: 101, cleavable by uPA) QRGRSA (SEQ ID: 102, cleavable by tPA) GAGSLLKSRMVPNFNAG (Sequence ID: 103, cleavable by cathepsin B) TQGAAA (Sequence ID: 104, cleavable by cathepsin B) GAAAAA (Sequence ID: 105, cleavable by cathepsin B) GAGAAG (Sequence ID: 106, cleavable by cathepsin B) AAAAAG (Sequence ID: 107, cleavable by cathepsin B) LCGAAI (Sequence ID: 108, cleavable by cathepsin B) FAQALG (Sequence ID: 109, cleavable by cathepsin B) LLQANP (Sequence ID: 110, cleavable by cathepsin B) LAAANP (Sequence ID: 111, cleavable by cathepsin B) LYGAQF (Sequence ID: 112, cleavable by cathepsin B) LSQAQG (Sequence ID: 113, cleavable by cathepsin B) ASAASG (Sequence ID: 114, cleavable by cathepsin B) FLGASL (Sequence ID: 115, cleavable by cathepsin B) AYGATG (Sequence ID: 116, cleavable by cathepsin B) LAQATG (Sequence ID: 117, cleavable by cathepsin B) GAGSGVVIATVIVITAG (Sequence ID: 118, cleavable by cathepsin L) APMAEGGG (Sequence ID: 119, cleavable by meprin α and meprin β) EAQGDKII (Sequence ID: 120, cleavable by meprin α and meprin β) LAFSDAGP (Sequence ID: 121, cleavable by meprin α and meprin β) YVADAPK (Sequence ID: 122, cleavable by meprin α and meprin β) RRRRR (Sequence ID: 123, can be cleaved by Fuulin) RRRRRR (Sequence ID: 124, can be cleaved by Fuhrin) GQSSRHRRAL (Sequence ID: 125, cleavable by Fuhrin) SSRHRRALD (Sequence ID: 126) RKSSIIIRMRDVVL (Sequence ID: 127, cleavable by plasminogen) SSSFDKGKYKKGDDA (Sequence ID: 128, cleavable by staphylokinase) SSSFDKGKYKRGDDA (Sequence ID: 129, cleavable by staphylokinase) IEGR (Sequence ID: 130, can be cut by Factor Xa) IDGR (Sequence ID: 131, can be cut by Factor Xa) GGSIDGR (Sequence ID: 132, can be cut by Factor Xa) GPQGIAGQ (Sequence ID: 133, cleavable by collagenase) GPQGLLGA (Sequence ID: 134, can be cleaved by collagenase) GIAGQ (Sequence ID: 135, cleavable by collagenase) GPLGIAG (Sequence ID: 136, cleavable by collagenase) GPEGLRVG (Sequence ID: 137, can be cleaved by collagenase) YGAGLGVV (Sequence ID: 138, cleavable by collagenase) AGLGVVER (Sequence ID: 139, cleavable by collagenase) AGLGISST (Sequence ID: 140, cleavable by collagenase) EPQALAMS (Sequence ID: 141, cleavable by collagenase) QALAMSAI (Sequence ID: 142, can be cleaved by collagenase) AAYHLVSQ (Sequence ID: 143, can be cleaved by collagenase) MDAFLESS (Sequence ID: 144, can be cleaved by collagenase) ESLPVVAV (SEQ ID: 145, cleavable by Collagenase) SAPAVESE (SEQ ID: 146, cleavable by Collagenase) DVAQFVLT (SEQ ID: 147, cleavable by Collagenase) VAQFVLTE (SEQ ID: 148, cleavable by Collagenase) AQFVLTEG (SEQ ID: 149, cleavable by Collagenase) PVQPIGPQ (SEQ ID: 150, cleavable by Collagenase) LVPRGS (SEQ ID: 151, cleavable by Thrombin) TSTSGRSANPRG (SEQ ID: 178, cleavable by uPA and MT - SP1)

[0107] In one embodiment of the present invention, a flexible linker is further added to either one or both ends of the protease cleavage sequence. The flexible linker at one end of the protease cleavage sequence can be referred to as the first flexible linker, and the flexible linker at the other end can be referred to as the second flexible linker. In certain embodiments, the protease cleavage sequence and the flexible linker include 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 movable linker is preferably a peptide linker. The first movable linker and the second movable linker are independently and optionally present, and are identical or different movable linkers containing at least one flexible amino acid (such as Gly). For example, the protease cleavage sequence contains a sufficient number of residues to obtain the desired protease accessibility (amino acids optionally 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, especially Gly, etc.).

[0108] A movable linker suitable for use at both ends of a protease cleavage sequence typically improves protease access to the protease cleavage sequence, thereby increasing the efficiency of protease cleavage. Suitable movable linkers are readily selectable, and suitable options can be chosen from a variety of lengths, including 3 to 12 amino acids, as well as 1 to 20 amino acids (e.g., Gly), 2 to 15 amino acids, 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 movable linker is a peptide linker with 1 to 7 amino acids.

[0109] Examples of movable linkers include, but are not limited to, glycine polymers (G)n, glycine-serine polymers (e.g., including (GS)n, (GSGGS:Sequence ID:27)n and (GGGS:Sequence ID:28)n, where n is at least an integer of 1), glycine-alanine polymers, alanine-serine polymers, and other movable linkers known in the prior art. Among these, glycine and glycine-serine polymers are attracting attention because these amino acids are relatively unstructured and easily function as neutral tethers between components. Examples of movable linkers made of glycine-serine polymers include, but are not limited to, the following: Ser Gly·Ser(GS) Ser·Gly(SG) Gly·Gly·Ser(GGS) Gly·Ser·Gly (GSG) Ser·Gly·Gly (SGG) Gly·Ser·Ser (GSS) Ser·Ser·Gly (SSG) Ser·Gly·Ser(SGS) Gly·Gly·Gly·Ser(GGGS, Sequence ID: 28) Gly·Gly·Ser·Gly (GGSG, Sequence ID: 29) Gly·Ser·Gly·Gly (GSGG, Sequence ID: 46) Ser·Gly·Gly·Gly (SGGG, Sequence ID: 47) Gly·Ser·Ser·Gly(GSSG, Sequence ID: 48) Gly·Gly·Gly·Gly·Ser(GGGGS, Sequence ID: 49) Gly·Gly·Gly·Ser·Gly (GGGSG, Sequence ID: 33) Gly·Gly·Ser·Gly·Gly(GGSGG, Sequence ID: 30) Gly·Ser·Gly·Gly·Gly(GSGGG, Sequence ID: 32) Gly·Ser·Gly·Gly·Ser(GSGGS, Sequence ID: 27) Ser·Gly·Gly·Gly·Gly (SGGGG, Sequence ID: 51) Gly·Ser·Ser·Gly·Gly (GSSGG, Sequence ID: 52) Gly·Ser·Gly·Ser·Gly(GSGSG, Sequence ID: 31) Ser·Gly·Gly·Ser·Gly (SGGSG, Sequence ID: 53) Gly·Ser·Ser·Ser·Gly(GSSSG, Sequence ID: 34) Gly·Gly·Gly·Gly·Gly·Ser(GGGGGS, Sequence ID: 50) Ser·Gly·Gly·Gly·Gly·Gly (SGGGGG, Sequence ID: 54) Gly·Gly·Gly·Gly·Gly·Gly·Ser(GGGGGGS, Sequence ID: 55) Ser·Gly·Gly·Gly·Gly·Gly·Gly (SGGGGGG, Sequence ID: 56) (Gly·Gly·Gly·Gly·Ser(GGGGS, Sequence ID: 49))n (Ser·Gly·Gly·Gly·Gly(SGGGG, Sequence ID: 51))n These are some examples.

[0110] In this specification, "association" can be rephrased as referring to a state in which, for example, two or more polypeptide regions interact. Generally, hydrophobic bonds, hydrogen bonds, ionic bonds, etc., are formed between the target polypeptide regions to create an aggregate. One common example of association is in antibodies, such as natural antibodies, where the heavy chain variable region (VH) and the light chain variable region (VL) are known to maintain a paired structure through non-covalent bonds between them.

[0111] In some embodiments of the present invention, the inhibitory domain of the transport portion associates with the antigen-binding domain. The inhibitory domain may be part of the transport portion or all of the transport portion. From another perspective, the portion of the transport portion that associates with the antigen-binding domain can be referred to as the inhibitory domain. In a more specific embodiment, an antigen-binding domain, which is a single-domain antibody, and an inhibitory domain, which is VL, VH, or VHH, form an association such as antibody VH and antibody VL. In an even more specific embodiment, an antigen-binding domain, which is a single-domain antibody, and an inhibitory domain, which is VL, VH, or VHH, form an association such as antibody VH and antibody VL, and in the state in which such an association is formed, the antigen-binding activity of the single-domain antibody is suppressed by the VL, VH, or VHH by the inhibitory domain sterically inhibiting the binding of the antigen-binding domain to the antigen, or by altering the steric structure of the antigen-binding site of the antigen-binding domain. In this embodiment in which VHH is used as the single-domain antibody, if the CDR3, which is the main antigen-binding site of VHH, or a site near it, is located at the interface where it associates with the inhibitory domain, it is thought that the binding of VHH to the antigen is sterically inhibited by the inhibitory domain. Furthermore, the association between the repressive domain and the antigen-binding domain can be resolved, for example, by cleaving the cleavage site. Resolution of the association can be rephrased as, for example, the resolution of the interaction state of two or more polypeptide regions. This can be resolved by resolving all interactions between two or more polypeptide regions, or by resolving some of the interactions between two or more polypeptide regions.

[0112] In this specification, "interface" usually refers to the association surface during association (interaction), and the amino acid residues forming the interface usually refer to one or more amino acid residues contained in the polypeptide region that are subject to the association, more preferably amino acid residues that approach each other during the association and participate in the interaction. Specifically, this interaction includes non-covalent bonds such as hydrogen bonds, electrostatic interactions, and salt bridges formed between amino acid residues that approach each other during the association.

[0113] In this specification, "amino acid residues forming an interface" refers, in more detail, to amino acid residues contained within a polypeptide region that constitutes an interface. A polypeptide region constituting an interface refers, for example, to a polypeptide region in an antibody, ligand, receptor, substrate, etc., that is responsible for selective binding within or between molecules. Specifically, in an antibody, examples include the heavy chain variable region and the light chain variable region, and in some embodiments of the present invention, examples include the antigen-binding domain and the repression domain. Examples of amino acid residues that form an interface include, but are not limited to, amino acid residues that come into close proximity during association. These amino acid residues that come into close proximity during association can be identified, for example, by analyzing the three-dimensional structure of a polypeptide and examining the amino acid sequence of the polypeptide region that forms the interface during the association of the polypeptide.

[0114] In some embodiments of the present invention, amino acid residues involved in association in the antigen-binding domain or in the repressive domain can be modified to promote association between the antigen-binding domain and the repressive domain. In more specific embodiments, amino acid residues that form an interface with the repressive domain in the antigen-binding domain or in the repressive domain can be modified. In preferred embodiments, modification of the interface-forming amino acid residues is a method of introducing mutations in amino acid residues at the interface such that two or more amino acid residues forming the interface have heterogeneous charges. Modifications of amino acid residues that result in heterogeneous charges include modification from a positively charged amino acid residue to a negatively charged amino acid residue or an uncharged amino acid residue, modification from a negatively charged amino acid residue to a positively charged amino acid residue or an uncharged amino acid residue, and modification from an uncharged amino acid residue to a positively or negatively charged amino acid residue. Such amino acid modifications are for the purpose of promoting association, and the location and type of amino acid are not limited as long as the objective of promoting association is achieved. Modifications include, but are not limited to, substitutions.

[0115] In some embodiments of the present invention, the antigen-binding domain VHH is associated with the repressive domain VL. Examples of amino acid residues in VHH that are involved in association with VL include those that form the interface between VHH and VL. Examples of amino acid residues in VHH that are involved in association with VL include, but are not limited to, those at positions 37, 44, 45, and 47 (J. Mol. Biol. (2005) 350, 112-125). The association between VHH and VL is promoted, thereby suppressing the activity of VHH. At the same time, examples of amino acid residues in VL that are involved in association with VHH include those that form the interface between VHH and VL.

[0116] To facilitate the association of VHH and VL, the amino acid residues in VHH that are involved in the association with VL can be modified. Examples of such amino acid substitutions, but not limited to, include F37V, Y37V, E44G, Q44G, R45L, H45L, G47W, F47W, L47W, T47W, or / and S47W. Furthermore, it is also possible to use VHH that has the amino acid residues 37V, 44G, 45L, or / and 47W from the beginning without modifying any residues in VHH. Furthermore, to the extent that the objective of promoting the association of VHH and VL is achieved, it is possible to modify the amino acid residues in VL that are involved in the association with VHH, rather than the amino acids in VHH, and it is also possible to introduce amino acid modifications to both VHH and VL.

[0117] In some further embodiments of the present invention, VHH can be used as the antigen-binding domain and VH or VHH can be used as the repression domain, and the antigen-binding domain and the repression domain can be associated. To promote the association between the antigen-binding domain VHH and the repression domain VH or VHH, amino acid residues in the antigen-binding domain VHH that are involved in the association with the repression domain VH or VHH can be identified and these amino acid residues can be modified. Alternatively, amino acid residues in the repression domain VH or VHH that are involved in the association with the antigen-binding domain VHH can be identified and these amino acid residues can be modified.

[0118] Furthermore, when using a single-domain antibody other than VHH as the antigen-binding domain, the amino acid residues involved in association within the antigen-binding domain or repressive domain can be identified and modified in the same way.

[0119] In some embodiments of the present invention, the transport portion and the antigen-binding domain are fused via a linker. In a more specific embodiment, the transport portion and the antigen-binding domain are fused via a linker containing a cleavage site. In another specific embodiment, the transport portion and the antigen-binding domain are fused via a linker, and the resulting fusion protein contains a cleavage site.

[0120] In another embodiment of the present invention, the transport portion and the antigen-binding domain are fused without a linker. In a more specific embodiment, an amino bond is formed between the N-terminal amino acid of the transport portion and the C-terminal amino acid of the antigen-binding domain to form a fusion protein. The formed fusion protein contains a cleavage site. In a particular embodiment, one to several amino acids at the N-terminus of the transport portion and / or one to several amino acids at the C-terminus of the antigen-binding domain are modified, and the N-terminus of the transport portion and the C-terminus of the antigen-binding domain are fused to form a cleavage site near the fusion site. More specifically, for example, the four amino acids at the C-terminus of the antigen-binding domain can be made into an LSGR sequence, and the four amino acids at the N-terminus of the transport portion can be made into an SDNH sequence to form a cleavage site.

[0121] In some embodiments of the present invention, the cleavage site of the polypeptide comprising the transport portion and the antigen-binding domain comprises a protease cleavage sequence. The protease cleavage sequence may be located at any part of the polypeptide, as long as it releases the antigen-binding domain upon cleavage by the protease and does not cause the antigen-binding activity of the released antigen-binding domain to be lost.

[0122] In some embodiments of the present invention, the transport portion includes an antibody constant region, the N-terminus of the antibody constant region and the C-terminus of the antigen-binding domain are fused via or without a linker. In a particular embodiment, the protease cleavage sequence is located within the antibody constant region contained in the transport portion. In this case, the protease cleavage sequence should be located within the antibody constant region so that the antigen-binding domain can be released when cleaved by the protease. In a specific embodiment, the protease cleavage sequence is located within the antibody heavy chain constant region contained in the transport portion, 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 contained in the transport portion, 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.

[0123] In some embodiments of the present invention, the antigen-binding domain is a single-domain antibody, and the C-terminus of the single-domain antibody and the N-terminus of the transport portion are fused via or without a linker. In a particular embodiment, the protease cleavage sequence is located within the monodomain antibody. In a more specific embodiment, the monodomain antibody is a monodomain antibody or VHH made from VH, and the protease cleavage sequence is located on the transporter side of the monodomain antibody from amino acid 35b (Kabat numbering), preferably on the transporter side of amino acid 95 (Kabat numbering), and more preferably on the transporter side of amino acid 109 (Kabat numbering). In another specific embodiment, the monodomain antibody is a monodomain antibody made from VL, and the protease cleavage sequence is located on the transporter side of the monodomain antibody from amino acid 32 (Kabat numbering), preferably on the transporter side of amino acid 91 (Kabat numbering), and more preferably on the transporter side of amino acid 104 (Kabat numbering).

[0124] In some embodiments of the present invention, the transport portion includes an antibody constant region, the antigen-binding domain is a single-domain antibody, and the antibody constant region and the single-domain antibody are fused via or without a linker. In a more specific embodiment, the N-terminus of the antibody constant region and the C-terminus of the single-domain antibody are fused via or without a linker. In another specific embodiment, the C-terminus of the antibody constant region and the N-terminus of the single-domain antibody are fused via or without a linker. In a particular embodiment, the protease cleavage sequence is located within the antibody constant region contained in the transport region. In a more specific embodiment, the protease cleavage sequence is located on the single-domain antibody side of amino acid 140 (EU numbering) in the antibody heavy chain constant region, preferably on the single-domain antibody side of amino acid 122 (EU numbering) in the antibody heavy chain constant region. In another specific embodiment, the protease cleavage sequence is located on the antigen-binding domain side of amino acid 130 (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. In a particular embodiment, the protease cleavage sequence is located within a single domain. In a more specific embodiment, the single-domain antibody is a single-domain antibody or VHH made from VH, and the protease cleavage sequence is located closer to the antibody constant region than amino acid 35b (Kabat numbering) of the single-domain antibody, preferably closer to the antibody constant region than amino acid 95 (Kabat numbering), and more preferably closer to the antibody constant region than amino acid 109 (Kabat numbering). In another specific embodiment, the single-domain antibody is a single-domain antibody made from VL, and the protease cleavage sequence is located closer to the antibody constant region than amino acid 32 (Kabat numbering) of the single-domain antibody, preferably closer to the antibody constant region than amino acid 91 (Kabat numbering), and more preferably closer to the antibody constant region than amino acid 104 (Kabat numbering). In a particular embodiment, the protease cleavage sequence is located near the boundary between the antigen-binding domain and the transporter. The area near the boundary between the antigen-binding domain and the transporter refers to the region before and after the site where the antigen-binding domain and the transporter are linked, which does not significantly affect the secondary structure of the antigen-binding domain. In a more specific embodiment, the antigen-binding domain is linked to the antibody constant region contained within the transport portion, and the protease cleavage sequence is located near the boundary between the antigen-binding domain and the antibody constant region. The vicinity of the boundary between the antigen-binding domain and the antibody constant region can refer to the vicinity of the boundary between the antigen-binding domain and the antibody heavy chain constant region, or the vicinity of the boundary between the antigen-binding domain and the antibody light chain constant region. When the antigen-binding domain is a single-domain antibody or VHH created from VH and is linked to the antibody heavy chain constant region, the vicinity of the boundary between the antigen-binding domain and the antibody constant region can refer to the area 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 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 created from VH and is linked to the constant region of the antibody light chain, the vicinity of the boundary between the antigen-binding domain and the constant region of the antibody light chain can refer to the area between amino acid 101 (Kabat numbering) of the single-domain antibody and amino acid 130 (EU numbering) (Kabat numbering 130) of the constant region of the antibody light chain, preferably between amino acid 109 (Kabat numbering) of the single-domain antibody and amino acid 113 (EU numbering) (Kabat numbering 113) of the constant region of the antibody light chain. When the antigen-binding domain is a single-domain antibody created from VL, the vicinity of the boundary between the antigen-binding domain and the constant region is from amino acid 96 (Kabat numbering) of the single-domain antibody, preferably from amino acid 104 (Kabat numbering) of the single-domain antibody.

[0125] In some embodiments of the present invention, the polypeptide is an IgG antibody-like molecule. Examples of such embodiments, but not limited to them, include, an embodiment in which the transport portion includes the constant region of an IgG antibody, and a single-domain antibody, which is the antigen-binding domain, replaces the VH of the IgG antibody, while the antigen-binding activity is suppressed by the VL; or an embodiment in which the transport portion includes the constant region of an IgG antibody, and a single-domain antibody, which is the antigen-binding domain, replaces the VL of the IgG antibody, while the antigen-binding activity is suppressed by the VH; or an embodiment in which the transport portion includes the constant region of an IgG antibody, and a single-domain antibody, which is the antigen-binding domain, replaces one of the VH / VL of the IgG antibody, while another single-domain antibody that suppresses the antigen-binding activity of the antigen-binding domain replaces the other of the VH / VL of the IgG antibody.

[0126] As used herein, the term "IgG antibody-like molecule" is used to define a molecule having a substantially similar structure to an IgG antibody, specifically a portion substantially similar to the structure of a constant domain or constant region, and a portion substantially similar to the structure of an IgG antibody, specifically a variable domain or variable region, and having a substantially similar three-dimensional structure to an IgG antibody. However, as used herein, an "IgG antibody-like molecule" is not limited to exhibiting antigen-binding activity while maintaining a structure similar to an IgG antibody.

[0127] The polypeptide may contain one or more antigen-binding domains. The inhibitory domains that suppress the antigen-binding activity of each of the antigen-binding domains may also consist of one or more inhibitory domains. The antigen-binding domains may each associate with the inhibitory domains. The antigen-binding domains may each be fused with the transporter. The antigen-binding domains may each be able to be released from the polypeptide. The cleavage sites for releasing the antigen-binding domains may be multiple, corresponding to each antigen-binding domain.

[0128] When the polypeptide is an IgG antibody-like molecule, the embodiment in which antigen-binding domains are provided in the regions corresponding to the two variable regions of an IgG antibody, as shown in Figure 7, would be understandable to anyone skilled in the art who is familiar with the present invention. Whether the antigen-binding domains incorporated in both arms have similar or different antigen-binding specificities, this embodiment would be easily understood by anyone skilled in the art who is familiar with the present invention, and it is clear that it does not deviate from the scope of the present invention.

[0129] In some embodiments of the present invention, the antigen-binding domain is further linked to a second antigen-binding domain. Examples of a second antigen-binding domain, though not limited to these, include: single-domain antibodies, antibody fragments, a module called the A domain of about 35 amino acids contained in Avimer, a cell membrane protein present in vivo (International Publication WO2004 / 044011, WO2005 / 040229), Adnectin containing the 10Fn3 domain, a protein-binding domain in fibronectin, a glycoprotein expressed on the cell membrane (International Publication WO2002 / 032925), Affibody (International Publication WO1995 / 001937), which uses an IgG-binding domain as a scaffold to form a bundle of three helices consisting of 58 amino acids of Protein A, and DARPins (Designed Ankyrin Repeat), which are regions exposed on the molecular surface of ankyrin repeats (ARs) that have a structure in which a turn containing 33 amino acid residues and two antiparallel helical and loop subunits are repeatedly stacked. Examples include proteins (International Publication WO2002 / 020565), Anticalin (International Publication WO2003 / 029462), which consists of four loop regions supporting one side of a barrel structure twisted towards the center by eight highly conserved antiparallel strands in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL), and recessed regions of parallel sheet structures within a horseshoe-shaped structure in which leucine-rich repeat (LRR) modules are repeatedly stacked, which are part of the acquired immune system of jawless fish such as lampreys and hagfish (International Publication WO2008 / 016854). In preferred embodiments, the second antigen-binding domain has a different antigen-binding specificity from 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 having different antigen-binding specificities, and the linked antigen-binding domain and the second antigen-binding domain are detachable from the polypeptide, forming a bispecific antigen-binding molecule after detachment. Examples of such bispecific antigen-binding molecules, but not limited to these, include, for example, a bispecific antigen-binding molecule in which the antigen-binding domain specifically binds to a target cell surface antigen and the second antigen-binding domain specifically binds to an immune cell surface antigen; a bispecific antigen-binding molecule in which the antigen-binding domain and the second antigen-binding domain bind to different subunits of the same antigen; and a bispecific antigen-binding molecule in which the antigen-binding domain and the second antigen-binding domain bind to different epitopes in the same antigen. Such bispecific antigen-binding molecules are considered useful in treating diseases caused by target cells because they can recruit immune cells to the vicinity of target cells. The antigen-binding activity of the second antigen-binding domain may or may not be suppressed by the transporter. Furthermore, the second antigen-binding domain may or may not form association with a part of the transporter's structure. 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 suppressed, and even if the second antigen-binding domain does not form association with a part of the transporter's structure, the antigen-binding activity of the antigen-binding domain cannot be exerted when the antigen-binding domain is not released, and the bispecific antigen-binding molecule in which the antigen-binding domain and the second antigen-binding domain are linked cannot exert the function of bispecifically binding to two types of antigens. Figure 8 illustrates one embodiment in which the antigen-binding domain is further linked to a second antigen-binding domain.

[0130] In this specification, the term "specificity" refers to the property that one molecule of a specifically binding molecule does not substantially bind to any molecule other than the one or more target molecules it binds to. It is also used when an antigen-binding domain has specificity to an epitope contained in a particular antigen. It is also used when an antigen-binding domain has specificity to a particular epitope among several epitopes contained in a certain antigen. Here, "substantially not binding" is determined according to the method described in the section on binding activity, and means that the binding activity of the specific binding molecule to molecules other than the target molecules is 80% or less, usually 50% or less, preferably 30% or less, and particularly preferably 15% or less of the binding activity to the target molecules.

[0131] The present invention also relates to a pharmaceutical composition (drug) comprising the polypeptide of the present invention and a pharmaceutically acceptable carrier.

[0132] As used herein, “treatment” (and its grammatical derivatives, e.g., “to treat,” “to treat,” etc.) means a clinical intervention intended to modify the natural course of the individual being treated, and may be carried out for preventive purposes or during the course of a clinical condition. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, reduction of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction of the rate of disease progression, recovery or mitigation of the disease state, and remission or improved prognosis. In some embodiments, the polypeptides of the present invention are used to delay the onset of disease or to slow the progression of disease.

[0133] In this invention, a pharmaceutical composition generally refers to an agent for the treatment or prevention of a disease, or for examination and diagnosis. Furthermore, in this invention, the term "pharmaceutical composition containing a polypeptide" can be rephrased as "a method for treating a disease, comprising administering a polypeptide to a target for treatment," or as "the use of a polypeptide in the manufacture of a pharmaceutical for treating a disease." Additionally, the term "pharmaceutical composition containing a polypeptide" can be rephrased as "the use of a polypeptide for treating a disease."

[0134] The pharmaceutical compositions of the present invention can be formulated using methods known to those skilled in the art. For example, they can be administered parenterally in the form of sterile solutions with water or other pharmaceutically acceptable liquids, or as injectable suspensions. For example, they can be formulated by mixing them with pharmacokinetically acceptable carriers or media, specifically sterile water or saline solution, vegetable oil, emulsifiers, suspensions, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, etc., in a unit dose form generally required for pharmaceutical practice. The amount of active ingredient in these formulations is set to obtain an appropriate volume within the indicated range.

[0135] Sterile compositions for injection can be formulated in accordance with standard formulation procedures using a vehicle such as distilled water for injection. Examples of aqueous solutions for injection include physiological saline, glucose, and isotonic solutions containing other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). Appropriate solubilizers, such as alcohols (ethanol, etc.), polyalcohols (propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (polysorbate 80™, HCO-50, etc.), may be used in combination.

[0136] Examples of oily solutions include sesame oil and soybean oil, and benzyl benzoate and / or benzyl alcohol may also be used as solubilizers. Furthermore, buffers (e.g., phosphate buffer and sodium acetate buffer), analgesics (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants may be added. The prepared injection solution is usually filled into appropriate ampoules.

[0137] The pharmaceutical composition of the present invention is preferably administered by parenteral administration. For example, compositions in the form of injection, nasal administration, pulmonary administration, or transdermal administration may be administered. For example, it may be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc.

[0138] The method of administration may be appropriately selected depending on the patient's age and symptoms. The dosage of the pharmaceutical composition containing polypeptide may be set, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight per dose. Alternatively, for example, a dosage of 0.001 to 100,000 mg per patient may be set, but the present invention is not necessarily limited to these values. The dosage and method of administration will vary depending on the patient's weight, age, symptoms, etc., but a person skilled in the art can set an appropriate dosage and method of administration considering these conditions.

[0139] The present invention also relates to a method for producing a polypeptide comprising a transport portion having an inhibitory domain and an antigen-binding domain. One method for producing the polypeptide of the present invention involves obtaining an antigen-binding domain having antigen-binding activity, linking the antigen-binding domain and a transporter portion to form a polypeptide precursor such that the antigen-binding activity of the antigen-binding domain is suppressed by an inhibitory domain, and then inserting a cleavage site into the polypeptide precursor or modifying a part of the polypeptide precursor into a cleavage site. It is sufficient to introduce a cleavage site into the polypeptide precursor, and the method of introducing the cleavage site can be either insertion of a cleavage site or modification of a part of the polypeptide precursor. Furthermore, it will be obvious to those skilled in the art who have read this specification that both means can be combined to introduce a modified site into the polypeptide precursor, and this will not deviate from the scope of the present invention. Furthermore, as another method for producing the polypeptide of the present invention, an antigen-binding domain having antigen-binding activity is obtained, and the antigen-binding domain and the transport portion are linked via a cleavage site to form a polypeptide such that the antigen-binding activity of the antigen-binding domain is suppressed by the inhibitory domain. When linking the antigen-binding domain and the transport portion via a cleavage site, the cleavage site may be sandwiched between the antigen-binding domain and the transport portion, or a part of the antigen-binding domain and / or a part of the transport portion may be modified and used as part of the cleavage site.

[0140] The following polypeptide manufacturing method describes 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.

[0141] In one embodiment of the present invention, a method for producing a polypeptide comprising a transport portion having an inhibitory domain and an antigen-binding domain is as follows: (a) A step to obtain a single-domain antibody that binds to a target antigen; (b) A step of forming a polypeptide precursor by linking the single-domain antibody obtained in step (a) with the transport domain such that the antigen-binding activity of the single-domain antibody is suppressed by the inhibitory domain of the transport domain; (c) A step of introducing a protease cleavage sequence into the polypeptide precursor; This is a manufacturing method that includes [something].

[0142] In one embodiment of the present invention, a method for producing a polypeptide comprising a transport portion having an inhibitory domain and an antigen-binding domain is as follows: (a) A step to obtain a single-domain antibody that binds to a target antigen; (b) A step of forming a polypeptide precursor by linking the single-domain antibody obtained in step (a) with the transport domain such that the antigen-binding activity of the single-domain antibody is suppressed by the inhibitory domain of the transport domain; (c) A step of introducing a protease cleavage sequence near the boundary between the monodomain antibody and the transport portion; This is a manufacturing method that includes [something].

[0143] In one embodiment of the present invention, a method for producing a polypeptide comprising a transport portion having an inhibitory domain and an antigen-binding domain is as follows: (a) A step to obtain a single-domain antibody that binds to a target antigen; (b) A step of forming a polypeptide by linking the single-domain antibody obtained in step (a) with the transport portion via a protease cleavage sequence such that the antigen-binding activity of the single-domain antibody is suppressed by the inhibitory domain of the transport portion; This is a manufacturing method that includes [something].

[0144] In a particular embodiment, a method for producing a polypeptide comprising a transport portion having an inhibitory domain and an antigen-binding domain further includes the following steps: (d) A step of confirming that the binding activity of the monodomain antibody incorporated in the polypeptide or polypeptide precursor to the target antigen is weakened or lost; This is a manufacturing method that includes [the specified component]. In this invention, "weakened binding activity" means that the binding activity to the target antigen is reduced compared to before binding, and the degree of reduction is not specified.

[0145] In a particular embodiment, a method for producing a polypeptide comprising a transport portion having an inhibitory domain and an antigen-binding domain further includes the following steps: (e) A step of releasing the monodomain antibody by cleaving the protease cleavage sequence with a protease, and confirming that the released monodomain antibody binds to the antigen; This is a manufacturing method that includes [something].

[0146] In one embodiment of the present invention, a method for producing a polypeptide that is an IgG antibody-like molecule comprising a transport portion having an inhibitory domain and an antigen-binding domain is as follows: (a) A step to obtain a single-domain antibody that binds to a target antigen; (b) A step of forming an IgG antibody-like molecular precursor into which the single-domain antibody has been introduced, by associating the single-domain antibody obtained in step (a) with the VL of the IgG antibody instead of the VH, or by associating the single-domain antibody with the VH instead of the VL of the IgG antibody, so as to suppress the antigen-binding activity of the single-domain antibody obtained in step (a); (c) A step of introducing a protease cleavage sequence into the IgG antibody-like molecular precursor into which the monodomain antibody has been introduced; This is a manufacturing method that includes [something].

[0147] In one embodiment of the present invention, a method for producing a polypeptide that is an IgG antibody-like molecule comprising a transport portion having an inhibitory domain and an antigen-binding domain is as follows: (a) A step to obtain a single-domain antibody that binds to a target antigen; (b) A step of forming an IgG antibody-like molecular precursor into which the single-domain antibody has been introduced, by associating the single-domain antibody obtained in step (a) with the VL of the IgG antibody instead of the VH, or by associating the single-domain antibody with the VH instead of the VL of the IgG antibody, so as to suppress the antigen-binding activity of the single-domain antibody obtained in step (a); (c) A step of introducing a protease cleavage sequence near the boundary between the monodomain antibody and the antibody constant region in the IgG antibody-like molecular precursor; This is a manufacturing method that includes [something].

[0148] In one embodiment of the present invention, a method for producing a polypeptide that is an IgG antibody-like molecule comprising a transport portion having an inhibitory domain and an antigen-binding domain is as follows: (a) A step to obtain a single-domain antibody that binds to a target antigen; (b) A step of forming an IgG antibody-like molecule into which the monodomain antibody obtained in step (a) is introduced, by linking the monodomain antibody 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 as to suppress the antigen-binding activity of the monodomain antibody; This is a manufacturing method that includes [something].

[0149] In a particular embodiment, a method for producing a polypeptide that is an IgG antibody-like molecule comprising a transport portion having an inhibitory domain and an antigen-binding domain further involves the following steps: (d) A step of confirming that the binding activity of the monodomain antibody introduced into the IgG antibody-like molecule or the IgG antibody-like molecule precursor to the target antigen is weakened or lost; This is a manufacturing method that includes [the specified component]. In the present invention, "weakened binding activity" means that the binding activity to the target antigen is reduced compared to before association or linkage, regardless of the degree of reduction.

[0150] In a particular embodiment, a method for producing a polypeptide that is an IgG antibody-like molecule comprising a transport portion having an inhibitory domain and an antigen-binding domain further involves the following steps: (e) A step of releasing the monodomain antibody by cleaving the protease cleavage sequence with a protease, and confirming that the released monodomain antibody binds to the target antigen; This is a manufacturing method that includes [something].

[0151] When using VH / VL / VHH as an inhibitory domain, one method to suppress the antigen-binding activity of a single-domain antibody with the inhibitory domain in the transport portion 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 in question and comparing the antigen-binding activity of the single-domain antibody before and after association. Another method to suppress the antigen-binding activity of a single-domain antibody at specific VH / VL / VHH domains is to substituted amino acid residues in the single-domain antibody that are involved in association with VH / VL / VHH to promote association, or to use a single-domain antibody whose amino acid residues are already capable of promoting association. This allows for the preparation of a single-domain antibody / suppressive domain pair where the difference in antigen-binding activity before and after association is at a desired level.

[0152] In one embodiment of the present invention, a method for producing an IgG antibody-like molecular polypeptide comprising a transport portion having an inhibitory domain and an antigen-binding domain is as follows: (a) A step of producing a modified single-domain antibody in which an amino acid residue involved in association with antibody VH in a single-domain antibody is substituted, or an amino acid residue involved in association with antibody VL in a single-domain antibody is substituted, thereby maintaining the binding activity of the single-domain antibody to the target antigen; (b) A step of forming an IgG antibody-like molecular precursor into which the modified single-domain antibody has been introduced, by associating the modified single-domain antibody with antibody VL or with antibody VH in such a way as to suppress the antigen-binding activity of the modified single-domain antibody prepared in step (a); (c) A step of introducing a protease cleavage sequence into an IgG antibody-like molecular precursor into which the modified single-domain antibody has been introduced; This is a manufacturing method that includes [something].

[0153] In one embodiment of the present invention, a method for producing a polypeptide that is an IgG antibody-like molecule comprising a transport portion having an inhibitory domain and an antigen-binding domain is as follows: (a) A step of producing a modified single-domain antibody in which an amino acid residue involved in association with antibody VH in a single-domain antibody is substituted, or an amino acid residue involved in association with antibody VL in a single-domain antibody is substituted, thereby maintaining the binding activity of the single-domain antibody to the target antigen; (b) A step of forming an IgG antibody-like molecular precursor into which the modified single-domain antibody has been introduced, by associating the modified single-domain antibody with antibody VL or with antibody VH in such a way as to suppress the antigen-binding activity of the modified single-domain antibody prepared in step (a); (c) A step of introducing a protease cleavage sequence near the boundary between the modified single-domain antibody and the constant region of the IgG antibody-like molecular precursor; This is a manufacturing method that includes [something].

[0154] In one embodiment of the present invention, a method for producing a polypeptide that is an IgG antibody-like molecule comprising a transport portion having an inhibitory domain and an antigen-binding domain is as follows: (a) A step of producing a modified single-domain antibody in which an amino acid residue involved in association with antibody VH in a single-domain antibody is substituted, or an amino acid residue involved in association with antibody VL in a single-domain antibody is substituted, thereby maintaining the binding activity of the single-domain antibody to the target antigen; (b) A step of forming an IgG antibody-like molecule into which the modified single-domain antibody has been introduced, by 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 by linking the modified single-domain antibody to the light chain constant region of an IgG antibody via a protease cleavage sequence, in order to suppress the antigen-binding activity of the modified single-domain antibody; This is a manufacturing method that includes [something].

[0155] In a particular embodiment, a method for producing a polypeptide that is an IgG antibody-like molecule comprising a transport portion having an inhibitory domain and an antigen-binding domain further involves the following steps: (d) A step of 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; This is a manufacturing method that includes [the specified component]. In the present invention, "weakened binding activity" means that the binding activity to the target antigen is reduced compared to before association or linkage, regardless of the degree of reduction.

[0156] In a particular embodiment, a method for producing a polypeptide that is an IgG antibody-like molecule comprising a transport portion having an inhibitory domain and an antigen-binding domain further involves the following steps: (e) A step of releasing the modified single-domain antibody by cleaving the protease cleavage sequence with a protease, and confirming that the released modified single-domain antibody binds to the target antigen; This is a manufacturing method that includes [something].

[0157] The present invention also relates to polynucleotides that encode polypeptides comprising a transport portion having an inhibitory domain and an antigen-binding domain.

[0158] The polynucleotides in this invention are typically loaded (inserted) into a suitable vector and introduced into host cells. The vector is not particularly limited as long as it stably retains the inserted nucleic acid. For example, if E. coli is used as the host, the pBluescript vector (Stratagene) is preferred as a cloning vector, but various commercially available vectors can be used. When using a vector for the purpose of producing the polypeptide of this invention, an expression vector is particularly useful. The expression vector is not particularly limited as long as it is a vector that expresses the polypeptide in vitro, in E. coli, in cultured cells, or in living organisms. For example, the pBEST vector (Promega) is preferred for in vitro expression, the pET vector (Invitrogen) is preferred for E. coli, the pME18S-FL3 vector (GenBank Accession No. AB009864) is preferred for cultured cells, and the pME18S vector (Mol Cell Biol. 8:466-472 (1988)) is preferred for living organisms. The DNA of the present invention can be inserted into a vector by conventional methods, for example, by a ligase reaction using restriction enzyme sites (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 11.4-11.11).

[0159] There are no particular restrictions on the host cells used, and various host cells can be used depending on the purpose. Examples of cells used to express polypeptides include bacterial cells (e.g., Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, Bacillus subtilis), fungal cells (e.g., yeast, Aspergillus), insect cells (e.g., Drosophila S2, Spodoptera SF9), animal cells (e.g., CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells), and plant cells. Vector introduction into host cells can be performed by known methods such as calcium phosphate precipitation, electroporation (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), lipofectamine method (GIBCO-BRL), and microinjection.

[0160] To cause polypeptides expressed in host cells to be secreted into the lumen of the endoplasmic reticulum, the pericellular lumen, or the extracellular environment, appropriate secretory signals can be incorporated into the target polypeptide. These signals may be endogenous or heterologous to the target polypeptide.

[0161] In the above manufacturing method, if the polypeptide of the present invention is secreted into the culture medium, the culture medium is recovered. If the polypeptide of the present invention is produced inside cells, the cells are first lysed, and then the polypeptide is recovered.

[0162] To recover and purify the polypeptide of the present invention from recombinant cell cultures, known methods can be used, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyl apatite chromatography, and lectin chromatography.

[0163] Furthermore, monodomain antibodies are used as antigen-binding domains in several embodiments of the present invention, in which the antigen-binding activity of the monodomain antibody is suppressed by association with a specific VL, or by association with a specific VH, or by association with a specific VHH. The present invention also relates to a method for screening such monodomain antibodies.

[0164] To suppress the antigen-binding activity of single-domain antibodies, VL / VH / VHH sequences with known sequences, such as those registered in the IMGT or Kabat database, can be used. Additionally, sequences newly identified as VL / VH / VHH from human antibody libraries can also be used. By combining these sequences to prepare proteins and measuring their binding activity using the aforementioned method, VL / VH / VHH sequences that suppress the binding activity of single-domain antibodies can be selected.

[0165] In some embodiments of the present invention, VL / VH / VHH sequences having human antibody germline sequences can be used to suppress the antigen-binding activity of single-domain antibodies. For example, when using VL as the suppression domain, VL sequences having a kappa chain framework sequence or VL sequences having a lambda chain framework sequence can be used. Furthermore, VL sequences having modified framework sequences, such as framework sequences combining kappa chain framework sequences and lambda chain framework sequences, can also be used.

[0166] One embodiment of the present invention, the following steps: (a) A step to obtain a monodomain antibody having target antigen-binding activity; (b) A step of associating the single-domain antibody obtained in step (a) with a specific VL; (c) A step to confirm that the binding activity of the monodomain antibody associated with a specific VL in step (b) to the antigen has been weakened or lost; The present invention provides a method for screening single-domain antibodies whose antigen-binding activity is suppressed by association with a specific VL, including [specific VL]. In this invention, "weakened binding activity" means that the binding activity to the target antigen is reduced compared to before association, regardless of the degree of reduction.

[0167] One embodiment of the present invention, the following steps: (a) A step to obtain a monodomain antibody having target antigen-binding activity; (b) A step of associating the single-domain antibody obtained in step (a) with a specific VH; (c) A step to confirm that the binding activity of the monodomain antibody associated with a specific VH in step (b) to the antigen has been weakened or lost; The present invention provides a method for screening single-domain antibodies whose antigen-binding activity is suppressed by association with a specific VH, including [specific VH]. In this invention, "weakened binding activity" means that the binding activity to the target antigen is reduced compared to before association, regardless of the degree of reduction.

[0168] One embodiment of the present invention, the following steps: (a) A step to obtain a monodomain antibody having target antigen-binding activity; (b) A step of associating the single-domain antibody obtained in step (a) with a specific VHH; (c) A step to confirm that the binding activity of the monodomain antibody associated with a specific VHH in step (b) to the antigen has been weakened or lost; The present invention provides a method for screening single-domain antibodies whose antigen-binding activity is suppressed by association with a specific VHH, including [specific VHH]. In this invention, "weakened binding activity" means that the binding activity to the target antigen is reduced compared to before association, regardless of the degree of reduction.

[0169] An example of a method for associating a single-domain antibody with a specific VL / VH / VHH sequence is to design a molecule that uses the sequence of a single-domain antibody instead of one of the VH or VL sequences in an antibody or antibody fragment containing both VH and VL, such as a complete antibody, Fab, Fab', or (Fab)2, and then express a polypeptide having that sequence.

[0170] Furthermore, the present invention relates not only to screening for monodomain antibodies whose antigen-binding activity is suppressed by association with a specific VL, a specific VH, or a specific VHH, but also to a method for producing monodomain antibodies whose antigen-binding activity is suppressed by promoting association between the monodomain antibody and a specific VL / VH / VHH, thereby promoting association with a specific VL, a specific VH, or a specific VHH.

[0171] One embodiment of the present invention, the following steps: (a) A step of producing a modified single-domain antibody in which an amino acid residue involved in association with the antibody VL is substituted in the single-domain antibody, thereby maintaining the binding activity of the single-domain antibody against the target antigen; The present invention provides a method for producing a single-domain antibody whose antigen-binding activity is suppressed by association with a specific VL, including [specific VL].

[0172] In a particular embodiment, the following steps are further taken: (b) A step of associating the modified single-domain antibody prepared in step (a) with a specific VL; (c) A step to confirm that the antigen-binding activity of the modified single-domain antibody associated with the VL is weakened or lost; The present invention provides a method for producing a single-domain antibody whose antigen-binding activity is suppressed by association with a specific VL, including [specific VL]. In this invention, "weakened binding activity" means that the binding activity to the target antigen is reduced compared to before association, regardless of the degree of reduction.

[0173] One embodiment of the present invention, the following steps: (a) A step of producing a modified single-domain antibody in which an amino acid residue involved in association with antibody VH is substituted in the single-domain antibody, thereby maintaining the binding activity of the single-domain antibody against the target antigen; This invention provides a method for producing a single-domain antibody whose antigen-binding activity is suppressed by association with a specific VH, including [specific VH].

[0174] In a particular embodiment, the following steps are further taken: (b) A step of associating the modified single-domain antibody prepared in step (a) with a specific VH; (c) A step to confirm that the antigen-binding activity of the modified single-domain antibody associated with the VH has been weakened or lost; The present invention provides a method for producing a single-domain antibody whose antigen-binding activity is suppressed by association with a specific VH, including [specific VH]. In this invention, "weakened binding activity" means that the binding activity to the target antigen is reduced compared to before association, regardless of the degree of reduction.

[0175] One embodiment of the present invention, the following steps: (a) A step of producing a modified single-domain antibody in which an amino acid residue involved in association with VHH is substituted in the single-domain antibody, thereby maintaining the binding activity of the single-domain antibody to the target antigen; This invention provides a method for producing a single-domain antibody whose antigen-binding activity is suppressed by association with a specific VHH, including [specific VHH].

[0176] In a particular embodiment, the following steps are further taken: (b) A step of associating the modified single-domain antibody prepared in step (a) with a specific VHH; (c) A step to confirm 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 by association with a specific VHH, including [specific VHH]. In this invention, "weakened binding activity" means that the binding activity to the target antigen is reduced compared to before association, regardless of the degree of reduction.

[0177] The process of associating a single-domain antibody with a specific VL / VH / VHH involves designing an antibody or antibody fragment containing both VH and VL, such as a complete antibody, Fab, Fab', (Fab)2, etc., which uses the sequence of a single-domain antibody instead of one of the VH or VL sequences, and then expressing a polypeptide having that sequence.

[0178] According to one embodiment of the present invention, a monodomain antibody 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 monodomain antibody is linked to a first association-supporting domain.

[0179] As an embodiment of the "library" in this specification, it is possible to provide a library that can efficiently obtain single-domain antibodies whose antigen-binding activity is suppressed or lost upon association with a specific VL / VH / VHH.

[0180] In this specification, "library" means a set of fusion polypeptides having different sequences, or a set of nucleic acids or polynucleotides encoding these fusion polypeptides. The fusion polypeptides contained in the library are not a single sequence, but rather fusion polypeptides with sequences that differ from each other.

[0181] In this specification, the term "differently sequenced" 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 portions in the individual fusion polypeptides in the library are different. That is, 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 12Therefore, by applying known techniques such as the ribosome display method, the library size can be reduced to 10 14 It is possible to expand up to [a certain size]. However, the actual number of phage particles used during panning selection of a phage library is typically 10 to 10,000 times larger than the library size. This excess multiplier, also called the "library equivalent number," indicates that there may be 10 to 10,000 individual clones having the same amino acid sequence. Therefore, the term "different sequences" in this invention means that the sequences of individual polypeptides in the library, excluding the library equivalent number, are different from each other, or more specifically, that there may be 10 different sequences of polypeptides. 6 from 10 14 Molecules, preferably 10 7 from 10 12 It means that molecules exist.

[0182] Furthermore, the term "multiple" in the description of the present invention as a library mainly consisting of multiple fusion polypeptides means that, for example, the polypeptides, polynucleotide molecules, vectors, or viruses of the present invention usually refer to a collection of two or more types of the substance. For example, if two or more substances differ from each other with respect to a particular trait, it means that there are two or more types of that substance. An example is a mutant amino acid observed at a particular amino acid position in the amino acid sequence. For example, if there are two or more polypeptides of the present invention that are substantially the same, preferably identical in sequence, except for a particular mutant amino acid at a very diverse range of amino acid positions exposed on the surface, then there are multiple polypeptides of the present invention. In another example, if there are two or more polynucleotide molecules of the present invention that are substantially the same, preferably identical in sequence, except for the bases encoding a particular mutant amino acid at a very diverse range of amino acid positions exposed on the surface, then there are multiple polynucleotide molecules of the present invention.

[0183] As a screening method for fusion polypeptides using binding activity as an indicator, a panning method using phage vectors is also suitably employed. A fusion polypeptide can be formed by linking a gene encoding a single-domain antibody with a gene encoding the IgG antibody CH1 domain or the light chain constant region in an appropriate embodiment. By inserting the gene encoding the fusion polypeptide into a phage vector, a phage expressing the fusion polypeptide on its surface can be obtained. After contact between this phage and the desired antigen, the phage bound to the antigen can be recovered, thereby recovering the DNA encoding the fusion polypeptide having the desired binding activity. By repeating this operation as needed, fusion polypeptides with the desired binding activity can be enriched.

[0184] In addition to phage display, other techniques for obtaining fusion polypeptides by panning using libraries include techniques using cell-free translation systems, techniques for presenting fusion polypeptides on the surface of cells or viruses, and techniques using emulsions. For example, techniques using cell-free translation systems include ribosome display, which forms a complex of mRNA and translated protein via ribosomes by removing stop codons, cDNA display, mRNA display, which covalently bonds gene sequences and translated proteins using compounds such as puromycin, and CIS display, which forms a complex of genes and translated proteins using nucleic acid-binding proteins. Furthermore, techniques for presenting fusion polypeptides on the surface of cells or viruses, in addition to phage display, may include E. coli display, Gram-positive bacterium display, yeast display, mammalian cell display, and virus display. Techniques using emulsions may include in vitro virus display, which encapsulates genes and translation-related molecules in an emulsion. These methods are already publicly known (Nat Biotechnol. 2000 Dec;18(12):1287-92, Nucleic Acids Res. 2006;34(19):e127, Proc Natl Acad Sci US A. 2004 Mar 2;101(9):2806-10, Proc Natl Acad Sci US A. 2004 Jun 22;101(25):9193-8, Protein Eng Des Sel. 2008 Apr;21(4):247-55, Proc Natl Acad Sci US A. 2000 Sep 26;97(20):10701-5, MAbs. 2010 Sep-Oct;2(5):508-18, Methods Mol Biol. 2012;911:183-98).

[0185] As a method for obtaining a target single-domain antibody from a library containing multiple fusion polypeptides, each consisting of a single-domain antibody linked to a first association support domain, an association partner, which consists of a suppressor domain linked to a second association support domain, can be used. In this specification, "first association-supporting domain" and "second association-supporting domain" refer to domains that can interact with each other through hydrophobic bonds, hydrogen bonds, ionic bonds, etc., and form aggregates. Preferred examples of the first and second association-supporting domains, but not limited to these, include, for example, the CH1 domains of the light chain constant region (CL) and heavy chain constant region of an antibody.

[0186] The first and second association-supporting domains interact with each other, and the fusion polypeptide and its association partner can form an association regardless of the degree of association between the single-domain antibody and the suppressor domain.

[0187] In another embodiment of the present invention, a library is provided comprising a plurality of fusion polypeptides in which a single-domain antibody and the constant region of an IgG antibody light chain are linked, wherein the single-domain antibody contains a single-domain antibody whose antigen-binding activity is suppressed or lost upon association with a specific VL / VH / VHH, and a method is provided for screening single-domain antibodies from the library whose antigen-binding activity is suppressed or lost upon association with a specific VL / VH / VHH.

[0188] In one specific embodiment, as shown in Figures 9A(1),(2),(3), 9B, and 9C, (1) A fusion polypeptide, consisting of a single-domain antibody linked to the first association support domain, is displayed on the surface of a phage or other organism using a display method such as phage display; (2) Prepare an association partner by linking the inhibitory domain and the second association support domain, and associate the fusion polypeptide with the association partner. In this state where the fusion polypeptide and the association partner are associated, select a fusion polypeptide that does not bind to the target antigen or whose antigen-binding activity is below a certain value; (3) The association between the monodomain antibody in the fusion polypeptide selected in (2) and the repressive domain in the association partner is resolved, and a fusion polypeptide is selected that binds to the target antigen or has an antigen-binding activity above a certain value while the monodomain antibody is not associated with the repressive domain. Here, as a method to resolve the association between the single-domain antibody and the repressive domain, methods such as cleaving near the boundary between the repressive domain and the second association-supporting domain of the association partner shown in Figure 9B, or cleaving near the boundary between the single-domain antibody and the first association-supporting domain of the fusion polypeptide shown in Figure 9C, can be used.

[0189] A further embodiment of the present invention provides a method for comparing the difference in binding activity of a single-domain antibody when the single-domain antibody and the repressor domain are expressed in a dissociated / unassociated state, as shown in Figures 9A to 9C, with the difference in binding activity of a single-domain antibody when the single-domain antibody and the repressor domain are expressed together / when the single-domain antibody is expressed without the repressor domain being expressed together, as shown in Figure 9D. As shown in Figure 9D(1), by simultaneously expressing a single-domain antibody and an inhibitory domain to form an association and selecting a fusion polypeptide containing a single-domain antibody that does not bind to the antigen or has antigen-binding activity below a certain value in that state, and as shown in Figure 9D(2) / (2') / (2''), by expressing a single-domain antibody without simultaneously expressing the inhibitory domain and selecting a fusion polypeptide containing a single-domain antibody that binds to the antigen or has antigen-binding activity above a certain value in that state, it is possible to screen for single-domain antibodies whose antigen-binding activity is suppressed or lost by associating with a specific inhibitory domain, such as VH / VL / VHH, from a library containing multiple fusion polypeptides in which a single-domain antibody is linked to a first association-supporting domain. By expressing a single-domain antibody without simultaneously expressing the inhibitory domain, selecting polypeptides containing single-domain antibodies that bind to the antigen or have antigen-binding activity above a certain value in that state, and then simultaneously expressing the single-domain antibody and the inhibitory domain to form an association, and then selecting polypeptides containing single-domain antibodies that do not bind to the antigen or have antigen-binding activity below a certain value in that state, it is possible to screen for single-domain antibodies whose antigen-binding activity is suppressed or lost by associating with a specific inhibitory domain, such as VH / VL / VHH, from a library containing multiple fusion polypeptides in which single-domain antibodies are linked to a first association-supporting domain.Furthermore, by expressing a single-domain antibody without simultaneously expressing the repressive domain (expressing only the single-domain antibody, or expressing only a fusion polypeptide containing the single-domain antibody and the first association support domain, or associating a fusion polypeptide containing the single-domain antibody and the first association domain with only the second association support domain), as shown in Figure 9D(2) / (2') / (2''), and selecting a fusion polypeptide containing the single-domain antibody that binds to the antigen or has an antigen-binding activity above a certain value, and then selecting a fusion polypeptide from the selected fusion polypeptide that simultaneously expresses the single-domain antibody and the repressive domain to form an association, as shown in Figure 9D(1), and then selecting a fusion polypeptide containing the single-domain antibody that does not bind to the antigen or has an antigen-binding activity below a certain value, it is possible to screen for single-domain antibodies whose antigen-binding activity is suppressed or lost by associating with a specific repressive domain, such as VH / VL / VHH, from a library containing multiple fusion polypeptides linking single-domain antibodies and the first association support domain. "Antigen binding activity below a certain value" can refer to antigen binding activity that falls below a certain standard when measured by the method illustrated in this specification, for example. "Antigen binding activity above a certain value" can similarly refer to antigen binding activity that exceeds a certain standard when measured by the method illustrated in this specification, for example. A fusion polypeptide with antigen binding activity above a certain value binds to the antigen more strongly than a fusion polypeptide with antigen binding activity below a certain value.

[0190] The fusion polypeptide selected in (3) above contains a single-domain antibody that has no or weak antigen-binding activity when associated with the repressive domain, and has or has strong antigen-binding activity when not associated with the repressive domain. By analyzing the sequence of the fusion polypeptide selected in this way, the sequence of the single-domain antibody contained therein can also be elucidated, and the said single-domain antibody can be manufactured.

[0191] A key aspect of screening fusion polypeptides containing a target mono-domain antibody using fusion polypeptides and association partners is comparing the suppressive domain of the mono-domain antibody with its antigen-binding activity in both the associated and unassociated states. As shown in Figures 9(A), (2'), and (3'), a method can also be used to obtain fusion polypeptides containing a target mono-domain antibody by first confirming the antigen-binding activity of the displayed fusion polypeptides, selecting those that bind to the antigen or have antigen-binding activity above a certain value, and then associating these fusion polypeptides with association partners. Finally, fusion polypeptides that do not bind to the antigen in the associated state or have antigen-binding activity below a certain value can be selected.

[0192] The following describes several embodiments in which the IgG antibody CH1 domain is used as the first association support domain and the IgG antibody CL is used as the second association support domain. A library containing multiple fusion polypeptides, each linked to a single-domain antibody and the CH1 domain of an IgG antibody, can be used to screen for fusion polypeptides containing the desired single-domain antibody.

[0193] In some embodiments of the present invention, a library is provided comprising a plurality of fusion polypeptides in which a single-domain antibody and an IgG antibody CH1 domain are linked, wherein the single-domain antibody contains a single-domain antibody whose antigen-binding activity is suppressed or lost upon association with a specific VL / VH / VHH, and a method is provided for screening fusion polypeptides from the library containing a single-domain antibody whose antigen-binding activity is suppressed or lost upon association with a specific VL / VH / VHH.

[0194] In a particular embodiment, a method is provided for screening a library containing multiple fusion polypeptides in which a single-domain antibody is linked to the CH1 domain of an IgG antibody, for fusion polypeptides containing a single-domain antibody whose antigen-binding activity is suppressed or lost upon association with a specific VL. Specifically, the following steps are taken: (a) A step of in vitro displaying the fused polypeptide of the library according to the present invention; (b) A step of preparing an association partner by fusing a specific VL with the constant region of an IgG antibody light chain; (c) A step of 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 the antigen when the single-domain antibody and the VL are associated, or whose antigen-binding activity is below a certain value; (d) A step of selecting a fusion polypeptide in which the single-domain antibody contained in the fusion polypeptide selected in step (c) binds to the antigen without the VL being associated, or in which the antigen-binding activity is above a certain value; A screening method for single-domain antibodies, including those containing such antibodies, is provided.

[0195] The association partner prepared in step (b) further includes a protease cleavage sequence, and in step (d), the association between the monodomain antibody and the VL is dissolved by protease treatment, making it possible to confirm the antigen-binding activity of the monodomain antibody in a state where the monodomain antibody and VL are not associated. The position of the protease cleavage sequence in the association partner is not limited as long as the association between the monodomain antibody and VL is dissolved during cleavage. As an example of the position of the protease cleavage sequence, it can be located near the boundary between the VL of the association partner and the constant region of the IgG antibody light chain, preferably between amino acid 96 (Kabat numbering) of the VL and amino acid 130 (EU numbering) (Kabat numbering 130) of the antibody light chain constant region, and more preferably between amino acid 104 (Kabat numbering) of the VL and amino acid 113 (EU numbering) (Kabat numbering 113) of the antibody light chain constant region. Alternatively, instead of using an association partner containing a protease cleavage sequence, it is possible to introduce a protease cleavage sequence into the fusion polypeptide in the library, thereby dissociating the monodomain antibody from the VL by cleavage of the fusion polypeptide by the protease. The position of the protease cleavage sequence in the fusion polypeptide is not limited, as long as the association between the monodomain antibody and the VL is dissociated upon cleavage, and the antigen-binding activity of the monodomain antibody is maintained after cleavage. For example, the protease cleavage sequence can be located near the boundary between the monodomain antibody and the IgG antibody CH1 domain in the fusion polypeptide.

[0196] Furthermore, in step (d), it is also possible to display the full-length or single-domain antibody portion of the fusion polypeptide selected in step (c) again, and confirm the antigen-binding activity of the single-domain antibody without association between the single-domain antibody and VL.

[0197] In a particular embodiment, a method is provided for screening a library containing multiple fusion polypeptides, each consisting of a single-domain antibody linked to the constant region of an IgG antibody light chain, for fusion polypeptides containing a single-domain antibody whose antigen-binding activity is suppressed or lost upon association with a specific VH. Specifically, the following steps are taken: (a) A step of in vitro displaying the fused polypeptide of the library according to the present invention; (b) A step of preparing an association partner in which a specific VH and the IgG antibody CH1 domain are fused; (c) A step of 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 the antigen when the monodomain antibody and the VH are associated, or whose antigen-binding activity is below a certain value; (d) A step of selecting a fusion polypeptide in which the single-domain antibody contained in the fusion polypeptide selected in step (c) binds to the antigen without associating with the VH, or in which the antigen-binding activity is above a certain value; A method for screening fusion polypeptides containing a single-domain antibody is provided.

[0198] The association partner prepared in step (b) further includes a protease cleavage sequence, and in step (d), the association between the monodomain antibody and the VH is dissolved by protease treatment, making it possible to confirm the antigen-binding activity of the monodomain antibody in a state where the monodomain antibody and VH are not associated. The position of the protease cleavage sequence in the association partner is not limited as long as the association between the monodomain antibody and VH is dissolved during cleavage. As an example of the position of the protease cleavage sequence, it can be located near the boundary between the VH of the association partner and the CH1 domain of the IgG antibody, preferably between amino acid 101 (Kabat numbering) of VH and amino acid 140 (EU numbering) of the antibody heavy chain constant region, and more preferably between amino acid 109 (Kabat numbering) of VH and amino acid 122 (EU numbering) of the antibody heavy chain constant region. Alternatively, instead of using an association partner containing a protease cleavage sequence, it is possible to introduce a protease cleavage sequence into the fusion polypeptide in the library, thereby dissociating the monodomain antibody from VH when the fusion polypeptide is cleaved by the protease. The location of the protease cleavage sequence in the fusion polypeptide is not limited, as long as the association between the monodomain antibody and VH is dissociated upon cleavage, and the antigen-binding activity of the monodomain antibody is maintained after cleavage. For example, the protease cleavage sequence can be located near the boundary between the monodomain antibody and the constant region of the IgG antibody light chain in the fusion polypeptide.

[0199] Furthermore, in step (d), it is also possible to display the full-length or monodomain antibody portion of the fusion polypeptide selected in step (c) again, and confirm the antigen-binding activity of the monodomain antibody without association between the monodomain antibody and VH.

[0200] The amino acids included in the amino acid sequence described in this invention may undergo post-translational modifications (for example, modification to pyroglutamic acid by pyroglutamylation of the N-terminal glutamine is a modification well known to those skilled in the art), but even when amino acids are modified post-translation in this way, they are naturally still included in the amino acid sequence described in this invention.

[0201] It will be understood by those skilled in the art that any combination of one or more embodiments described herein is included in the present invention, insofar as it does not contradict the common technical knowledge of those skilled in the art. [Examples]

[0202] The following are examples of the methods and compositions of the present invention. In light of the general description above, it will be understood that various other embodiments may be implemented.

[0203] Example 1: Challenges of existing protease-activating antibodies A method has been reported for producing antibodies that only exhibit antigen-binding activity after being cleaved by proteases expressed at lesion sites such as cancerous or inflammatory tissues. These antibodies, called Probodies, are antibody molecules in which the antigen-binding activity of the antibody is inhibited by linking a peptide that masks the antigen-binding site of the antibody to the antibody with a linker that is cleaved by a protease expressed at the lesion site, as shown in Figure 1 (Non-Patent Literature 18). When the linker constituting the Probodycetes is cleaved by a protease expressed at the target disease site, the mask peptide dissociates, and an antibody molecule with restored antigen-binding activity is produced, which can then bind to the antigen in the target diseased tissue. Probodies are thought to expand the therapeutic window by selectively binding to antigens at target disease sites through the mechanism described above. However, since antibody cleavage by proteases in probodies is irreversible, antibodies cleaved at the disease site can return to the bloodstream, potentially distributing to normal tissues via the bloodstream and binding to antigens expressed in those tissues. Probodies activated by proteases, like unactivated probodies, possess an Fc region and therefore have long-lasting blood retention. Consequently, antibodies activated by proteases expressed at disease sites may remain in the bloodstream for extended periods. Furthermore, even if a protease is elevated in the disease site, such proteases are also expressed at low levels in normal tissues, and free proteases produced at the disease site may leak into the bloodstream (The Chinese-German Journal of Clinical Oncology Jun. 2004, Vol. 3, No. 2 P78-P80). Therefore, Probodies can be activated by such free proteases. Consequently, Probodies may be activated even outside the disease site, and such activated Probodies will similarly remain in the bloodstream for a long time. Thus, Probodies are continuously activated in the disease site, normal tissues, and bloodstream, and if activated Probodies have long-term bloodstream retention, they may accumulate in the blood. Activated Probodies accumulated in the bloodstream may bind to antigens expressed in normal tissues, potentially causing side effects (Figure 2). Although the antigen-binding activity of probody is inhibited by the mask peptide linked to the antibody by a linker, the antigen-binding activity is not completely inhibited. Probody exists in an equilibrium state between the state in which the linker-linked mask peptide is bound to the antigen-binding site and the state in which it is dissociated, and the molecule in the dissociated state can bind to the antigen (Figure 3). In fact, the anti-EGFR probody described in Non-Patent Literature 17 has binding activity to EGFR even before the linker is cleaved by protease. Although the binding activity is increased 30-100 times after the linker is cleaved by protease, the pre-activation probody also has 1 / 30-1 / 100 of the binding activity of the activated probody. Therefore, if the pre-activation probody is present at a high concentration, it may cause side effects by binding to antigens expressed in normal tissue. Furthermore, Probody uses artificial peptides to mask the antigen-binding site of antibodies. Because these artificial peptides have sequences not found in natural human proteins, they may be immunogenic in humans. Immunogenicity is known to reduce the effects of antibody drugs by inducing anti-drug antibodies (Blood. 2016 Mar 31;127(13):1633-41.). Furthermore, anti-drug antibodies against Probody can be considered in the following forms: anti-drug antibodies against the antibody-mask peptide complex (Probody before activation), anti-drug antibodies against the antibody after the mask peptide has dissociated (activated Probody), and anti-drug antibodies against the mask peptide (mask peptide dissociated from activated Probody). Of these, anti-drug antibodies against mask peptides (anti-mask peptide antibodies) may activate Probody even without protease cleavage by binding to the mask peptide of Probody before activation (Figure 4). Probody activated by anti-mask peptide antibodies may exert side effects by binding to antigens expressed in normal tissue.

[0204] Example 2: Concept of a protease-activating polypeptide using a single-domain antibody As shown in Example 1, the Probody technology has the following problems. 1. Probodies activated by cleavage by proteases have a long-lasting presence in the bloodstream. 2. The probody, even before cleavage by protease, possesses binding activity to the antigen. 3. The mask peptide is an artificial, non-human sequence and may induce anti-mask peptide antibodies. To provide antibody drugs that overcome these challenges and exert activity at the site of disease, we believe it is useful to satisfy the following conditions. 1. The antigen-binding domain, activated by cleavage by protease, has a short half-life in the blood. 2. Minimize the antigen-binding activity of the molecule before cleavage by protease. 3. Do not use mask peptides that have artificial non-human sequences. As an example of a polypeptide that satisfies the above conditions, we devised the molecule shown in Figure 5. Polypeptides in a state where the antigen-binding domain and the transporter are linked have a long half-life, and the antigen-binding activity of the antigen-binding domain is suppressed, so they do not bind to the antigen (A). After the antigen-binding domain is released, the antigen-binding activity is restored, and the half-life is also short (B). The polypeptides shown in Figure 5 have various variations, but when using IgG antibody-like molecules, they can be manufactured using the manufacturing method exemplified in Figure 6. First, a single-domain antibody (e.g., VH or VHH) that binds to the target antigen is obtained (A). The obtained single-domain antibody is swapped with one of the VH or VL segments of an IgG antibody having a germline sequence, and associated with the other VH or VL segment to form an IgG antibody-like molecule (B). A protease cleavage sequence is introduced into the IgG antibody-like molecule (C). An example of the introduction site is near the boundary between the introduced single-domain antibody (VH or VHH) and the constant region (CH1 or CL). Monodomain antibodies possess antigen-binding activity when existing as a single domain, but lose this activity when forming a variable region with VL / VH / VHH, etc. Since VL / VH are natural human antibody sequences with germline sequences, the risk of immunogenicity is low, and the possibility of induction of anti-drug antibodies that recognize VL / VH is extremely low. Furthermore, when using VHH to form a variable region with a monodomain antibody, humanizing VHH can reduce the risk of immunogenicity and decrease the possibility of induction of anti-drug antibodies that recognize the humanized VHH. Monodomain antibodies are released when the protease-cleavage sequence inserted into the IgG antibody-like molecule is cleaved by a protease. The released monodomain antibody possesses antigen-binding activity. The IgG antibody-like molecule before protease cleavage has a structure similar to a typical IgG molecule and therefore has long blood retention, whereas the monodomain antibody released by protease cleavage does not possess an Fc region and has a molecular weight of approximately 13 kDa, so it is rapidly eliminated by renal excretion. In fact, while the half-life of full-length IgG is about 2-3 weeks (Blood. 2016 Mar 31;127(13):1633-41.), the half-life of single-domain antibodies is about 2 hours (Antibodies 2015, 4(3), 141-156). Therefore, antigen-binding molecules activated by proteases have a shorter half-life in the blood and are less likely to bind to antigens in normal tissue. If a single-domain antibody is VL, a similar concept can be achieved by introducing a protease cleavage sequence, for example, near the boundary between VL and CL.

[0205] Example 3: Preparation of protease-activating polypeptide using VHH that binds to IL6R 3-1 Preparation of polypeptides incorporating VHH that binds to IL6R An expression vector encoding IL6R90-G1m (SEQ ID NO: 2), which is a VHH (Very Highly Hydrogenated Hemoglobin) that binds to and neutralizes human IL6R as described in International Publication WO2010 / 115998, fused to the constant region (CH1-hinge-CH2-CH3) of human IgG1, was prepared by methods known to those skilled in the art. Expression vectors encoding various subclasses of light chains (variable region-constant region) having human germline sequences, namely VK1-39-k0MT (SEQ ID NO: 3), VK2-28-k0MT (SEQ ID NO: 4), VK3-20-k0MT (SEQ ID NO: 5), VL1-40-lamL (SEQ ID NO: 6), VL1-44-lamL (SEQ ID NO: 7), VL2-14-lamL (SEQ ID NO: 8), VL3-21-lamL (SEQ ID NO: 9), k0 (SEQ ID NO: 10), and lamL (SEQ ID NO: 11), were prepared by methods known to the art. IgG antibody-like molecules: IL6R90-G1m / VK1-39-k0MT (heavy chain SEQ ID NO: 2, light chain SEQ ID NO: 3), IL6R90-G1m / VK2-28-k0MT (heavy chain SEQ ID NO: 2, light chain SEQ ID NO: 4), IL6R90-G1m / VK3-20-k0MT (heavy chain SEQ ID NO: 2, light chain SEQ ID NO: 5), IL6R90-G1m / VL1-40-lamL (heavy chain SEQ ID NO: 2, light chain SEQ ID NO: 6), IL6R90-G1m / VL1-44-la mL (heavy chain SEQ ID NO: 2, light chain SEQ ID NO: 7), IL6R90-G1m / VL2-14-lamL (heavy chain SEQ ID NO: 2, light chain SEQ ID NO: 8), IL6R90-G1m / VL3-21-lamL (heavy chain SEQ ID NO: 2, light chain SEQ ID NO: 9), IL6R90-G1m / k0 (heavy chain SEQ ID NO: 2, light chain SEQ ID NO: 10), and IL6R90-G1m / lamL (heavy chain SEQ ID NO: 2, light chain SEQ ID NO: 11) were expressed transiently using FreeStyle293 cells (Invitrogen) by methods known to those skilled in the art, and purified using protein A by methods known to those skilled in the art.

[0206] 3-2 Evaluation of IL6R binding of polypeptides incorporating VHH that binds to human IL6R The binding activity of IL6R90-G1m / VK1-39-k0MT, IL6R90-G1m / VK2-28-k0MT, IL6R90-G1m / VK3-20-k0MT, IL6R90-G1m / VL1-40-lamL, IL6R90-G1m / VL1-44-lamL, IL6R90-G1m / VL2-14-lamL, IL6R90-G1m / VL3-21-lamL, IL6R90-G1m / k0, and IL6R90-G1m / lamL to human IL6R was evaluated by the following method. The recombinant human IL-6R used as the antigen was prepared as follows: A CHO constant expression strain of soluble human IL-6R (hereinafter also referred to as hsIL-6R, IL6R, or IL-6R), consisting of the amino acid sequence from the 1st to the 357th amino acid position from the N-terminus, as reported in J. Immunol. 152, 4958-4968 (1994), was constructed using methods known to those skilled in the art, cultured, and expressed. From the resulting culture supernatant, hsIL-6R was purified using two steps: Blue Sepharose 6 FF column chromatography and gel filtration column chromatography. The fraction eluted as the main peak in the final step was used as the final purified product. The binding of each molecule to hsIL-6R was evaluated using OctetHTX (ForteBio). Specifically, each molecule was bound to Biosensor / Protein A (ProA) (ForteBio, 18-5013), and hsIL-6R was applied to evaluate the binding at 30°C. Figure 10 shows a sensorgram representing the time-dependent binding amount measured by OctetHTX. IL6R90-G1m / k0 and IL6R90-G1m / lamL, which lacked VL, bound to hsIL-6R. However, IL6R90-G1m / VK1-39-k0MT, IL6R90-G1m / VK2-28-k0MT, IL6R90-G1m / VK3-20-k0MT, IL6R90-G1m / VL1-40-lamL, IL6R90-G1m / VL1-44-lamL, and IL6R90-G1m / VL2-14-lamL, which formed a variable region with VL, were unable to bind to hsIL-6R. This suggests that VHH, which has binding activity to human IL6R, can lose its IL6R binding activity by associating with VL to form a variable region.

[0207] 3-3 Introduction of a protease cleavage sequence into a polypeptide incorporating VHH that binds to IL6R We investigated inserting a protease cleavage sequence near the boundary between IL6R90, an anti-human IL6R VHH, and CH1. Six heavy chains, as shown in Figure 11, were designed by inserting peptide sequence A (SEQ ID NO: 12), a sequence reported to be cleaved by urokinase (uPA), which is cancer-specific, and MT-SP1, at three locations near the boundary between IL6R90 and CH1, with and without a glycine-serine linker. Expression vectors encoding IL6R90H1001 (SEQ ID NO: 13), IL6R90H1002 (SEQ ID NO: 14), IL6R90H1003 (SEQ ID NO: 15), IL6R90H1004 (SEQ ID NO: 16), IL6R90H1005 (SEQ ID NO: 17), and IL6R90H1006 (SEQ ID NO: 18) were prepared by methods known to the art. Using these heavy chains and VK1-39-k0MT (SEQ ID NO: 3) as the light chain, IgG antibody-like molecules such as IL6R90H1001 / VK1-39-k0MT (heavy chain SEQ ID NO: 13, light chain SEQ ID NO: 3), IL6R90H1002 / VK1-39-k0MT (heavy chain SEQ ID NO: 14, light chain SEQ ID NO: 3), and IL6R90H1003 / VK1-39-k0MT (heavy chain SEQ ID NO: 15. Light chain sequence number: 3), IL6R90H1004 / VK1-39-k0MT (heavy chain sequence number: 16, light chain sequence number: 3), IL6R90H1005 / VK1-39-k0MT (heavy chain sequence number: 17, light chain sequence number: 3), and IL6R90H1006 / VK1-39-k0MT (heavy chain sequence number: 18, light chain sequence number: 3) were expressed transiently using FreeStyle293 cells (Invitrogen) by methods known to those skilled in the art, and purified using protein A by methods known to those skilled in the art.

[0208] 3-4 Activation of polypeptides containing protease cleavage sequences by protease cleavage We cleaved IL6R90H1001 / VK1-39-k0MT, IL6R90H1002 / VK1-39-k0MT, IL6R90H1003 / VK1-39-k0MT, IL6R90H1004 / VK1-39-k0MT, IL6R90H1005 / VK1-39-k0MT, and IL6R90H1006 / VK1-39-k0MT with protease and verified whether VHH, which has binding activity to IL6R, was released. Soluble human IL6R was prepared by methods known to those skilled in the art. The prepared soluble human IL6R was biotinylated by methods known to those skilled in the art. To add biotin to the C-terminus of soluble human IL-6R (also called hsIL-6R or soluble human IL-6R, SEQ ID NO: 35), a gene fragment encoding a specific sequence to which biotin is added by biotin ligase (AviTag sequence, SEQ ID NO: 36) was linked downstream of the gene fragment encoding hsIL-6R via a linker. The gene fragment encoding the protein with hsIL-6R and the AviTag sequence linked (hsIL6R-Avitag, SEQ ID NO: 37) was incorporated into an animal cell expression vector, and the constructed plasmid vector was introduced into FreeStyle293 cells (Invitrogen) using 293Fectin (Invitrogen). At this time, a gene expressing EBNA1 (SEQ ID NO: 57) and a gene expressing biotin ligase (BirA, SEQ ID NO: 58) were introduced simultaneously, and biotin was added to further biotin-label hsIL-6R-Avitag. Cells into which the gene had been introduced according to the procedure described above were cultured at 37°C and 8% CO2, and the target protein (hsIL-6R-BAP1) was secreted into the culture supernatant. This cell culture medium was filtered through a 0.22 μm bottle-top filter to obtain the culture supernatant. Following the manufacturer's protocol, a column (anti-human IL-6R antibody column) was prepared by immobilizing anti-human IL-6R antibody on HiTrap NHS-activated HP (GE Healthcare). The culture supernatant was applied to the anti-human IL-6R antibody column equilibrated with TBS, and the adsorbed hsIL-6R was eluted with 2 M Arginine, pH 4.0. Next, the anti-human IL-6R antibody column eluate, diluted with the same buffer, was applied to a SoftLink Avidin column (Promega) equilibrated with TBS, and hsIL-6R-BAP1 was eluted with 5 mM biotin, 50 mM Tris-HCl, pH 8.0, and 2 M Arginine, pH 4.0. The aggregates of hsIL-6R-BAP1 were removed from this eluate by gel filtration chromatography using Superdex200 (GE Healthcare), and purified hsIL-6R-BAP1 was obtained by replacing the buffer with D-PBS and 0.05% CHAPS. Recombinant human matryptase / ST14 catalytic domain (R&D Systems, 3946-SE-010) was used as the protease, and the mixture was reacted for 20 hours under conditions of 12.5 nM protease, 100 ug / mL IgG antibody-like molecule, PBS, and 37°C. The results of the evaluation of protease cleavage by reduced SDS-PAGE are shown in Figure 12. As a result, it was confirmed that in IL6R90H1002 / VK1-39-k0MT, IL6R90H1004 / VK1-39-k0MT, IL6R90H1005 / VK1-39-k0MT, and IL6R90H1006 / VK1-39-k0MT, the protease cleavage sequence was cleaved by the protease near the boundary between the VHH and the heavy chain constant region. Next, the binding of VHH released by protease treatment to IL6R was evaluated using OctetHTX (ForteBio). Specifically, hsIL-6R-BAP1 was bound to a streptavidin sensor (ForteBio, 18-5021), and cleaved IgG antibody-like molecules were applied to evaluate binding at 30°C. Figure 13 shows a sensorgram representing the time-dependent binding amount measured by OctetHTX. As a result, binding was confirmed in IL6R90H1002 / VK1-39-k0MT, IL6R90H1004 / VK1-39-k0MT, IL6R90H1005 / VK1-39-k0MT, and IL6R90H1006 / VK1-39-k0MT. IL6R90-G1m / k0 and IL6R90-G1m / lamL bind in a divalent state and therefore bind by avidity, while free VHH binds by affinity. As a result, protease-treated IL6R90H1002 / VK1-39-k0MT, IL6R90H1004 / VK1-39-k0MT, IL6R90H1005 / VK1-39-k0MT, and IL6R90H1006 / VK1-39-k0MT showed a faster dissociation rate from IL6R compared to IL6R90-G1m / k0 and IL6R90-G1m / lamL. Furthermore, because VHH has a smaller molecular weight compared to IL6R90-G1m / k0 and IL6R90-G1m / lamL, its binding amount (response) is lower. These results confirm that IL6R90H1002 / VK1-39-k0MT, IL6R90H1004 / VK1-39-k0MT, IL6R90H1005 / VK1-39-k0MT, and IL6R90H1006 / VK1-39-k0MT do not show binding activity to IL6R in their original form. However, protease treatment cleaves peptide sequence A, which is inserted near the boundary between VHH and the heavy chain constant region, resulting in the release of the VHH domain, which can then bind to IL6R. From this, it can be said that we were able to actually create the molecule based on the concept described in Example 2.

[0209] Example 4: Production of a protease-activating polypeptide using modified VHH that binds to IL6R. 4-1 Evaluation of IL6R binding of polypeptides incorporating VHH that binds to IL6R An expression vector encoding 20A11-G1m (SEQ ID NO: 38) was prepared by fusing 20A11 (SEQ ID NO: 19), a VHH having binding and neutralizing activity to IL6R as described in International Publication WO2010 / 115998, to the constant region (CH1-hinge-CH2-CH3) of human IgG1, in the same manner as in Example 3, using methods known to those skilled in the art. Using this heavy chain and VK1-39-k0MT (SEQ ID NO: 3), VK2-28-k0MT (SEQ ID NO: 4), VK3-20-k0MT (SEQ ID NO: 5), VL1-40-lamL (SEQ ID NO: 6), VL1-44-lamL (SEQ ID NO: 7), VL2-14-lamL (SEQ ID NO: 8), and VL3-21-lamL (SEQ ID NO: 9) as light chains, the same method as in Example 3 was used to produce The lipeptides 20A11-G1m / VK1-39-k0MT, 20A11-G1m / VK2-28-k0MT, 20A11-G1m / VK3-20-k0MT, 20A11-G1m / VL1-40-lamL, 20A11-G1m / VL1-44-lamL, 20A11-G1m / VL2-14-lamL, and 20A11-G1m / VL3-21-lamL were expressed and purified. In the same manner as in Example 3, the following were obtained: 20A11-G1m / VK1-39-k0MT (heavy chain sequence number: 38, light chain sequence number: 3), 20A11-G1m / VK2-28-k0MT (heavy chain sequence number: 38, light chain sequence number: 4), 20A11-G1m / VK3-20-k0MT (heavy chain sequence number: 38, light chain sequence number: 5), and 20A11-G1m / VL1-40-lamL (heavy chain sequence number: 38). Figure 14 shows the results of evaluating the binding of IL6R to 20A11-G1m / VL1-44-lamL (sequence number: 38, light chain sequence number: 6), 20A11-G1m / VL2-14-lamL (sequence number: 38, light chain sequence number: 7), 20A11-G1m / VL2-14-lamL (sequence number: 38, light chain sequence number: 8), and 20A11-G1m / VL3-21-lamL (sequence number: 38, light chain sequence number: 9). As a result, none of the light chains used in this example caused the loss of IL6R binding activity of 20A11 by associating with the heavy chain fused with the constant region (CH1-hinge-CH2-CH3) of the human germline IgG1. One possible reason for this is that 20A11 and the VL used in this embodiment do not form a stable variable region.

[0210] 4-2 Introduction of amino acid modifications to the VHH-VL interface site in polypeptides incorporating VHH that retains antigen binding To form a stable variable region between 20A11 and VL, mutations were introduced into the amino acids at the interface between 20A11 and VL. An expression vector encoding 20A11hu-G1m (SEQ ID NO: 39) was prepared by fusing 20A11hu (SEQ ID NO: 20), which was created by substituting the 37th F with V (F37V), the 45th R with L, and the 47th G with W (all using Kabat numbering), to the constant region (CH1-hinge-CH2-CH3) of human IgG1 in the same manner as in Example 3, using methods known to those skilled in the art. Using this heavy chain and VK1-39-k0MT (sequence number: 3), VK2-28-k0MT (sequence number: 4), VK3-20-k0MT (sequence number: 5), VL1-40-lamL (sequence number: 6), VL1-44-lamL (sequence number: 7), VL2-14-lamL (sequence number: 8), and VL3-21-lamL (sequence number: 9) as light chains, polypeptides 20A11hu-G1m / VK1-39-k0MT (heavy chain sequence number: 39, light chain sequence number: 3) and 20A11hu-G1m / VK2-28-k0MT (heavy chain sequence number: 39, light chain sequence number: 3) were created. Expression and purification of 20A11hu-G1m / VK3-20-k0MT (heavy chain sequence number: 39, light chain sequence number: 5), 20A11hu-G1m / VL1-40-lamL (heavy chain sequence number: 39, light chain sequence number: 6), 20A11hu-G1m / VL1-44-lamL (heavy chain sequence number: 39, light chain sequence number: 7), 20A11hu-G1m / VL2-14-lamL (heavy chain sequence number: 39, light chain sequence number: 8), and 20A11hu-G1m / VL3-21-lamL (heavy chain sequence number: 39, light chain sequence number: 9) were carried out using the same method as in Example 3.

[0211] 4-3 Evaluation of IL6R binding of polypeptides incorporating VHH with amino acid modifications introduced at the interface site between VHH and VL The binding of the obtained 20A11hu-G1m / VK1-39-k0MT, 20A11hu-G1m / VK2-28-k0MT, 20A11hu-G1m / VK3-20-k0MT, 20A11hu-G1m / VL1-40-lamL, 20A11hu-G1m / VL1-44-lamL, 20A11hu-G1m / VL2-14-lamL, and 20A11hu-G1m / VL3-21-lamL to IL6R at 30°C or 25°C was evaluated using the same method as in Example 3. The results are shown in Figure 15. As a result, it was shown that 20A11hu-G1m / VK1-39-k0MT, 20A11hu-G1m / VK2-28-k0MT, 20A11hu-G1m / VK3-20-k0MT, 20A11hu-G1m / VL1-40-lamL, 20A11hu-G1m / VL1-44-lamL, and 20A11hu-G1m / VL2-14-lamL cannot bind to IL6R. These results indicate that by modifying 20A11hu, which retained its IL6R binding activity even when associated with VL used in Example 3, by changing the amino acids present at the interface between VHH and VL to 37V, 45L, and 47W (Kabat numbering), a stable variable region can be formed between VHH and VL, thereby causing VHH to lose its IL6R binding activity.

[0212] 4-4 Introduction of a protease cleavage sequence into a polypeptide incorporating VHH with amino acid modifications introduced to the VL interface site Using the same method as in Example 3, heavy chains 20A11huH1001 (SEQ ID NO: 40), 20A11huH1002 (SEQ ID NO: 41), 20A11huH1004 (SEQ ID NO: 42), and 20A11huH1006 (SEQ ID NO: 43) were prepared by inserting a protease cleavage sequence (SEQ ID NO: 12) or a protease cleavage sequence linked to a movable linker (SEQ ID NO: 44) near the boundary between 20A11hu and CH1. Using these heavy chains and VK1-39-k0MT (SEQ ID NO: 3) as the light chain, the polypeptides 20A11huH1001 / VK1-39-k0MT (heavy chain SEQ ID NO: 40, light chain SEQ ID NO: 3), 20A11huH1002 / VK1-39-k0MT (heavy chain SEQ ID NO: 41, light chain SEQ ID NO: 3), 20A11huH1004 / VK1-39-k0MT (heavy chain SEQ ID NO: 42, light chain SEQ ID NO: 3), and 20A11huH1006 / VK1-39-k0MT (heavy chain SEQ ID NO: 43, light chain SEQ ID NO: 3) were expressed and purified using the same method as in Example 3.

[0213] 4-5 Activation of polypeptides containing protease cleavage sequences by protease cleavage 20A11huH1001 / VK1-39-k0MT, 20A11huH1002 / VK1-39-k0MT, 20A11huH1004 / VK1-39-k0MT, and 20A11huH1006 / VK1-39-k0MT were cleaved with protease in the same manner as in Example 3, and the degree of cleavage was evaluated by reduced SDS-PAGE. The results are shown in Figure 16. As a result, it was confirmed that in 20A11huH1002 / VK1-39-k0MT, 20A11huH1004 / VK1-39-k0MT, and 20A11huH1006 / VK1-39-k0MT, the area near the boundary between VHH and CH1 was cleaved by protease. Next, the binding of VHH and IL6R released by protease treatment at 30°C or 25°C was evaluated using the same method as in Example 3. The Octet sensorgram is shown in Figure 17. As a result, binding to IL6R was confirmed in 20A11huH1002 / VK1-39-k0MT, 20A11huH1004 / VK1-39-k0MT, and 20A11huH1006 / VK1-39-k0MT, where cleavage near the boundary between VHH and CH1 was confirmed by protease treatment. These results confirm that even if antigen-binding activity is not immediately lost when a polypeptide incorporating VHH is associated with a specific VL, antigen-binding activity can be lost by introducing association-promoting mutations into the amino acids present at the interface between VHH and VL. These results demonstrate that, in addition to the method of combining pre-obtained VHH with a light chain as in Example 3, the molecule of the concept described in Example 2 can also be produced by combining VHH with a light chain in which amino acids involved in association with the light chain have been substituted.

[0214] Example 5: Production of protease-activating polypeptides using immunoalpaca-derived VHH 5-1 Obtaining VHH derived from immune alpaca Alpacas were immunized with IL6R, CD3, and PlexinA1 using methods known to those skilled in the art, and PBMCs were collected after 4 and 8 weeks. The VHH gene was amplified from the collected PBMCs according to the method described in J. Immunol. Methods (2007) 324, 13. The amplified VHH gene fragment was linked to the gene3 gene and inserted into a phagemide vector. The phagemide vector containing the VHH fragment was introduced into E. coli by electroporation, and phages presenting VHH were obtained using methods known to those skilled in the art. The binding of the obtained phages to IL6R, CD3, or PlexinA1 was evaluated by ELISA, and the sequences of the binding clones were analyzed using methods known to those skilled in the art to identify the VHH that binds to the antigens.

[0215] 5-2 Enrichment of VHH bound to CD3 VHHs that bind to human CD3 were identified from the VHH library constructed in Example 5-1. As antigens, biotin-labeled human CD3ε and human CD3δ were ligated to the constant region of a human antibody (human CD3ed-Fc), and VHHs capable of binding to human CD3 were enriched. Human CD3ed-Fc was prepared as follows: An animal cell expression vector containing genes encoding the amino acid sequences shown in SEQ ID NO: 59, SEQ ID NO: 60, and BirA (SEQ ID NO: 58) was introduced into FreeStyle293 cells (Invitrogen). After introduction, L-biotin was added to perform biotinylation in the culture medium, and the cells were cultured with shaking at 37°C according to the protocol, and the supernatant was collected after 4 to 5 days. From the supernatant, a Protein A column (Eshmuno A (Merck)) was used to obtain proteins fused with the constant region of the antibody. Furthermore, to obtain only the CD3εδ heterodimer, an Anti-FLAG M2 column was used to fractionate the CD3εδ heterodimer (referred to as human CD3ed-Fc) to which the constant region of the antibody was fused. Subsequently, gel filtration chromatography (Superdex200, GE Healthcare) was performed to isolate the target CD3εδ heterodimer (referred to as human CD3ed-Fc). Phage production was induced from E. coli containing the constructed phage display phagemide. A phage library solution was obtained by adding 2.5 M NaCl / 10% PEG to the culture medium of the phage-producing E. coli, precipitating the phage population, and then diluting the precipitate with TBS. Next, BSA was added to the phage library solution to a final concentration of 4% BSA. For the panning method, the common method of panning using antigens immobilized on magnetic beads was referenced (J. Immunol. Methods. (2008) 332 (1-2), 2-9, J. Immunol. Methods. (2001) 247 (1-2), 191-203, Biotechnol. Prog. (2002) 18 (2) 212-20, Mol. Cell Proteomics (2003) 2 (2), 61-9). NeutrAvidin-coated beads (FG beads NeutrAvidin) or Streptavidin-coated beads (Dynabeads MyOne Streptavidin T1) were used as magnetic beads. Specifically, 100 pmol of biotin-labeled antigen was added to the prepared phage library solution, and the phage library solution was contacted with the antigen at room temperature for 60 minutes. Magnetic beads blocked with BSA were added, and the antigen-phage complex was bound to the magnetic beads at room temperature for 15 minutes. The beads were washed twice with 0.5 mL of TBST (TBS containing 0.1% Tween20, TBS manufactured by TaKaRa), and then washed once more with 0.5 mL of TBS. Subsequently, 0.5 mL of 1 mg / mL of trypsin was added to the beads, and after being suspended at room temperature for 15 minutes, the beads were immediately separated using a magnetic stand, and the phage solution was recovered. The recovered phage solution was added to 20 mL of E. coli strain ER2738 in the logarithmic growth phase (OD600 0.4-0.5). The E. coli was slowly cultured with agitation at 37°C for 1 hour to infect the E. coli with the phages. Infected E. coli were seeded onto 225 mm x 225 mm plates. Next, a phage library was prepared by recovering phages from the culture medium of the seeded E. coli. This cycle, called panning, was repeated twice. In the second panning, the beads were washed three times with TBST, followed by two times with TBS. Additionally, 4 nmol of human CD3ed-Fc was added during phage binding.

[0216] 5-3 Preparation of a protease-activated IgG antibody-like molecule incorporating VHH that binds to CD3 The nucleotide sequences encoding the VHH sequences (Table 1) of human CD3-binding clones obtained from Example 5-1 or 5-2 were ligated to the nucleotide sequences encoding the protease cleavage site and constant region using the method described in Example 3, inserted into an animal cell expression vector, and used as the heavy chain of an IgG antibody-like molecule.

[0217] [Table 1]

[0218] The protease-activated IgG antibody-like molecules shown in Table 2 below were expressed transiently using FreeStyle293 cells (Invitrogen) by methods known to those skilled in the art, and purified using methods known to those skilled in the art with protein A.

[0219] [Table 2]

[0220] 5-4 Activation of protease-activated IgG antibody-like molecules by protease cleavage The IgG antibody-like molecules prepared in Example 5-3 were cleaved with protease in the same manner as in Example 3, and the degree of cleavage was evaluated by reduced SDS-PAGE. The results are shown in Figure 18. The protease concentration was 25 nM, and OctetRED (ForteBio) was used for measurement. As a result, it was confirmed that the protease cleavage sequence in the IgG antibody-like molecule was cleaved by the protease. Next, the binding of VHH released by protease treatment to CD3 was evaluated using the same method as in Example 3. The Octet sensorgram is shown in Figure 19. As a result, in bC3edL1R1N160H01-G1mISHI01 / VK1-39-k0MT, bC3edL1R1N161H01-G1mISHI01 / VK1-39-k0MT, and bC3edL1R1N164H01-G1mISHI01 / VK1-39-k0MT, the IgG antibody-like molecules before protease treatment did not show antigen binding, while antigen binding was confirmed after protease treatment. Furthermore, when IgG-like molecules containing protease cleavage sites similar to those of the IgG antibody-like molecules described in Table 2 were created for VHH that bind to multiple CD3s using a method similar to that of the VHH described in Table 1, antigen binding was confirmed after protease treatment. These results indicate that, in addition to the polypeptides shown in Examples 3 and 4, by incorporating a protease cleavage sequence, an IgG antibody-like molecule can be formed in which the protease cleavage sequence is cleaved by protease treatment, releasing an antigen-binding domain, and the released antigen-binding domain can bind to an antigen.

[0221] Example 6: Polypeptide in which a protease cleavage sequence is introduced into the light chain. Similar to Example 3, VK1-39P-2-Pk0MT (SEQ ID NO: 67), VK1-39P-1-Pk0MT (SEQ ID NO: 68), VK1-39P-Pk0MT (SEQ ID NO: 69), VK1-39P+2-Pk0MT (SEQ ID NO: 70), VK1-39P+3-Pk0MT (SEQ ID NO: 71), VK1-39P+4-Pk0MT (SEQ ID NO: 72), and VK1-39P+5-Pk0MT (SEQ ID NO: 73) were prepared by incorporating protease cleavage sequences at each position of the light chain. Expression and purification of IgG antibody-like molecules using these light chains and IL6R90-G1m (SEQ ID NO: 2) as the heavy chain were carried out in the same manner as in Example 3. The protease concentration was 25 nM. IL6R90-G1m / VK1-39-k0MT (heavy chain SEQ ID NO: 2, light chain SEQ ID NO: 3) was used as an IgG antibody-like molecule without the introduction of a cleavage sequence. Next, the prepared IgG antibody-like molecules were cleaved with protease in the same manner as in Example 3, and the degree of cleavage was evaluated by reduced SDS-PAGE. The results are shown in Figure 20. As a result, it was confirmed that the protease cleavage sequence was cleaved by protease in VK1-39P+2-Pk0MT (SEQ ID NO: 70), VK1-39P+3-Pk0MT (SEQ ID NO: 71), VK1-39P+4-Pk0MT (SEQ ID NO: 72), and VK1-39P+5-Pk0MT (SEQ ID NO: 73). Furthermore, the binding of VHH and IL6R exposed by protease treatment was evaluated in the same manner as in Example 3. The Octet sensorgram is shown in Figure 21. As a result, even when the cleavage sequence was introduced into the light chain, binding was observed by protease treatment, indicating that it is possible to obtain a protease-activated polypeptide in which the antigen-binding domain is exposed and exhibits antigen-binding ability when the light chain is cleaved with protease after introducing the protease cleavage sequence into the light chain.

[0222] Example 7 A library containing a heavy chain having an antigen-binding domain and a light chain into which a protease cleavage sequence has been introduced, and acquisition of a protease-activated polypeptide from the library by phage display. As confirmed in Example 6, even when a protease cleavage sequence is introduced into the light chain of a protease-activated polypeptide, the antigen-binding domain is exposed after light chain cleavage and binds to the antigen. Therefore, a heavy chain containing an antigen-binding domain, such as a single-domain antibody, and a light chain into which a protease cleavage sequence has been introduced are incorporated into a phagemide, and the phage is made to display it. Multiple phage display phagemides containing different types of antigen-binding domains are constructed, and phages are produced from E. coli holding these phagemides. A phage library solution is obtained by adding 2.5 M NaCl / 10% PEG to the culture medium of the E. coli that produced the phages, precipitating the phage population, and then diluting the resulting population with TBS. BSA is added to the phage library solution to a final concentration of 4% BSA. Protease-activated polypeptides are obtained from the phage library prepared as described above by panning. As a panning method, the panning method using antigens immobilized on magnetic beads, a common method, is referred to (J. Immunol. Methods. (2008) 332 (1-2), 2-9, J. Immunol. Methods. (2001) 247 (1-2), 191-203, Biotechnol. Prog. (2002) 18 (2) 212-20, Mol. Cell Proteomics (2003) 2 (2), 61-9). Before protease addition, phages that did not bind to the magnetic beads immobilized with the antigen are collected, and after protease addition, phages that bound to the magnetic beads immobilized with the antigen are collected. NeutrAvidin-coated beads (Sera-Mag SpeedBeads NeutrAvidin-coated, FG beads NeutrAvidin) or Streptavidin-coated beads (Dynabeads M-280 Streptavidin) are used as magnetic beads. Clones that bind to the antigen may be selected from the recovered phages using the phage ELISA described in the previous section, or the antibody gene may be subcloned into an animal expression vector and expressed in animal cells, and the binding activity before and after protease treatment may be compared to select the binding clone.

[0223] Example 8: Library containing heavy and light chains having antigen-binding domains, and acquisition of heavy chains from the library whose antigen-binding ability is controlled by the light chain by phage display. As confirmed in Example 3, the antigen-binding ability of the heavy chain containing the antigen-binding domain is controlled by the association of the light chain. Therefore, heavy chains that lose antigen-binding ability when associated with the light chain, and that exhibit antigen-binding ability when presented alone or when presented with the constant region of the heavy chain and light chain, are obtained by phage display. A heavy chain containing an antigen-binding domain, such as a single-domain antibody, is incorporated into a phagemide and presented by a phage. Multiple phage-display phagemides containing different types of antigen-binding domains are constructed, and phages are produced from E. coli holding these phagemides. A phage library is obtained by adding 2.5 M NaCl / 10% PEG to the culture medium of the phage-producing E. coli, precipitating the phage population, and then diluting the resulting population with TBS. BSA is added to the phage library to a final concentration of 4% BSA. From the phage library prepared as described above, heavy chains that exhibit antigen-binding ability when presenting only the heavy chain or the heavy chain and light chain constant region, and lose antigen-binding ability when the heavy chain associates with the light chain variable region, are obtained by panning. As the panning method, the panning method using an antigen immobilized on magnetic beads described in Example 5 is referred to. From the phage library presenting the heavy chain or the heavy chain and light chain constant region, phages bound to magnetic beads on which the antigen is immobilized are recovered. The recovered phages are used to infect E. coli, and phages presenting the heavy chain and light chain are produced using a helper phage that expresses the light chain. From the culture medium of E. coli on which phage production has taken place, phages presenting the heavy chain and light chain containing the antigen-binding domain are obtained by the method described above. From the population of phages presenting the heavy chain and light chain, phages that do not bind to magnetic beads on which the antigen is immobilized are recovered. As shown in Figure 9D, panning may also be performed by alternating the order of retrieval: first, a population of phages displaying only the heavy chain bound to the magnetic beads to which the antigen is immobilized, or a population of phages displaying both the heavy chain and the constant region of the light chain, and second, a population of phages displaying the heavy chain and the light chain that do not bind to the magnetic beads to which the antigen is immobilized. In addition to the method of expressing the light chain using helper phages, it is also possible to incorporate the region encoding the light chain into the same phagemide as the heavy chain, as is usually done, and then incorporate either only the constant region of the light chain or the gene encoding the entire light chain for each panning. From the recovered phages, clones that bind to the antigen may be selected using the phage ELISA described in the previous section, or the antibody gene may be subcloned into an animal expression vector and expressed using animal cells, and the binding activity before and after protease treatment may be compared to select the binding clone.

[0224] Example 9: Acquisition of VHH with antigen-binding ability controlled by light chain using phage display method and preparation of IgG antibody-like molecules containing it. In Example 3, it was confirmed that the antigen-binding ability of VHH, which is included in the heavy chain as a substitute for VH, is controlled by association with the light chain. Therefore, VHH that loses its antigen-binding ability when associated with a specific light chain, and exhibits antigen-binding ability when the heavy chain alone or when the heavy chain and the constant region of the light chain are presented, i.e., when not associated with the variable region of the light chain, was obtained from a phage library in which VHH derived from immunoalpaca PBMCs was linked to CH1, and an IgG antibody-like molecule containing this VHH was constructed.

[0225] 9-1 Construction of a light chain expression helper phage incorporating a light chain expression unit Based on the method described in International Publication No. WO2015 / 046554, light chain expressing helper phages were constructed by incorporating the promoter, signal sequence, antibody light chain variable region and light chain constant region genes, or light chain constant region genes, into the helper phage genome. E. coli infected with these helper phages can express the antibody light chain variable region and light chain constant region, or only the light chain constant region. Specifically, the genome of the helper phage M13KO7TC, constructed using the method described in International Publication No. WO2015 / 046554, was extracted, and a light chain expression unit was introduced. The light chain genes used for introduction included a gene encoding the light chain variable region and the light chain constant region (VK1-39-k0MTdC, SEQ ID NO: 152), or a gene encoding the light chain constant region (k0MTdC, SEQ ID NO: 153). The lac promoter - pelB signal sequence - light chain gene were inserted into M13KO7TC / SacI using the method described above, and then introduced into E. coli strain ER2738 by electroporation. The obtained E. coli were cultured, and helper phages were purified by PEG precipitation using 2.5 M NaCl / 10% PEG added to the culture supernatant. The titers of the obtained helper phages M13KO7TC-Vk1-39-k0MTdC and M13KO7TC-k0MTdC were confirmed using a general plaque formation method.

[0226] 9-2 Preparation of a library containing multiple VHH-CH1 units Alpacas were immunized with four immunogens—the extracellular domain of human IL6R, human CD3εγ heterodimer, monkey CD3εγ heterodimer, and the cellular domain of human Plexin A1—using methods known to those skilled in the art, and PBMCs were collected after 4 weeks. The CD3εγ heterodimer was prepared based on Journal of Molecular Biology (2000) 302:899-916. The VHH gene was amplified from the collected PBMCs based on the method described in J. Immunol. Methods (2007) 324, 13. The amplified VHH gene fragment was linked to the CH1-gene3 gene and inserted into a phagemide vector to prepare a library containing multiple VHH-CH1 molecules linked to CH1.

[0227] 9-3 Method for generating a phage population that presents the VHH-CH1 / full-length light chain or the VHH-CH1 / constant light chain region. By introducing a phagemide vector containing the gene encoding VHH-CH1 into E. coli using electroporation, culturing the resulting E. coli, and infecting it with the helper phage M13KO7TC-Vk1-39-k0MTdC prepared in Example 9-1, the VHH-CH1 expressed from the phagemide vector and the full-length light chain expressed from the helper phage form a Fab structure, and a population of phages can be created that displays VHH-CH1 / full-length light chain (VHH-CH1 / Vk1-39-k0MTdC) on the surface of the phagemide containing the gene encoding VHH-CH1. Furthermore, by culturing E. coli into which a phagemide vector containing the gene encoding VHH-CH1 has been inserted, and infecting it with the helper phage M13KO7TC-k0MTdC prepared in Example 9-1, the VHH-CH1 expressed from the phagemide vector and the light chain constant region expressed from the helper phage form a structure in which VHH-CH1 and CL associate, thereby creating a phage population that presents VHH-CH1 / light chain constant region (VHH-CH1 / k0MTdC). The phages can be purified by adding 2.5 M NaCl / 10% PEG to the culture supernatant and using the PEG precipitation method. The titer of the obtained phages can be confirmed using a general plaque formation method.

[0228] 9-4 Obtaining VHH-CH1 from a VHH-CH1 phage library, including PlexinA1 VHH, which inhibits antigen binding upon association with the light chain variable region and exhibits antigen-binding ability when the light chain variable region is absent. From the VHH-CH1 library prepared in Example 9-2, VHH-CH1 containing VHH that exhibits antigen-binding ability when the light chain variable region is absent and whose antigen-binding ability is inhibited by association with the light chain variable region was obtained by panning. As the antigen, biotin-labeled human Plexin A1 prepared in the reference example was used. The panning method involves the following steps: (1) Using the VHH-CH1 phage library prepared in Example 9-2, a population of phages presenting the VHH-CH1 / light chain constant region (VHH-CH1 / k0MTdC) is created using the method in Example 9-3, and phages bound to magnetic beads with immobilized antigens are recovered from this population. (2) A population of phages presenting VHH-CH1 / full-length light chain (VHH-CH1 / Vk1-39-k0MTdC) is created from the recovered phages using the method of Example 9-3, and phages that do not bind to the magnetic beads on which the antigen is immobilized are recovered from among them; (3) Repeat steps (1) and (2) with the recovered phages to recover the desired phages; The procedure was carried out accordingly. Panning revealed that binding to PlexinA1 was inhibited by ass...

Claims

1. A polypeptide comprising an antigen-binding domain, a transport portion, and a cleavage site, wherein the transport portion has an inhibitory domain that suppresses the antigen-binding activity of the antigen-binding domain, and the antigen-binding domain has a shorter half-life in the blood than the transport portion. The polypeptide wherein the antigen-binding domain includes or is a single-domain antibody, the inhibitory domain of the transport portion is VHH, antibody VH, or antibody VL, and the antigen-binding activity of the single-domain antibody is inhibited by the VHH, antibody VH, or antibody VL.

2. The polypeptide according to claim 1, wherein the antigen-binding domain is detachable from the polypeptide, and the antigen-binding activity of the antigen-binding domain increases upon detachment from the polypeptide compared to before detachment.

3. The polypeptide according to claim 1 or claim 2, wherein the antigen-binding activity of the antigen-binding domain is suppressed by association between the antigen-binding domain and the inhibitory domain of the transport portion.

4. The polypeptide according to any one of claims 1 to 3, wherein the cleavage of the cleavage site allows the antigen-binding domain to be released from the polypeptide, and / or the association between the antigen-binding domain and the repressive domain of the transport portion is resolved.

5. The polypeptide according to any one of claims 1 to 4, wherein the cleavage site includes a protease cleavage sequence.

6. The polypeptide according to any one of claims 1 to 5, wherein the cleavage site is cleaved by a target tissue-specific protease.

7. The polypeptide according to any one of claims 1 to 6, wherein the molecular weight of the antigen-binding domain is 60 kDa or less.

8. The polypeptide according to any one of claims 1 to 7, wherein the transport portion has FcRn binding activity, and the antigen-binding domain has no FcRn binding activity or has weaker FcRn binding activity than the transport portion.

9. The polypeptide according to any one of claims 1 to 8, wherein the transport portion has an FcRn binding region.

10. The polypeptide according to any one of claims 1 to 9, wherein the transport portion includes an antibody constant region.

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

12. The polypeptide according to claim 10 or claim 11, wherein the antibody constant region of the polypeptide is an IgG antibody constant region.

13. The polypeptide according to any one of claims 1 to 12, wherein the polypeptide is an IgG antibody-like molecule.

14. The polypeptide according to claim 10, wherein the polypeptide has a protease cleavage sequence, and the protease cleavage sequence is located near the boundary between the antigen-binding domain and the antibody constant region.

15. A pharmaceutical composition comprising the polypeptide described in any one of claims 1 to 14.

16. A method for producing a polypeptide according to any one of claims 1 to 14.

17. A method for producing a polypeptide comprising an antigen-binding domain, a transport portion, and a protease cleavage sequence, comprising the following steps: the transport portion has an inhibitory domain that suppresses the antigen-binding activity of the antigen-binding domain, the antigen-binding domain comprises or is a monodomain antibody, the inhibitory domain of the transport portion is VHH, antibody VH, or antibody VL, and the antigen-binding domain has a shorter blood half-life than the transport portion; the method: (a) A step to obtain a monodomain antibody that binds to a target antigen; (b) A step of forming a polypeptide precursor by linking the single-domain antibody obtained in step (a) with the transport portion such that the antigen-binding activity of the single-domain antibody is suppressed by the inhibitory domain of the transport portion; and (c) A step of introducing a protease cleavage sequence near the boundary between the monodomain antibody and the transport portion.

18. A method for producing a polypeptide comprising an antigen-binding domain, a transport portion, and a protease cleavage sequence, comprising the following steps: the transport portion has an inhibitory domain that suppresses the antigen-binding activity of the antigen-binding domain, the antigen-binding domain comprises or is a monodomain antibody, the inhibitory domain of the transport portion is VHH, antibody VH, or antibody VL, and the antigen-binding domain has a shorter blood half-life than the transport portion; the method: (a) A step of obtaining a monodomain antibody that binds to a target antigen; and (b) A step of forming a polypeptide by linking the single-domain antibody obtained in step (a) with the transport portion via a protease cleavage sequence such that the antigen-binding activity of the single-domain antibody is suppressed by the inhibitory domain of the transport portion.

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