Polypeptides comprising an antigen binding domain and a transport portion

Abstract

The present invention relates to: a polypeptide comprising an antigen-binding domain and a transport portion, wherein the transport portion has an inhibitory domain for inhibiting the antigen-binding activity of the antigen-binding domain, and the polypeptide has a longer half-life than the antigen-binding domain existing alone; a method for preparing and screening the polypeptide; a pharmaceutical composition containing the polypeptide; a method for preparing and screening a single-domain antibody, the antigen-binding activity of the single-domain antibody being inhibited by associating with a specific VL / VH / VHH; and a library of fusion polypeptides comprising single-domain antibodies, the antigen-binding activity of the single-domain antibody being inhibited by associating with a specific VL / VH / VHH.

Description

Technical Field

[0001] The present invention relates to a polypeptide comprising an antigen-binding domain and a transport moiety and having a longer half-life than the antigen-binding domain alone, wherein the transport moiety has an inhibitory domain that inhibits the antigen-binding activity of the antigen-binding domain, methods for preparing and screening such polypeptides, pharmaceutical compositions containing such polypeptides, methods for preparing and screening single-domain antibodies whose antigen-binding activity can be inhibited by associating with a specific VL, VH, or VHH, and libraries of fusion polypeptides each comprising a single-domain antibody whose antigen-binding activity can be inhibited by associating with a specific VL, VH, or VHH. Background Art

[0002] Because antibodies are highly stable in plasma and cause few side effects, they have attracted attention as pharmaceuticals. Among them, many IgG-type antibody drugs have been launched, and a large number of antibody drugs are currently under development (NPL 1 and 2).

[0003] So far, rituximab (Rituxan) for CD20, cetuximab (cetuximab) for EGFR, trastuzumab (Herceptin) for HER2, etc. have been approved as cancer treatment drugs using antibody drugs (NPL 3). These antibody molecules bind to the antigens they express on cancer cells, thereby exerting cytotoxicity against cancer cells through ADCC activity, etc. It is known that this cytotoxic activity based on ADCC activity, etc. depends on the number of antigens expressed on the target cells of the therapeutic antibody (NPL 4). Therefore, from the perspective of the effect of therapeutic antibodies, high expression levels of the targeted antigen are preferred. However, if the antigen (although with a high expression level) is expressed in normal tissues, cytotoxicity based on ADCC activity, etc. is exerted against normal cells. Therefore, side effects have become a serious problem. Therefore, the antigen targeted by therapeutic antibodies as cancer treatment drugs is preferably specifically expressed on cancer cells. For example, antibody molecules against EpCAM, which is known to be a cancer antigen, are considered promising as cancer treatment drugs. However, it is known that EpCAM is also expressed in the pancreas. In fact, it has been reported in clinical trials that administration of anti-EpCAM antibodies leads to pancreatitis as a side effect due to cytotoxicity against the pancreas (NPL 5).

[0004] After the success of antibody drugs that exert cytotoxicity based on ADCC activity, second-generation improved antibody molecules that exert strong cytotoxic activity have been reported as a result of the following: for example, by removing trehalose from the N-linked oligosaccharides in the natural human IgG1 Fc region to enhance ADCC activity (NPL 6), or by enhancing the binding to FcγRIIIa via amino acid substitution in the natural human IgG1 Fc region and enhancing ADCC activity (NPL 7). Improved antibody molecules and small molecule antibodies have also been reported as antibody drugs that exert cytotoxic activity against cancer cells under a mechanism other than NK cell-mediated ADCC activity as described above, wherein the improved antibody molecules exert stronger cytotoxic activity, such as antibody drug conjugates (ADCs) containing antibodies conjugated to drugs with strong cytotoxic activity (NPL 8), wherein the small molecule antibodies exert cytotoxic activity against cancer cells by recruiting T to cancer cells (NPL 9).

[0005] Such antibody molecules that exert stronger cytotoxic activity also exert cytotoxic activity to cancer cells that express low levels of antigens, but also exert cytotoxic activity to normal tissues that express low levels of antigens, similar to cancer cells. In fact, compared with cetuximab (natural human IgG1 for EGFR), EGFR-BiTE (a bispecific antibody for CD3 and EGFR) can exert strong cytotoxic activity and anti-tumor effects on cancer cells by recruiting T cells to cancer cells. On the other hand, it has also been found that serious side effects occur by administering EGFR-BiTE to cynomolgus monkeys because EGFR is also expressed in normal tissues (NPL 10). In addition, since CD44v6 is also expressed in normal tissues, it has been found clinically that ADC bivatuzumab (bivatuzumab) DM1 (mertansine) containing DM1 conjugated to an antibody for CD44v6 can cause severe skin toxicity and hepatotoxicity, wherein the CD44v6 is highly expressed on cancer cells (NPL 11).

[0006] As mentioned above, the use of antibodies that can exert strong cytotoxic activity against cancer cells that express antigens at low levels requires that the target antigen be expressed in a highly cancer-specific manner. However, considering that HER2, the target antigen of trastuzumab, or EGFR, the target antigen of cetuximab, is also expressed in normal tissues, only a limited number of cancer antigens can be expressed in a highly cancer-specific manner. Therefore, although cytotoxic activity against cancer can be enhanced, adverse reactions attributable to cytotoxic effects on normal tissues may become a problem.

[0007] Recently, ipilimumab, which enhances tumor immunity by inhibiting CTLA4, which leads to cancer immunosuppression, has been shown to prolong overall survival in metastatic melanoma (NPL 12). However, despite enhancing tumor immunity, ipilimumab systemically inhibits CTLA4 and thus causes severe side effects similar to autoimmune diseases due to systemic activation of the immune system (NPL 13).

[0008] Meanwhile, antibody drugs that exert therapeutic effects by inhibiting inflammatory cytokines in inflammation or autoimmune diseases are known as antibody drugs for diseases other than cancer (NPL 14). For example, infliximab (Remicade) and adalimumab (Humira) that target TNF, and tocilizumab (Actemra) that targets IL-6R are known to have high therapeutic effects on rheumatoid arthritis. However, due to the systemic neutralization of these cytokines, infectious diseases are considered as side effects (NPL 15).

[0009] Various technologies have been developed as technologies applicable to second generation antibody drugs. For example, it has been reported that the technology (NPL 16) for improving effector function, antigen binding ability, pharmacokinetics or stability or reducing immunogenicity risk has been improved. However, there are still some reports on the technology for allowing antibody drug specificity to act on target tissue to solve side effects as described above. The reported technology includes a method, which includes: via a joint cut by a protease expressed at lesion site (such as cancerous tissue or inflammatory tissue) by antibody and masking peptide connection, thereby masking the antigen binding site of antibody with masking peptide, and suppressing the antigen binding activity of the antibody; The masking peptide is dissociated by the protease cutting of the joint, so that antibody recovers its antigen binding activity, and becomes able to be combined with the antigen in target pathological tissue (NPL 17 and 18 and patent documentation 1).

[0010] Reference List

[0011] [Patent Document]

[0012] [Patent Document 1] WO2010 / 081173

[0013] [Non-patent literature]

[0014] [NPL 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

[0015] [NPL 2]The therapeutic antibodies market to 2008.Pavlou AK,BelseyMJ.,Eur.J.Pharm.Biopharm.(2005)59(3),389-396

[0016] [NPL 3]Monoclonal antibodies:versatile platforms for cancerimmunotherapy.Weiner LM,Surana R,Wang S.,Nat.Rev.Immunol.(2010)10(5),317-327

[0017] [NPL 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

[0018] [NPL 5]ING-1,a monoclonal antibody targeting Ep-CAM in patients withadvanced adenocarcinomas.de Bono JS,Tolcher AW,Forero A,Vanhove GF,TakimotoC,Bauer RJ,Hammond LA,Patnaik A,White ML,Shen S,Khazaeli MB,Rowinsky EK,LoBuglio AF,Clin.Cancer Res.(2004)10(22),7555-7565

[0019] [NPL 6]Non-fucosylated therapeutic antibodies as next-generationtherapeutic antibodies.Satoh M,Iida S,Shitara K.,Expert Opin.Biol.Ther.(2006)6(11),1161-1173

[0020] [NPL 7]Optimizing engagement of the immune system by anti-tumorantibodies:an engineer's perspective.Desjarlais JR,Lazar GA,Zhukovsky EA,ChuSY.,Drug Discov.Today(2007)12(21-22),898-910

[0021] [NPL 8]Antibody-drug conjugates:targeted drug delivery forcancer.Alley SC,Okeley NM,Senter PD.,Curr.Opin.Chem.Biol.(2010)14(4),529-537

[0022] [NPL 9]BiTE:Teaching antibodies to engage T-cells for cancertherapy.Baeuerle PA,Kufer P,Bargou R.,Curr.Opin.Mol.Ther.(2009)11(1),22-30

[0023] [NPL 10]T cell-engaging BiTE antibodies specific for EGFR potentlyeliminate KRAS-and BRAF-mutated colorectal cancer cells.Lutterbuese R,Raum T,Kischel R,Hoffmann P,Mangold S,Rattel B,Friedrich M,Thomas O,Lorenczewski G,Rau D,Schaller E,Herrmann I,Wolf A,Urbig T,Baeuerle PA,Kufer P.,Proc.Natl.Acad.Sci.U.S.A.(2010)107(28),12605-12610

[0024] [NPL 11]Phase I trial with the CD44v6-targeting immunoconjugate bivatuzumab mertansine in head and neck squamous cell carcinoma. Riechelmann H, Sauter A, Golze W, Hanft G, Schroen C, Hoermann K, Erhardt T, Gronau S., Oral Oncol. (2008) 44(9), 823-829

[0025] [NPL 12]Ipilimumab in the treatment of melanoma. Trinh VA, Hwu WJ., Expert Opin. Biol. Ther., (2012) April 14 (doi:10.1517 / 14712598.2012.675325)

[0026] [NPL 13]IPILIMUMAB - A NOVEL IMMUNOMODULATING THERAPY CAUSING AUTOIMMUNE HYPOPHYSITIS: A CASE REPORT AND REVIEW. Juszczak A, Gupta A, Karavitaki N, Middleton MR, Grossman A., Eur. J. Endocrinol. (2012) April 10 (doi:10.1530 / EJE-12-0167)

[0027] [NPL 14]The Japanese experience with biologic therapies for rheumatoid arthritis. Takeuchi T, Kameda H., Nat. Rev. Rheumatol. (2010) 6(11), 644-652

[0028] [NPL 15]Current evidence for the management of rheumatoid arthritis with biological disease-modifying antirheumatic drugs: a systematic literature review informing the EULAR recommendations for the management of RA. Nam JL, Winthrop KL, van Vollenhoven RF, Pavelka K, Valesini G, Hensor EM, Worthy G, Landewe R, Smolen JS, Emery P, Buch MH., Ann. Rheum. Dis. (2010) 69(6), 976-986

[0029] [NPL 16]Antibody engineering for the development of therapeutic antibodies. Kim SJ, Park Y, Hong HJ., Mol. Cells. (2005) 20(1), 17-29

[0030] [NPL 17]Tumor-specific activation of an EGFR-targeting probody enhances therapeutic index. Desnoyers LR, Vasiljeva O, Richardson JH, Yang A, Menendez EE, Liang TW, Wong C, Bessette PH, Kamath K, Moore SJ, Sagert JG, Hostetter DR, Han F, Gee J, Flandez J, Markham K, Nguyen M, Krimm M, Wong KR, Liu S, Daugherty PS, West JW, Lowman HB. Sci Transl Med. 2013 October 16;5(207):207ra144.

[0031] [NPL 18]Probody therapeutics for targeting antibodies to diseased tissue. Polu KR, Lowman HB. Expert Opin Biol Ther. 2014 August; 14(8):1049-53. SUMMARY OF THE INVENTION

[0033] [Technical Issues]

[0034] The present inventors have considered that the technology of dissociating the masking peptide that inhibits the antigen-binding activity of the antibody by protease cleavage so that the antibody can restore its antigen-binding activity may cause side effects as described above. This is because since protease cleavage is irreversible, the antibody cleaved at the lesion site can be distributed to normal tissues through the bloodstream.

[0035] The present invention is based on such an idea. One object of the present invention is to provide a pharmaceutical composition with reduced side effects for the treatment of a disease, and an active ingredient thereof. Another object of the present invention is to provide a method for screening and preparing the pharmaceutical composition and the active ingredient.

[0036] [Solution to the problem]

[0037] The present inventors have conducted diligent research and have therefore developed a polypeptide comprising an antigen-binding domain and a transport portion having a longer half-life than the antigen-binding domain alone, wherein the transport portion has an inhibitory domain that inhibits the binding activity of the antigen-binding domain. It is believed that the use of this polypeptide can restore the antigen-binding activity of the antigen-binding domain in diseased tissues and exert the antigen-binding activity in diseased tissues. In addition, due to the difference in half-life between the polypeptide comprising the antigen-binding domain whose antigen-binding activity is inhibited and the polypeptide comprising the antigen-binding domain whose antigen-binding activity is restored, the systemic distribution of the activated form of the antigen-binding domain can be inhibited. In addition, the present inventors have discovered that the polypeptide or a pharmaceutical composition comprising the polypeptide can be used for the treatment of diseases, and have also discovered that: the polypeptide or the pharmaceutical composition can be used for the treatment of diseases comprising the administration of the polypeptide; the polypeptide can be used to prepare a medicament for the treatment of diseases. The present inventors further developed methods for screening and preparing polypeptides, methods for preparing and screening single-domain antibodies whose antigen-binding activity can be inhibited by association with a specific VL, VH or VHH, and libraries comprising single-domain antibodies whose antigen-binding activity can be inhibited by association with a specific VL, VH or VHH, thereby completing the present invention.

[0038] The present invention is based upon these findings and specifically encompasses the exemplary embodiments described below.

[0039] (1) A polypeptide comprising an antigen-binding domain and a transport moiety, wherein the transport moiety has an inhibitory domain that inhibits the antigen-binding activity of the antigen-binding domain, and the antigen-binding domain has a shorter half-life in blood than the transport moiety.

[0040] (2) The polypeptide according to (1), wherein the molecular weight of the antigen-binding domain is smaller than the molecular weight of the transport moiety.

[0041] (3) The polypeptide according to (1) or (2), wherein the molecular weight of the antigen-binding domain is 60 kDa or less.

[0042] (4) The polypeptide according to any one of (1) to (3), wherein the transport moiety has FcRn-binding activity, and the antigen-binding domain does not have FcRn-binding activity or has weaker FcRn-binding activity than that of the transport moiety.

[0043] (5) The polypeptide according to any one of (1) to (4), wherein the antigen-binding domain can be released from the polypeptide, and the antigen-binding domain released from the polypeptide has higher antigen-binding activity than before release.

[0044] (6) The polypeptide according to any one of (1) to (5), wherein the inhibitory domain of the transport moiety associates with the antigen-binding domain, thereby inhibiting the antigen-binding activity of the antigen-binding domain.

[0045] (7) The polypeptide according to (5), wherein the polypeptide comprises a cleavage site, wherein the cleavage site is cleaved so that the antigen-binding domain becomes releasable from the polypeptide.

[0046] (8) The polypeptide according to (6), wherein the polypeptide comprises a cleavage site, wherein the cleavage site is cleaved, thereby eliminating the association of the inhibitory domain of the trafficking moiety with the antigen-binding domain.

[0047] (9) The polypeptide according to (7) or (8), wherein the cleavage site comprises a protease cleavage sequence.

[0048] (10) The polypeptide according to (9), wherein the protease is a target tissue-specific protease.

[0049] (11) The polypeptide according to (10), wherein the target tissue is cancer tissue or inflammatory tissue.

[0050] (12) The polypeptide according to (9), wherein the protease is at least one protease selected from matriptase, urokinase (uPA) and metalloprotease.

[0051] (13) The polypeptide according to (12), wherein the protease is at least one protease selected from MT-SP1, uPA, MMP-2, MMP-9, ADAMTS5, MMP-7 and MMP-13.

[0052] (14) The polypeptide according to (9), wherein the protease cleavage sequence comprises one or more sequences selected from the group consisting of SEQ ID NOs: 12, 25, 26, 78 to 81, 83, 84, 91, 168 to 178, 193 to 195, 833 to 852 and 1062 to 1081 and the sequences shown in Table 1.

[0053] (15) The polypeptide according to any one of (9) to (14), wherein the first flexible linker is further attached to one end of the protease cleavage sequence.

[0054] (16) The polypeptide according to (15), wherein a second flexible linker is further attached to the other end of the protease cleavage sequence.

[0055] (17) The polypeptide according to (15), wherein the first flexible linker is a flexible linker composed of a glycine-serine polymer.

[0056] (18) The polypeptide according to (16), wherein the second flexible linker is a flexible linker composed of a glycine-serine polymer.

[0057] (19) The polypeptide according to any one of (1) to (18), wherein the antigen-binding domain comprises a single domain antibody or is a single domain antibody, and wherein the inhibitory domain of the transport moiety inhibits the antigen-binding activity of the single domain antibody.

[0058] (20) The polypeptide according to (19), wherein the single-domain antibody is VHH, VH having antigen-binding activity itself, or VL having antigen-binding activity itself.

[0059] (21) The polypeptide according to any one of (1) to (20), wherein the antigen-binding domain comprises a single domain antibody, and the inhibitory domain of the transport moiety is VHH, antibody VH or antibody VL, wherein the antigen-binding activity of the single domain antibody is inhibited by VHH, antibody VH or antibody VL.

[0060] (22) The polypeptide according to any one of (1) to (21), wherein the antigen-binding domain comprises a single domain antibody, and the inhibitory domain of the transport moiety is VHH, antibody VH or antibody VL, wherein the antigen-binding activity of the single domain antibody is inhibited by its association with VHH, antibody VH or antibody VL.

[0061] (23) The polypeptide according to any one of (19) to (22), wherein the single-domain antibody is VHH or VH that itself has antigen-binding activity, and the inhibitory domain of the transport portion is antibody VL, wherein the antigen-binding activity of the VHH or the VH that itself has antigen-binding activity is inhibited by its association with the antibody VL.

[0062] (24) The polypeptide according to any one of (19) to (23), wherein the single domain antibody is a VHH, wherein the VHH has an amino acid substitution at at least one position selected from amino acid positions 37, 44, 45 and 47 (all according to Kabat numbering).

[0063] (25) The polypeptide according to any one of (19) to (23), wherein the single domain antibody is a VHH, wherein the VHH contains at least one amino acid selected from amino acids 37V, 44G, 45L and 47W (all according to Kabat numbering).

[0064] (26) A polypeptide according to any one of (19) to (23), wherein the single domain antibody is a VHH, wherein the VHH contains at least one amino acid substitution selected from the group consisting of amino acid substitutions F37V, Y37V, E44G, Q44G, R45L, H45L, G47W, F47W, L47W, T47W and S47W (all according to Kabat numbering).

[0065] (27) A polypeptide according to any one of (19) to (23), wherein the single domain antibody is a VHH, wherein the VHH has an amino acid substitution at at least one group of positions selected from position 37 / 44, position 37 / 45, position 37 / 47, position 44 / 45, position 44 / 47, position 45 / 47, position 37 / 44 / 45, position 37 / 44 / 47, position 37 / 45 / 47, position 44 / 45 / 47 and position 37 / 44 / 45 / 47 (all according to Kabat numbering).

[0066] (28) A polypeptide according to any one of (19) to (23), wherein the single domain antibody is a VHH, wherein the VHH contains at least one set of amino acids selected from 37V / 44G, 37V / 45L, 37V / 47W, 44G / 45L, 44G / 47W, 45L / 47W, 37V / 44G / 45L, 37V / 44G / 47W, 37V / 45L / 47W, 44G / 45L / 47W and 37V / 44G / 45L / 47W (all according to Kabat numbering).

[0067] (29) The polypeptide according to any one of (19) to (23), wherein the single domain antibody is a VHH, wherein the VHH contains at least one set of amino acid substitutions selected from F37V / R45L, F37V / G47W, R45L / G47W and F37V / R45L / G47W (all according to Kabat numbering).

[0068] (30) The polypeptide according to any one of (19) to (22), wherein the single-domain antibody is a VL that itself has antigen-binding activity, and the inhibitory domain of the transport portion is an antibody VH, wherein the antigen-binding activity of the VL that itself has antigen-binding activity is inhibited by its association with the antibody VH.

[0069] (31) The polypeptide according to any one of (1) to (30), wherein the transport moiety has an FcRn-binding region.

[0070] (32) The polypeptide according to any one of (1) to (31), wherein the transport moiety comprises an antibody constant region.

[0071] (33) The polypeptide according to (32), wherein the antibody constant region of the transport moiety and the antigen-binding domain are fused with or without a linker.

[0072] (34) The polypeptide according to (32), wherein the transport portion comprises an antibody heavy chain constant region, wherein the antibody heavy chain constant region and the antigen-binding domain are fused with or without a linker.

[0073] (35) The polypeptide according to (32), wherein the transport portion comprises an antibody light chain constant region, wherein the antibody light chain constant region and the antigen-binding domain are fused with or without a linker.

[0074] (36) A polypeptide according to (34), wherein in the polypeptide, the N-terminus of the antibody heavy chain constant region of the transport portion and the C-terminus of the antigen-binding domain are fused via a linker or not, and the polypeptide further has a protease cleavage sequence, wherein the protease cleavage sequence is located within the sequence of the antigen-binding domain, or on a side closer to the antigen-binding domain than the amino acid at position 122 (EU numbering) in the antibody heavy chain constant region.

[0075] (37) A polypeptide according to (35), wherein in the polypeptide, the N-terminus of the antibody light chain constant region of the transport portion and the C-terminus of the antigen-binding domain are fused via a linker or not, and the polypeptide further has a protease cleavage sequence, wherein the protease cleavage sequence is located within the sequence of the antigen-binding domain, or on a side of the antibody light chain constant region that is closer to the antigen-binding domain than the amino acid at position 113 (EU numbering) (Kabat numbering position 113).

[0076] (38) A polypeptide according to any one of (33) to (35), wherein in the polypeptide, the N-terminus of the antibody constant region of the transport portion and the C-terminus of the antigen-binding domain are fused via a linker or not, the antigen-binding domain is a single domain antibody prepared from VH or VHH, and the polypeptide further has a protease cleavage sequence, wherein the protease cleavage sequence is located within the sequence of the antibody constant region, or on a side closer to the antibody constant region than the amino acid at position 109 (EU numbering) in the single domain antibody of the antigen-binding domain.

[0077] (39) A polypeptide according to (33), wherein in the polypeptide, the N-terminus of the antibody constant region of the transport portion and the C-terminus of the antigen-binding domain are fused via a linker or not, and the polypeptide further has a protease cleavage sequence, wherein the protease cleavage sequence is located near the boundary between the antigen-binding domain and the antibody constant region.

[0078] (40) A polypeptide according to (34), wherein in the polypeptide, the N-terminus of the antibody heavy chain constant region of the transport portion and the C-terminus of the antigen-binding domain are fused via a linker or not, and the polypeptide further has a protease cleavage sequence, wherein the protease cleavage sequence is located near the boundary between the antigen-binding domain and the antibody heavy chain constant region.

[0079] (41) A polypeptide according to (35), wherein in the polypeptide, the N-terminus of the antibody light chain constant region of the transport portion and the C-terminus of the antigen-binding domain are fused via a linker or not, and the polypeptide further has a protease cleavage sequence, wherein the protease cleavage sequence is located near the boundary between the antigen-binding domain and the antibody light chain constant region.

[0080] (42) A polypeptide according to (40), wherein the antigen-binding domain is a single domain antibody prepared from VH or VHH, and the protease cleavage sequence is located anywhere between amino acid position 109 (Kabat numbering) of the single domain antibody of the antigen-binding domain and amino acid position 122 (EU numbering) of the constant region of the antigen heavy chain.

[0081] (43) A polypeptide according to (41), wherein the antigen-binding domain is a single domain antibody prepared from VH or VHH, and the protease cleavage sequence is located at any position between the amino acid at position 109 (Kabat numbering) of the single domain antibody of the antigen-binding domain and the amino acid at position 113 (EU numbering) of the constant region of the antigen light chain (Kabat numbering position 113).

[0082] (44) A polypeptide according to (40), wherein the antigen binding domain is a single domain antibody prepared from VL, and the protease cleavage sequence is located at any position between the amino acid at position 104 (Kabat numbering) of the single domain antibody of the antigen binding domain and the amino acid at position 122 (EU numbering) of the constant region of the antibody heavy chain.

[0083] (45) The polypeptide according to (41), wherein the antigen-binding domain is a single domain antibody prepared from VL, and the protease cleavage sequence is located at any position between the amino acid at position 109 (Kabat numbering) of the single domain antibody of the antigen-binding domain and the amino acid at position 113 (EU numbering) of the antibody light chain constant region (Kabat numbering position 113).

[0084] (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.

[0085] (47) The polypeptide according to any one of (1) to (46), wherein the polypeptide is an IgG antibody-like molecule.

[0086] (48) The polypeptide according to any one of (1) to (47), wherein when the antigen-binding domain is measured in an unreleased state by using BLI (Biolayer Interferometry) (Octet), no binding of the antigen-binding domain to the antigen is observed.

[0087] (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.

[0088] (50) The polypeptide according to (49), wherein the second antigen-binding domain has an antigen-binding specificity different from that of the antigen-binding domain.

[0089] (51) The polypeptide according to (49) or (50), wherein the second antigen-binding domain comprises a second single domain antibody.

[0090] (52) A polypeptide according to (51), wherein the antigen-binding domain is a single domain antibody, the second antigen-binding domain is a second single domain antibody, and the antigen-binding domain and the second antigen-binding domain are capable of being released from the polypeptide, wherein the single domain antibody and the second single domain antibody form a bispecific antigen-binding molecule in the released state of the antigen-binding domain and the second antigen-binding domain.

[0091] (53) The polypeptide according to any one of (49) to (52), wherein the second antigen-binding domain is directed against HER2 or GPC3 as a target antigen.

[0092] (54) The polypeptide according to any one of (1) to (53), wherein the polypeptide further has an additional antigen-binding domain different from the antigen-binding domain, wherein the antigen-binding activity of the additional antigen-binding domain is also inhibited by its linkage to the transport portion of the polypeptide.

[0093] (55) The polypeptide according to (54), wherein the additional antigen-binding domain and the antigen-binding domain differ in antigen-binding specificity.

[0094] (56) The polypeptide according to any one of (1) to (55), wherein the antigen-binding domain is an antigen-binding domain against plexin A1, IL-6R, or CD3 as a target antigen.

[0095] (57) A pharmaceutical composition comprising the polypeptide according to any one of (1) to (56).

[0096] (58) A method for preparing the polypeptide according to any one of (1) to (56).

[0097] (59) The preparation method according to (58), comprising the following steps:

[0098] (a) obtaining a single domain antibody that binds to a target antigen;

[0099] (b) linking the single domain antibody obtained in step (a) to a transport moiety such that the antigen binding activity of the single domain antibody is inhibited by the inhibitory domain of the transport moiety to form a polypeptide precursor; and

[0100] (c) Introducing a protease cleavage sequence into the polypeptide precursor.

[0101] (60) The preparation method according to (58), comprising the following steps:

[0102] (a) obtaining a single domain antibody that binds to a target antigen;

[0103] (b) linking the single domain antibody obtained in step (a) to a transport moiety such that the antigen binding activity of the single domain antibody is inhibited by the inhibitory domain of the transport moiety to form a polypeptide precursor; and

[0104] (c) A protease cleavage sequence is introduced near the boundary between the single domain antibody and the transport moiety.

[0105] (61) The preparation method according to (58), comprising the following steps:

[0106] (a) obtaining a single domain antibody that binds to a target antigen; and

[0107] (b) linking the single domain antibody obtained in step (a) to a transport moiety via a protease cleavage sequence, such that the antigen binding activity of the single domain antibody is inhibited by the inhibitory domain of the transport moiety, to form a polypeptide.

[0108] (62) The preparation method according to any one of (59) to (61), further comprising the following steps:

[0109] (d) confirming that the binding activity of the single domain antibody incorporated into the polypeptide or polypeptide precursor to the target antigen is weakened or lost.

[0110] (63) The preparation method according to any one of (59) to (62), further comprising the following steps:

[0111] (e) releasing the single domain antibody by protease cleavage of the protease cleavage sequence and confirming that the released single domain antibody binds to the antigen.

[0112] (64) The preparation method according to (58), wherein the polypeptide is an IgG antibody-like molecule.

[0113] (65) The preparation method according to (64), comprising the following steps:

[0114] (a) obtaining a single domain antibody that binds to a target antigen;

[0115] (b) allowing the single domain antibody obtained in step (a) to associate with VL as a substitute for VH of an IgG antibody, or allowing the single domain antibody to associate with VH as a substitute for VL of an IgG antibody, so that the antigen-binding activity of the single domain antibody is suppressed, to form an IgG antibody-like molecule precursor comprising the single domain antibody; and

[0116] (c) Introducing a protease cleavage sequence into an IgG antibody-like molecule precursor comprising a single domain antibody.

[0117] (66) The preparation method according to (64), comprising the following steps:

[0118] (a) obtaining a single domain antibody that binds to a target antigen;

[0119] (b) allowing the single domain antibody obtained in step (a) to associate with VL as a substitute for VH of an IgG antibody, or allowing the single domain antibody to associate with VH as a substitute for VL of an IgG antibody, so that the antigen-binding activity of the single domain antibody is suppressed, to form an IgG antibody-like molecule precursor comprising the single domain antibody; and

[0120] (c) A protease cleavage sequence is introduced near the boundary between the single domain antibody and the antibody constant region in the IgG antibody-like molecule precursor.

[0121] (67) The preparation method according to (64), comprising the following steps:

[0122] (a) obtaining a single domain antibody that binds to a target antigen; and

[0123] (b) connecting the single domain antibody obtained in step (a) as a substitute for IgG antibody VH or VL to the IgG antibody heavy chain constant region or light chain constant region through a protease cleavage sequence, so that the antigen binding activity of the single domain antibody is inhibited to form an IgG antibody-like molecule comprising the single domain antibody.

[0124] (68) The preparation method according to any one of (65) to (67), further comprising the following steps:

[0125] (d) confirming that the binding activity of the single domain antibody contained in the IgG antibody-like molecule or the IgG antibody-like molecule precursor to the target antigen is weakened or lost.

[0126] (69) The preparation method according to any one of (65) to (68), further comprising the following steps:

[0127] (e) releasing the single domain antibody by protease cleavage of the protease cleavage sequence, and confirming that the released single domain antibody binds to the target antigen.

[0128] (70) The preparation method according to (64), comprising the following steps:

[0129] (a) replacing the amino acid residues involved in the association of the single domain antibody with the antibody VH, or replacing the amino acid residues involved in the association of the single domain antibody with the antibody VL, to prepare a variant single domain antibody that retains the binding activity of the single domain antibody to the target antigen;

[0130] (b) allowing the variant single domain antibody prepared in step (a) to associate with antibody VH, or allowing the variant single domain antibody to associate with antibody VL, such that the antigen-binding activity of the variant single domain antibody is inhibited, to form an IgG antibody-like molecule precursor comprising the variant single domain antibody; and

[0131] (c) Introducing a protease cleavage sequence into an IgG antibody-like molecule precursor comprising a variant single domain antibody.

[0132] (71) The preparation method according to (64), comprising the following steps:

[0133] (a) replacing the amino acid residues involved in association with the antibody VH in the single domain antibody, or replacing the amino acid residues involved in association with the antibody VL in the single domain antibody, to prepare a variant single domain antibody that retains the binding activity of the single domain antibody to the target antigen;

[0134] (b) allowing the variant single domain antibody prepared in step (a) to associate with antibody VH, or allowing the variant single domain antibody to associate with antibody VL, such that the antigen-binding activity of the variant single domain antibody is inhibited, to form an IgG antibody-like molecule precursor comprising the variant single domain antibody; and

[0135] (c) A protease cleavage sequence is introduced near the boundary between the variant single domain antibody and the constant region in the IgG antibody-like molecule precursor.

[0136] (72) The preparation method according to (64), comprising the following steps:

[0137] (a) replacing amino acid residues involved in association with antibody VH in a single domain antibody, or replacing amino acid residues involved in association with antibody VL in a single domain antibody, to prepare a variant single domain antibody that retains the binding activity of the single domain antibody to the target antigen; and

[0138] (b) linking the variant single domain antibody prepared in step (a) to the heavy chain constant region of an IgG antibody through a protease cleavage sequence, or linking the variant single domain antibody to the light chain constant region of an IgG antibody through a protease cleavage sequence, such that the antigen-binding activity of the variant single domain antibody is inhibited, to form an IgG antibody-like molecule comprising the variant single domain antibody.

[0139] (73) The preparation method according to any one of (70) to (72), further comprising the following steps:

[0140] (d) confirming that the binding activity of the variant single domain antibody contained in the IgG antibody-like molecule or the binding activity of the variant single domain antibody contained in the IgG antibody-like molecule precursor to the target antigen is weakened or lost.

[0141] (74) The preparation method according to any one of (70) to (73), further comprising the following steps:

[0142] (e) releasing the variant single domain antibody by cleaving the protease cleavage sequence with a protease, and confirming that the released variant single domain antibody binds to the target antigen.

[0143] (75) A polynucleotide encoding the polypeptide according to any one of (1) to (56).

[0144] (76) A vector comprising the polynucleotide according to (75).

[0145] (77) A host cell comprising the polynucleotide according to (75) or the vector according to (76).

[0146] (78) A method for producing the polypeptide according to any one of (1) to (56), comprising the step of culturing the host cell according to (77).

[0147] (79) A method for screening single-domain antibodies, the antigen-binding activity of which can be inhibited by its association with a specific VL, by its association with a specific VH, or by its association with a specific VHH.

[0148] (80) The screening method according to (79), wherein the method is a method for screening single-domain antibodies, wherein the antigen-binding activity of the single-domain antibody can be inhibited by its association with a specific VL.

[0149] (81) The screening method according to (80), comprising the following steps:

[0150] (a) obtaining a single domain antibody having target antigen binding activity;

[0151] (b) allowing the single domain antibody obtained in step (a) to associate with a specific VL; and

[0152] (c) confirming that the antigen-binding activity of the single domain antibody associated with the specific VL in step (b) is weakened or lost compared to that before the association.

[0153] (82) The screening method according to (80), comprising the following steps:

[0154] (a) allows the association of a single domain antibody with a specific VL;

[0155] (b) selecting an association product formed by VL and the single domain antibody based on the fact that the single domain antibody associated with the specific VL in step (a) has no binding activity against the antigen or has a binding activity of a predetermined value or lower; and

[0156] (c) confirming that the single domain antibody in the association product selected in step (b) has stronger antigen-binding activity in a state not associated with a specific VL than in a state associated with the VL.

[0157] (83) The screening method according to (79), wherein the method is a method for screening a single-domain antibody whose antigen-binding activity can be inhibited by its association with a specific VH.

[0158] (84) The screening method according to (83), comprising the following steps:

[0159] (a) obtaining a single domain antibody having target antigen binding activity;

[0160] (b) allowing the single domain antibody obtained in step (a) to associate with a specific VH; and

[0161] (c) confirming that the antigen-binding activity of the single-domain antibody associated with the specific VH in step (b) is weakened or lost compared to before the association.

[0162] (85) The screening method according to (83), comprising the following steps:

[0163] (a) allows the association of single-domain antibodies with specific VHs;

[0164] (b) selecting an association product formed by VH and the single domain antibody based on the fact that the single domain antibody associated with the specific VH in step (a) has no binding activity against the antigen or has a binding activity of a predetermined value or lower; and

[0165] (c) confirming that the single domain antibody in the association product selected in step (b) has stronger antigen-binding activity in a state not associated with a specific VH than in a state associated with the specific VH.

[0166] (86) The screening method according to (79), wherein the method is a method for screening a single-domain antibody whose antigen-binding activity can be inhibited by its association with a specific VHH.

[0167] (87) The screening method according to (86), comprising the following steps:

[0168] (a) obtaining a single domain antibody having target antigen binding activity;

[0169] (b) allowing the single domain antibody obtained in step (a) to associate with a specific VHH; and

[0170] (c) confirming that the antigen-binding activity of the single-domain antibody associated with the specific VHH in step (b) is weakened or lost compared to before the association.

[0171] (88) The screening method according to (86), comprising the following steps:

[0172] (a) allows the association of single-domain antibodies with specific VHHs;

[0173] (b) selecting an association product formed by VHH and the single domain antibody based on the fact that the single domain antibody associated with the specific VHH in step (a) has no binding activity against the antigen or has a binding activity of a predetermined value or lower; and

[0174] (c) confirming that the single domain antibody in the association product selected in step (b) has stronger antigen-binding activity in a state not associated with a specific VHH than in a state associated with the specific VHH.

[0175] (89) A method for preparing a single-domain antibody, wherein the antigen-binding activity of the single-domain antibody can be inhibited by its association with a specific VL, by its association with a specific VH, or by its association with a specific VHH.

[0176] (90) The preparation method according to (89), wherein the method is a method for preparing a single-domain antibody whose antigen-binding activity can be inhibited by its association with a specific VL.

[0177] (91) The manufacturing method according to (90), comprising the following steps:

[0178] (a) Substituting amino acid residues involved in association with the VL of the antibody in the single domain antibody to prepare a variant single domain antibody that retains the binding activity of the single domain antibody to the target antigen.

[0179] (92) The preparation method according to (91), further comprising the following steps:

[0180] (b) allowing the variant single domain antibody prepared in step (a) to associate with VL; and

[0181] (c) Confirming that the antigen-binding activity of the variant single-domain antibody associated with VL is weakened or lost compared to before association.

[0182] (93) The preparation method according to (89), wherein the method is a method for preparing a single-domain antibody whose antigen-binding activity can be inhibited by its association with a specific VH.

[0183] (94) The preparation method according to (93), comprising the following steps:

[0184] (a) Substituting amino acid residues involved in association with antibody VH in a single domain antibody to prepare a variant single domain antibody that retains the binding activity of the single domain antibody to the target antigen.

[0185] (95) The preparation method according to (94), further comprising the following steps:

[0186] (b) allowing the variant single domain antibody prepared in step (a) to associate with VH; and

[0187] (c) Confirming that the antigen-binding activity of the variant single-domain antibody associated with VH is weakened or lost compared to before association.

[0188] (96) The preparation method according to (89), wherein the method is a method for preparing a single-domain antibody whose antigen-binding activity can be inhibited by its association with a specific VHH.

[0189] (97) The preparation method according to (96), comprising the following steps:

[0190] (a) Substituting amino acid residues involved in VHH association in a single domain antibody to prepare a variant single domain antibody that retains the binding activity of the single domain antibody to the target antigen.

[0191] (98) The preparation method according to (97), further comprising the following steps:

[0192] (b) allowing the variant single domain antibody prepared in step (a) to associate with a VHH; and

[0193] (c) Confirming that the antigen-binding activity of the variant single-domain antibody in association with the VHH is weakened or lost compared to before association.

[0194] (99) A library of fusion polypeptides comprising a plurality of single domain antibodies, each single domain antibody being linked to a first association-maintaining domain, wherein the single domain antibodies include: a single domain antibody whose antigen-binding activity can be inhibited or lost by its association with a specific VL, a single domain antibody whose antigen-binding activity can be inhibited or lost by its association with a specific VH, or a single domain antibody whose antigen-binding activity can be inhibited or lost by its association with a specific VHH.

[0195] (100) The library according to (99), wherein the single domain antibody portion in each fusion polypeptide in the library includes a single domain antibody obtained from a Camelidae animal or a transgenic animal carrying a gene capable of producing the single domain antibody, or a humanized antibody thereof, a single domain antibody obtained by immunizing a Camelidae animal or a transgenic animal carrying a gene capable of producing the single domain antibody, or a humanized antibody thereof, or an artificially prepared single domain antibody derived from human antibody VH or VL.

[0196] (101) A library according to (99) or (100), which is a library comprising a plurality of single domain antibody fusion polypeptides, each single domain antibody being linked to a first association maintaining domain, wherein the single domain antibodies include single domain antibodies whose antigen binding activity can be inhibited or lost by their association with a specific VL.

[0197] (102) A library according to (99) or (100), which is a library comprising a plurality of single domain antibody fusion polypeptides, each single domain antibody being linked to a first association maintaining domain, wherein the single domain antibodies include single domain antibodies whose antigen binding activity can be inhibited or lost by their association with a specific VH.

[0198] (103) A library according to (99) or (100), which is a library comprising a plurality of single domain antibody fusion polypeptides, each single domain antibody being linked to a first association maintaining domain, wherein the single domain antibodies include single domain antibodies whose antigen binding activity can be inhibited or lost by their association with a specific VHH.

[0199] (104) A method for screening the library according to (99) or (100) to obtain a fusion polypeptide, wherein the fusion polypeptide comprises a single domain antibody whose antigen binding activity can be inhibited or may be lost by its association with a specific VL, a single domain antibody whose antigen binding activity can be inhibited or lost by its association with a specific VH, or a single domain antibody whose antigen binding activity can be inhibited or lost by its association with a specific VHH.

[0200] (105) A method for screening the library according to (101) to obtain a fusion polypeptide comprising a single domain antibody whose antigen binding activity can be inhibited or potentially lost by its association with a specific VL.

[0201] (106) The screening method according to (105), comprising the following steps:

[0202] (a) allowing for in vitro display of fusion polypeptides of the library;

[0203] (b) providing an association partner of a second association-maintaining domain fused to a specific VL;

[0204] (c) allowing each of the fusion polypeptides displayed in step (a) to associate with the association partner provided in step (b), and selecting a fusion polypeptide that does not bind to the antigen or has an antigen-binding activity of a predetermined value or lower in a state in which the single domain antibody is associated with the VL; and

[0205] (d) selecting, from the fusion polypeptides thus selected in step (c), a fusion polypeptide that binds to the antigen or has an antigen-binding activity of a predetermined value or higher in a state where the single domain antibody contained therein is not associated with the VL.

[0206] (107) The screening method according to (106), wherein the binding partner provided in step (b) further comprises a protease cleavage sequence, and step (d) comprises cleaving the binding partner by protease treatment, such that the association of the single domain antibody with the VL is abolished.

[0207] (108) The screening method according to (107), wherein the protease cleavage sequence of the association partner provided in step (b) is located near the boundary between the specific VL and the second association maintaining domain.

[0208] (109) The screening method according to (106), wherein the fusion polypeptide of the library further comprises a protease cleavage sequence, and step (d) comprises cleaving the fusion polypeptide by protease treatment so that the association of the single domain antibody with VL is abolished.

[0209] (110) The screening method according to (109), wherein the protease cleavage sequence contained in each fusion polypeptide is located near the boundary between the single domain antibody and the first association maintaining domain.

[0210] (111) The screening method according to (106), wherein step (d) comprises allowing the full length of the fusion polypeptide selected in step (c) or a portion thereof comprising a single domain antibody to be displayed again in vitro.

[0211] (112) The screening method according to (106), wherein step (d) includes allowing the full length of the fusion polypeptide selected in step (c) to be displayed again in vitro and selecting a fusion polypeptide that binds to the antigen or has an antigen-binding activity of a predetermined value or higher when associated only with the second association maintaining domain.

[0212] (113) A method for screening the library according to (102) to obtain a fusion polypeptide comprising a single domain antibody whose antigen binding activity can be inhibited or potentially lost by its association with a specific VH.

[0213] (114) The screening method according to (113), comprising the following steps:

[0214] (a) allowing for in vitro display of fusion polypeptides of the library;

[0215] (b) providing an association partner for a second association-maintaining domain fused to a specific VH;

[0216] (c) allowing each of the fusion polypeptides displayed in step (a) to associate with the association partner provided in step (b), and selecting a fusion polypeptide that does not bind to the antigen or has an antigen-binding activity of a predetermined value or lower in a state in which the single domain antibody is associated with the VH; and

[0217] (d) selecting, from the fusion polypeptides thus selected in step (c), a fusion polypeptide that binds to the antigen or has an antigen-binding activity of a predetermined value or higher in a state where the single domain antibody contained therein is not associated with VH.

[0218] (115) The screening method according to (114), wherein the binding partner provided in step (b) further comprises a protease cleavage sequence, and step (d) comprises cleaving the binding partner by protease treatment, such that the association of the single domain antibody with VH is abolished.

[0219] (116) The screening method according to (115), wherein the protease cleavage sequence of the association partner provided in step (b) is located near the boundary between the specific VH and the second association maintaining domain.

[0220] (117) The screening method according to (114), wherein the fusion polypeptide of the library further comprises a protease cleavage sequence, and step (d) comprises cleaving the fusion polypeptide by protease treatment so that the association of the single domain antibody with VH is abolished.

[0221] (118) The screening method according to (117), wherein the protease cleavage sequence contained in each fusion polypeptide is located near the boundary between the single domain antibody and the first association maintaining domain.

[0222] (119) The screening method according to (114), wherein step (d) comprises allowing the full length of the fusion polypeptide selected in step (c) or a portion thereof comprising a single domain antibody to be displayed again in vitro.

[0223] (120) The screening method according to (114), wherein step (d) includes allowing the full length of the fusion polypeptide selected in step (c) to be displayed again in vitro and selecting a fusion polypeptide that binds to the antigen or has an antigen-binding activity of a predetermined value or higher when associated only with the second association maintaining domain.

[0224] (121) A method for screening the library according to (103) to obtain a fusion polypeptide comprising a single domain antibody whose antigen binding activity can be inhibited or potentially lost by its association with a specific VHH.

[0225] (122) The screening method according to (121), comprising the following steps:

[0226] (a) allowing for in vitro display of fusion polypeptides of the library;

[0227] (b) providing an association partner to which a second association-maintaining domain is fused to a specific VHH;

[0228] (c) allowing each of the fusion polypeptides displayed in step (a) to associate with the association partner provided in step (b), and selecting a fusion polypeptide that does not bind to the antigen or has an antigen-binding activity of a predetermined value or lower in a state in which the single domain antibody is associated with a specific VHH; and

[0229] (d) selecting, from the fusion polypeptides thus selected in step (c), a fusion polypeptide that binds to the antigen or has an antigen-binding activity of a predetermined value or higher in a state where the single domain antibody contained therein is not associated with a VHH.

[0230] (123) The screening method according to (122), wherein the binding partner provided in step (b) further comprises a protease cleavage sequence, and step (d) comprises cleaving the binding partner by protease treatment, such that the association of the single domain antibody with the VHH is abolished.

[0231] (124) The screening method according to (123), wherein the protease cleavage sequence of the association partner provided in step (b) is located near the boundary between the specific VHH and the second association maintaining domain.

[0232] (125) The screening method according to (122), wherein the fusion polypeptide of the library further comprises a protease cleavage sequence, and step (d) comprises cleaving the fusion polypeptide by protease treatment so that the association of the single domain antibody with the VHH is abolished.

[0233] (126) The screening method according to (125), wherein the protease cleavage sequence contained in each fusion polypeptide is located near the boundary between the single domain antibody and the first association maintaining domain.

[0234] (127) The screening method according to (122), wherein step (d) comprises allowing the full length of the fusion polypeptide selected in step (c) or a portion thereof comprising a single domain antibody to be displayed again in vitro.

[0235] (128) The screening method according to (122), wherein step (d) includes allowing the full length of the fusion polypeptide selected in step (c) to be displayed again in vitro and selecting a fusion polypeptide that binds to the antigen or has an antigen-binding activity of a predetermined value or higher when associated only with the second association maintaining domain.

[0236] (129) The screening method according to any one of (106) to (112), (114) to (120) and (122) to (128), wherein the step of providing the association partner in step (b) is a step of displaying the association partner and the fusion polypeptide together.

[0237] (130) The library according to any one of (99) to (103), wherein the first association maintaining domain comprises an IgG antibody CH1 domain or an antibody light chain constant region.

[0238] (131) The screening method according to any one of (106) to (112), (114) to (120) and (122) to (128), wherein the first association-maintaining domain comprises an IgG antibody CH1 domain and the second association-maintaining domain comprises an antibody light chain constant region.

[0239] (132) The screening method according to any one of (106) to (112), (114) to (120) and (122) to (128), wherein the first association-maintaining domain comprises an antibody light chain constant region and the second association-maintaining domain comprises an IgG antibody CH1 domain.

[0240] (133) The screening method according to (105), comprising the following steps:

[0241] (a) allowing for in vitro display of fusion polypeptides of the library;

[0242] (b) providing an association partner of a second association-maintaining domain fused to a specific VL;

[0243] (c) selecting a fusion polypeptide comprising a single domain antibody that binds to an antigen or has an antigen-binding activity of a predetermined value or higher; and

[0244] (d) allowing the fusion polypeptide thus selected in step (c) to associate with the association partner provided in step (b), and selecting a fusion polypeptide that does not bind to the antigen or has an antigen-binding activity of a predetermined value or lower in a state in which the single domain antibody is associated with VL.

[0245] (134) The screening method according to (129), wherein step (d) comprises allowing the fusion polypeptide selected in step (c) to be displayed again in vitro.

[0246] (135) The screening method according to (133), wherein step (c) comprises allowing the fusion polypeptide to associate only with the second association maintaining domain, or confirming that the fusion polypeptide associates only with the second association maintaining domain, to bind to the antigen of the single domain antibody contained in the fusion polypeptide.

[0247] (136) The screening method according to (113), comprising the following steps:

[0248] (a) allowing for in vitro display of fusion polypeptides of the library;

[0249] (b) providing an association partner for a second association-maintaining domain fused to a specific VH;

[0250] (c) selecting a fusion polypeptide comprising a single domain antibody that binds to the antigen or has an antigen-binding activity of a predetermined value or higher; and

[0251] (d) allowing the fusion polypeptide thus selected in step (c) to associate with the association partner provided in step (b), and selecting a fusion polypeptide that does not bind to the antigen or has an antigen-binding activity of a predetermined value or lower in a state in which the single domain antibody is associated with VH.

[0252] (137) The screening method according to (136), wherein step (d) comprises allowing the fusion polypeptide selected in step (c) to be displayed again in vitro.

[0253] (138) The screening method according to (136), wherein step (c) comprises allowing the fusion polypeptide to associate only with the second association-maintaining domain, or confirming antigen binding of the single domain antibody contained in the fusion polypeptide that associates only with the second association-maintaining domain.

[0254] (139) The screening method according to (121), comprising the following steps:

[0255] (a) allowing for in vitro display of fusion polypeptides of the library;

[0256] (b) providing an association partner to which a second association-maintaining domain is fused to a specific VHH;

[0257] (c) selecting a fusion polypeptide comprising a single domain antibody that binds to the antigen or has an antigen-binding activity of a predetermined value or higher; and

[0258] (d) allowing the fusion polypeptide thus selected in step (c) to associate with the association partner provided in step (b), and selecting a fusion polypeptide that does not bind to the antigen or has an antigen-binding activity of a predetermined value or lower in a state in which the single domain antibody and VHH are associated.

[0259] (140) The screening method according to (139), wherein step (d) comprises allowing the fusion polypeptide selected in step (c) to be displayed again in vitro.

[0260] (141) The screening method according to (139), wherein step (c) comprises allowing the fusion polypeptide to associate only with the second association maintaining domain, or confirming antigen binding of the single domain antibody contained in the fusion polypeptide that associates only with the second association maintaining domain.

[0261] (142) The screening method according to any one of (133) to (141), wherein the step of allowing the fusion polypeptide to associate with the association partner in step (d) is a step of allowing the association partner and the fusion polypeptide to be displayed together.

[0262] (143) The screening method according to any one of (133) to (142), wherein the first association-maintaining domain comprises an IgG antibody CH1 domain, and the second association-maintaining domain comprises an antibody light chain constant region.

[0263] (144) The screening method according to any one of (133) to (142), wherein the first association-maintaining domain comprises an antibody light chain constant region, and the second association-maintaining domain comprises an IgG antibody CH1 domain.

[0264] Specifically, the present invention may also cover the following exemplary embodiments.

[0265] (B1) A polypeptide comprising an antigen-binding domain and a transport portion, wherein the transport portion has an inhibitory domain that inhibits the antigen-binding activity of the antigen-binding domain, and wherein the polypeptide has a protease cleavage sequence comprising one or more sequences selected from the group consisting of SEQ ID NOs: 833 to 852 and SEQ ID NOs: 1062 to 1081 and the sequences described in Table 1.

[0266] (B2) The polypeptide according to (B1), wherein the inhibition of the antigen-binding activity of the antigen-binding domain by the inhibitory domain in a state where the protease cleavage sequence has been cleaved by a protease is weaker than the inhibition of the antigen-binding activity of the antigen-binding domain by the inhibitory domain in a state where the protease cleavage sequence has been cleaved by a protease.

[0267] (B3) The polypeptide according to (B1) or (B2), wherein the antigen-binding domain has a shorter half-life in blood than the transport moiety.

[0268] (B4) The polypeptide according to any one of (B1) to (B3), wherein the molecular weight of the antigen-binding domain is smaller than the molecular weight of the transport moiety.

[0269] (B5) The polypeptide according to any one of (B1) to (B4), wherein the molecular weight of the antigen-binding domain is 60 kDa or less.

[0270] (B6) The polypeptide according to any one of (B1) to (B5), wherein the transport moiety has FcRn-binding activity, and the antigen-binding domain does not have FcRn-binding activity or has weaker FcRn-binding activity than the transport moiety.

[0271] (B7) The polypeptide according to any one of (B1) to (B6), wherein the antigen-binding domain can be released from the polypeptide, and the antigen-binding domain has higher antigen-binding activity when released from the polypeptide than when not released from the polypeptide.

[0272] (B8) The polypeptide according to any one of (B1) to (B7), wherein the antigen-binding activity of the antigen-binding domain is inhibited by association of the inhibitory domain of the trafficking moiety with the antigen-binding domain.

[0273] (B9) The polypeptide according to (B7), wherein the protease cleavage sequence is cleaved by a protease, so that the antigen-binding domain becomes capable of being released from the polypeptide.

[0274] (B10) The polypeptide of (B8), wherein the protease cleavage sequence is cleaved by a protease, thereby eliminating the association of the inhibitory domain of the transport moiety with the antigen binding domain.

[0275] (B11) The polypeptide according to any one of (B1) to (B10), wherein the protease is a target tissue-specific protease.

[0276] (B12) The polypeptide according to (B11), wherein the target tissue is cancer tissue or inflammatory tissue, and the protease is a cancer tissue-specific protease or an inflammatory tissue-specific protease.

[0277] (B13) The polypeptide according to any one of (B1) to (B12), wherein the protease is at least one protease selected from matriptase, urokinase (uPA), and metalloprotease.

[0278] (B14) The polypeptide according to any one of (B1) to (B12), wherein the protease is at least one protease selected from MT-SP1, uPA, MMP-2, MMP-9, ADAMTS5, MMP-7 and MMP-13.

[0279] (B15) The polypeptide according to any one of (B1) to (B14), wherein a first flexible linker is further attached to one end of the protease cleavage sequence.

[0280] (B16) The polypeptide according to (B15), wherein the first flexible linker is a flexible linker composed of a glycine-serine polymer.

[0281] (B17) The polypeptide according to (B15) or (B16), wherein a second flexible linker is further attached to the other end of the protease cleavage sequence.

[0282] (B18) The polypeptide according to (B17), wherein the second flexible linker is a flexible linker composed of a glycine-serine polymer.

[0283] (B19) The polypeptide of any one of (B1) to (B18), wherein the antigen binding domain comprises a single domain antibody or is a single domain antibody, and wherein the inhibitory domain of the transport moiety inhibits the antigen binding activity of the single domain antibody.

[0284] (B20) The polypeptide according to (B19), wherein the single domain antibody is VHH, VH having antigen-binding activity itself, or VL having antigen-binding activity itself.

[0285] (B21) A polypeptide according to any one of (B1) to (B20), wherein the antigen-binding domain comprises a single domain antibody, wherein the inhibitory domain of the transport moiety is VHH, antibody VH or antibody VL, and wherein the antigen-binding activity of the single domain antibody is inhibited by the VHH, antibody VH or antibody VL.

[0286] (B22) The polypeptide of any one of (B1) to (B21), wherein the antigen-binding domain comprises a single domain antibody, wherein the inhibitory domain of the transport moiety is VHH, antibody VH, or antibody VL, and wherein the antigen-binding activity of the single domain antibody is inhibited by its association with the VHH, antibody VH, or antibody VL.

[0287] (B23) The polypeptide according to any one of (B19) to (B22), wherein the single-domain antibody is a VHH or a VH that itself has antigen-binding activity, wherein the inhibitory domain of the transport portion is an antibody VL, and wherein the antigen-binding activity of the VHH or the VH that itself has antigen-binding activity is inhibited by its association with the antibody VL.

[0288] (B24) The polypeptide of any one of (B19) to (B23), wherein the single domain antibody is a VHH, and wherein the VHH has an amino acid substitution at at least one position selected from the amino acids at positions 37, 44, 45 and 47 (all according to Kabat numbering).

[0289] (B25) The polypeptide according to any one of (B19) to (B23), wherein the single domain antibody is a VHH, and wherein the VHH comprises at least one amino acid selected from amino acids 37V, 44G, 45L and 47W (all according to Kabat numbering).

[0290] (B26) The polypeptide of any one of (B19) to (B23), wherein the single domain antibody is a VHH, and wherein the VHH comprises at least one amino acid substitution selected from the group consisting of amino acid substitutions F37V, Y37V, E44G, Q44G, R45L, H45L, G47W, F47W, L47W, T47W and S47W (all according to Kabat numbering).

[0291] (B27) A polypeptide according to any one of (B19) to (B23), wherein the single domain antibody is a VHH, and wherein the VHH has amino acid substitutions at at least one group of positions selected from the following: position 37 / 44, position 37 / 45, position 37 / 47, position 44 / 45, position 44 / 47, position 45 / 47, position 37 / 44 / 45, position 37 / 44 / 47, position 37 / 45 / 47, position 44 / 45 / 47 and position 37 / 44 / 45 / 47 (all according to Kabat numbering).

[0292] (B28) A polypeptide according to any one of (B19) to (B23), wherein the single domain antibody is a VHH, and wherein the VHH comprises at least one group of amino acids selected from the group consisting of 37V / 44G, 37V / 45L, 37V / 47W, 44G / 45L, 44G / 47W, 45L / 47W, 37V / 44G / 45L, 37V / 44G / 47W, 37V / 45L / 47W, 44G / 45L / 47W and 37V / 44G / 45L / 47W (all according to Kabat numbering).

[0293] (B29) The polypeptide of any one of (B19) to (B23), wherein the single domain antibody is a VHH, and wherein the VHH comprises at least one set of amino acid substitutions selected from the group consisting of: F37V / R45L, F37V / G47W, R45L / G47W, and F37V / R45L / G47W (all according to Kabat numbering).

[0294] (B30) A polypeptide according to any one of (B19) to (B22), wherein the single-domain antibody is a VL that itself has antigen-binding activity, wherein the inhibitory domain of the transport portion is an antibody VH, and wherein the antigen-binding activity of the VL that itself has antigen-binding activity is inhibited by its association with the antibody VH.

[0295] (B31) The polypeptide according to any one of (B1) to (B30), wherein the transport moiety has an FcRn binding region.

[0296] (B32) The polypeptide of any one of (B1) to (B31), wherein the transport moiety comprises an antibody constant region.

[0297] (B33) The polypeptide according to (B32), wherein the antibody constant region of the transport moiety and the antigen-binding domain are fused with or without a linker.

[0298] (B34) The polypeptide according to (B32), wherein the transport moiety comprises an antibody heavy chain constant region, and wherein the antibody heavy chain constant region and the antigen binding domain are fused with or without a linker.

[0299] (B35) The polypeptide according to (B32), wherein the transport moiety comprises an antibody light chain constant region, and wherein the antibody light chain constant region and the antigen binding domain are fused with or without a linker.

[0300] (B36) A polypeptide according to (B34), wherein the N-terminus of the antibody heavy chain constant region of the transport portion and the C-terminus of the antigen-binding domain are fused with or without a linker, and wherein the protease cleavage sequence is located within the sequence of the antigen-binding domain, or on a side of the antigen-binding domain closer to the amino acid at position 122 (EU numbering) in the antibody heavy chain constant region.

[0301] (B37) A polypeptide according to (B35), wherein the N-terminus of the antibody light chain constant region of the transport portion and the C-terminus of the antigen-binding domain are fused with a linker or without a linker, and wherein the protease cleavage sequence is located within the sequence of the antigen-binding domain, or on a side of the antigen-binding domain closer to the amino acid at position 113 (EU numbering) (Kabat numbering position 113) in the antibody light chain constant region.

[0302] (B38) A polypeptide according to (B33) to (B36), wherein the N-terminus of the antibody constant region of the transport portion and the C-terminus of the antigen-binding domain are fused with or without a linker, wherein the antigen-binding domain is a single domain antibody prepared from VH, or is a VHH, and wherein the protease cleavage sequence is located within the sequence of the antibody constant region, or on a side of the antibody constant region closer to the amino acid at position 109 (Kabat numbering) in the single domain antibody of the antigen-binding domain.

[0303] (B39) A polypeptide according to (B33), wherein the N-terminus of the antibody constant region of the transport portion and the C-terminus of the antigen binding domain are fused with or without a linker, and wherein a protease cleavage sequence is located near the boundary between the antigen binding domain and the antibody constant region.

[0304] (B40) A polypeptide according to (B34), wherein the N-terminus of the antibody heavy chain constant region of the transport portion and the C-terminus of the antigen binding domain are fused with or without a linker, and wherein a protease cleavage sequence is located near the boundary between the antigen binding domain and the antibody heavy chain constant region.

[0305] (B41) A polypeptide according to (B35), wherein the N-terminus of the antibody light chain constant region of the transport portion and the C-terminus of the antigen binding domain are fused with or without a linker, and wherein a protease cleavage sequence is located near the boundary between the antigen binding domain and the antibody light chain constant region.

[0306] (B42) A polypeptide according to (B40), wherein the antigen binding domain is a single domain antibody prepared from VH, or is a VHH, and wherein the protease cleavage sequence is located between the amino acid at position 109 (Kabat numbering) of the single domain antibody of the antigen binding domain and the amino acid at position 122 (EU numbering) of the constant region of the antibody heavy chain.

[0307] (B43) A polypeptide according to (B41), wherein the antigen-binding domain is a single domain antibody prepared from VH, or is a VHH, and wherein the protease cleavage sequence is located between the amino acid at position 109 (Kabat numbering) of the single domain antibody of the antigen-binding domain and the amino acid at position 113 (EU numbering) (Kabat numbering position 113) of the antibody light chain constant region.

[0308] (B44) A polypeptide according to (B40), wherein the antigen binding domain is a single domain antibody prepared from VL, and wherein the protease cleavage sequence is located between the amino acid at position 104 (Kabat numbering) of the single domain antibody of the antigen binding domain and the amino acid at position 122 (EU numbering) of the constant region of the antibody heavy chain.

[0309] (B45) A polypeptide according to (B41), wherein the antigen binding domain is a single domain antibody prepared from VL, and wherein the protease cleavage sequence is located between the amino acid at position 109 (Kabat numbering) of the single domain antibody of the antigen binding domain and the amino acid at position 113 (EU numbering) (Kabat numbering position 113) of the antibody light chain constant region.

[0310] (B46) The polypeptide according to any one of (B32) to (B45), wherein the antibody constant region of the polypeptide is an IgG antibody constant region.

[0311] (B47) The polypeptide according to any one of (B1) to (B46), wherein the polypeptide is an IgG antibody-like molecule.

[0312] (B48) The polypeptide according to any one of (B1) to (B47), wherein no binding of the antigen-binding domain to the antigen is observed when the antigen-binding domain is measured in an unreleased state by using biolayer interferometry (BLI) (Octet).

[0313] (B49) The polypeptide according to any one of (B1) to (B48), wherein a second antigen-binding domain is further linked to the antigen-binding domain.

[0314] (B50) The polypeptide according to (B49), wherein the second antigen-binding domain has a different antigen-binding specificity than the antigen-binding domain.

[0315] (B51) The polypeptide according to (B49) or (B50), wherein the second antigen-binding domain comprises a second single domain antibody.

[0316] (B52) A polypeptide according to (B51), wherein the antigen-binding domain is a single domain antibody, wherein the second antigen-binding domain is a second single domain antibody, wherein the antigen-binding domain and the second antigen-binding domain are capable of being released from the polypeptide, and wherein the single domain antibody and the second single domain antibody form a bispecific antigen-binding molecule in a state in which the antigen-binding domain and the second antigen-binding domain are released.

[0317] (B53) The polypeptide according to any one of (B49) to (B52), wherein the second antigen-binding domain is directed against HER2 or GPC3 as a target antigen.

[0318] (B54) A polypeptide according to any one of (B1) to (B53), wherein the polypeptide further has an additional antigen-binding domain different from the antigen-binding domain, wherein the antigen-binding activity of the additional antigen-binding domain is also inhibited by its connection to the transport portion of the polypeptide.

[0319] (B55) The polypeptide according to (B54), wherein the additional antigen-binding domain and the antigen-binding domain have different antigen-binding specificities.

[0320] (B56) The polypeptide according to any one of (B1) to (B55), wherein the antigen-binding domain is an antigen-binding domain against Plexin A1, IL-6R, or CD3 as a target antigen.

[0321] (B57) A pharmaceutical composition comprising the polypeptide according to any one of (B1) to (B56).

[0322] (B58) A method for producing the polypeptide according to any one of (B1) to (B56).

[0323] (B59) The preparation method according to (B58), comprising the following steps:

[0324] (a) obtaining a single domain antibody that binds to a target antigen;

[0325] (b) linking the single domain antibody obtained in step (a) to a transport moiety such that the antigen binding activity of the single domain antibody is inhibited by the inhibitory domain of the transport moiety to form a polypeptide precursor; and

[0326] (c) introducing a protease cleavage sequence into the polypeptide precursor.

[0327] (B60) The preparation method according to (B58), comprising the following steps:

[0328] (a) obtaining a single domain antibody that binds to a target antigen;

[0329] (b) linking the single domain antibody obtained in step (a) to a transport moiety such that the antigen binding activity of the single domain antibody is inhibited by the inhibitory domain of the transport moiety to form a polypeptide precursor; and

[0330] (c) introducing a protease cleavage sequence near the boundary between the single domain antibody and the transport portion, wherein the protease cleavage sequence comprises one or more sequences selected from the group consisting of SEQ ID NOs: 833 to 852 and SEQ ID NOs: 1062 to 1081 and the sequences described in Table 1.

[0331] (B61) The preparation method according to (B58), comprising the following steps:

[0332] (a) obtaining a single domain antibody that binds to a target antigen; and

[0333] (b) linking the single domain antibody obtained in step (a) to a transport moiety via a protease cleavage sequence, such that the antigen-binding activity of the single domain antibody is inhibited by the inhibitory domain of the transport moiety to form a polypeptide, wherein the protease cleavage sequence comprises one or more sequences selected from the group consisting of SEQ ID NOs: 833 to 852 and SEQ ID NOs: 1062 to 1081 and the sequences described in Table 1.

[0334] (B62) The preparation method according to any one of (B59) to (B61), further comprising the following steps:

[0335] (d) confirming that the binding activity of the single domain antibody containing the polypeptide or polypeptide precursor to the target antigen is weakened or lost.

[0336] (B63) The preparation method according to any one of (B59) to (B62), further comprising the following steps:

[0337] (e) releasing the single domain antibody by cleaving the protease cleavage sequence with a protease, and confirming that the released single domain antibody binds to the antigen.

[0338] (B64) The preparation method according to (B58), wherein the polypeptide is an IgG antibody-like molecule.

[0339] (B65) The preparation method according to (B64), comprising the following steps:

[0340] (a) obtaining a single domain antibody that binds to a target antigen;

[0341] (b) allowing the single domain antibody obtained in step (a) to associate with VL as a substitute for VH of an IgG antibody, or allowing the single domain antibody to associate with VH as a substitute for VL of an IgG antibody, so that the antigen-binding activity of the single domain antibody is suppressed, to form an IgG antibody-like molecule precursor comprising the single domain antibody; and

[0342] (c) introducing a protease cleavage sequence into the IgG antibody-like molecule precursor comprising a single domain antibody, wherein the protease cleavage sequence comprises one or more sequences selected from the group consisting of SEQ ID NOs: 833 to 852 and SEQ ID NOs: 1062 to 1081 and the sequences described in Table 1.

[0343] (B66) The preparation method according to (B64), comprising the following steps:

[0344] (a) obtaining a single domain antibody that binds to a target antigen;

[0345] (b) allowing the single domain antibody obtained in step (a) to associate with VL as a substitute for VH of an IgG antibody, or allowing the single domain antibody to associate with VH as a substitute for VL of an IgG antibody, so that the antigen-binding activity of the single domain antibody is suppressed, to form an IgG antibody-like molecule precursor comprising the single domain antibody; and

[0346] (c) introducing a protease cleavage sequence near the boundary between the single domain antibody and the antibody constant region in the IgG antibody-like molecule precursor, wherein the protease cleavage sequence comprises one or more sequences selected from the group consisting of SEQ ID NOs: 833 to 852 and SEQ ID NOs: 1062 to 1081 and the sequences described in Table 1.

[0347] (B67) The preparation method according to (B64), comprising the following steps:

[0348] (a) obtaining a single domain antibody that binds to a target antigen; and

[0349] (b) using the single domain antibody obtained in step (a) as a substitute for IgG antibody VH or VL, and connecting it to the IgG antibody heavy chain constant region or light chain constant region through a protease cleavage sequence, so that the antigen binding activity of the single domain antibody is inhibited to form an IgG antibody-like molecule comprising the single domain antibody, wherein the protease cleavage sequence comprises one or more sequences selected from the following: sequences of SEQ ID NOs: 833 to 852 and SEQ ID NOs: 1062 to 1081 and the sequences described in Table 1.

[0350] (B68) The preparation method according to any one of (B65) to (B67), further comprising the following steps:

[0351] (d) confirming that the binding activity of the single domain antibody introduced into the IgG antibody-like molecule or the IgG antibody-like molecule precursor against the target antigen is weakened or lost.

[0352] (B69) The preparation method according to any one of (B65) to (B68), further comprising the following steps:

[0353] (e) releasing the single domain antibody by cleaving the protease cleavage sequence with a protease, and confirming that the released single domain antibody binds to the target antigen.

[0354] (B70) The preparation method according to (B64), comprising the following steps:

[0355] (a) replacing the amino acid residues involved in association with the antibody VH in the single domain antibody, or replacing the amino acid residues involved in association with the antibody VL in the single domain antibody, to prepare a variant single domain antibody that retains the binding activity of the single domain antibody to the target antigen;

[0356] (b) allowing the variant single domain antibody prepared in step (a) to associate with antibody VH, or by allowing the variant single domain antibody to associate with antibody VL, such that the antigen-binding activity of the variant single domain antibody is inhibited, to form an IgG antibody-like molecule precursor containing the variant single domain antibody; and

[0357] (c) introducing a protease cleavage sequence into the IgG antibody-like molecule precursor having the variant single domain antibody, wherein the protease cleavage sequence comprises one or more sequences selected from the group consisting of SEQ ID NOs: 833 to 852 and SEQ ID NOs: 1062 to 1081 and the sequences described in Table 1.

[0358] (B71) The preparation method according to (B64), comprising the following steps:

[0359] (a) replacing the amino acid residues involved in association with the antibody VH in the single domain antibody, or replacing the amino acid residues involved in association with the antibody VL in the single domain antibody, to prepare a variant single domain antibody that retains the binding activity of the single domain antibody to the target antigen;

[0360] (b) allowing the variant single domain antibody prepared in step (a) to associate with antibody VH, or allowing the variant single domain antibody to associate with antibody VL, such that the antigen-binding activity of the variant single domain antibody is inhibited, to form an IgG antibody-like molecule precursor containing the variant single domain antibody; and

[0361] (c) introducing a protease cleavage sequence near the boundary between the variant single domain antibody and the constant region in the IgG antibody-like molecule precursor, wherein the protease cleavage sequence comprises one or more sequences selected from the following: sequences of SEQ ID NO: 833 to 852 and SEQ ID NO: 1062 to 1081 and the sequences described in Table 1.

[0362] (B72) The preparation method according to (B64), comprising the following steps:

[0363] (a) replacing amino acid residues involved in association with antibody VH in a single domain antibody, or replacing amino acid residues involved in association with antibody VL in a single domain antibody, to prepare a variant single domain antibody that retains the binding activity of the single domain antibody to the target antigen; and

[0364] (b) linking the variant single-domain antibody prepared in step (a) to the heavy chain constant region of an IgG antibody via a protease cleavage sequence, or linking the variant single-domain antibody to the light chain constant region of an IgG antibody via a protease cleavage sequence, thereby inhibiting the antigen-binding activity of the variant single-domain antibody to form an IgG antibody-like molecule containing the variant single-domain antibody, wherein the protease cleavage sequence comprises one or more sequences selected from the following: sequences of SEQ ID NOs: 833 to 852 and SEQ ID NOs: 1062 to 1081 and the sequences described in Table 1.

[0365] (B73) The preparation method according to any one of (B70) to (B72), further comprising the following steps:

[0366] (d) confirming that the variant single domain antibody introduced into the IgG antibody-like molecule or IgG antibody-like molecule precursor has weakened or lost binding activity against the target antigen.

[0367] (B74) The preparation method according to any one of (B70) to (B73), further comprising the following steps:

[0368] (e) releasing the variant single domain antibody by cleaving the protease cleavage sequence with a protease, and confirming that the released variant single domain antibody binds to the target antigen.

[0369] (B75) A polynucleotide encoding the polypeptide of any one of (B1) to (B56).

[0370] (B76) A vector comprising the polynucleotide described in (B75).

[0371] (B77) A host cell comprising the polynucleotide of (B75) or the vector of (B76).

[0372] (B78) A method for producing the polypeptide according to any one of (B1) to (B56), comprising the step of culturing the host cell according to (B77). BRIEF DESCRIPTION OF THE DRAWINGS

[0373] [ Figure 1 ] Figure 1 The concept of Probody technology is illustrated. Probodies are antibody molecules whose antigen-binding activity is inhibited by linking the antibody to a peptide that masks the antibody's antigen-binding site via a linker that is cleaved by a protease expressed at the disease site.

[0374] [ Figure 2 ] Figure 2 The diagram illustrates the reasons why Probody may exhibit side effects. Activated Probody that accumulates in the blood may exhibit side effects by binding to antigens expressed in normal tissues.

[0375] [ Figure 3 ] Figure 3 The diagram illustrates the potential side effects of Probody. Probody exists in equilibrium between a state where the masking peptide, linked via a linker, is bound to the antigen-binding site and a state where the masking peptide is dissociated. The dissociated molecule can bind to the antigen.

[0376] [ Figure 4 ] Figure 4 The diagram illustrates the reasons why Probody may exhibit side effects. Anti-drug antibodies against masking peptides (anti-masking peptide antibodies) can bind to the masking peptide of Probody before activation, thereby activating Probody without protease cleavage.

[0377] [ Figure 5 ] Figure 5The concept of a polypeptide comprising an antigen-binding domain and a transport moiety is illustrated. (A) The polypeptide having the antigen-binding domain linked to the transport moiety has a long half-life and does not bind to the antigen. (B) The antigen-binding domain is released to bind to the antigen, for example by cleavage at the cleavage site, and the released antigen-binding domain has a short half-life.

[0378] [ Figure 6 ] Figure 6 The diagram illustrates one embodiment of a method for preparing a polypeptide of the present invention. In this embodiment, the polypeptide of interest is an IgG antibody-like molecule. (A) A single-domain antibody that binds to a target antigen is obtained. (B) The single-domain antibody is allowed to associate with the VL as a replacement for the VH of an IgG antibody, thereby inhibiting the antigen-binding activity of the single-domain antibody. (C) A protease cleavage sequence is introduced into an IgG antibody-like molecule precursor comprising the single-domain antibody.

[0379] [ Figure 7 ] Figure 7 An embodiment of the polypeptide of the present invention is illustrated. In this embodiment, the polypeptide is an IgG antibody-like molecule, and the antigen-binding domains are respectively based on the parts corresponding to the two variable regions of the IgG antibody. The two antigen-binding domains can have the same antigen-binding specificity or can have different antigen-binding specificities.

[0380] [ Figure 8 ] Figure 8 The diagram shows an embodiment in which a second antigen binding domain is further linked to the antigen binding domain of the invention. In this embodiment, the antigen binding domain and the second antigen binding domain form a bispecific antigen binding molecule after release. Figure 8 (A) is a diagram showing the polypeptide in an unreleased state. The antigen-binding activity of the antigen-binding domain is inhibited. Figure 8 (B) Schematic diagram of the release of a bispecific antigen-binding molecule formed by an antigen-binding domain and a second antigen-binding domain. Figure 8 (C) illustrates a bispecific antigen-binding molecule targeting, for example, a T cell surface antigen and a cancer cell surface antigen as an example of a released bispecific antigen-binding molecule.

[0381] [ Figure 9A ] Figure 9A An embodiment of a method for screening a fusion polypeptide from a library comprising a plurality of single-domain antibody fusion polypeptides, wherein each single-domain antibody is linked to a first association-maintaining domain, is illustrated, wherein the fusion polypeptide comprises a single-domain antibody whose antigen-binding activity can be inhibited or potentially lost by association with a specific inhibitory domain. Figure 9A(1) Schematic representation of a library comprising a plurality of single domain antibody fusion polypeptides, each single domain antibody being linked to a first association-maintaining domain. Figure 9A (2) The graph shows the antigen-binding activity of each single-domain antibody in a state where the fusion polypeptide is associated with an association partner. Fusion polypeptides comprising single-domain antibodies that do not bind to the target antigen or have an antigen-binding activity of a predetermined value or less in this associated state are selected. Figure 9A (3) The diagram shows that the association of the single domain antibody in the fusion polypeptide selected in (2) with the inhibitory domain in the binding partner is abolished, and the antigen-binding activity of the single domain antibody is confirmed. A fusion polypeptide comprising a single domain antibody is selected, wherein the single domain antibody binds to the target antigen in this non-associated state or has an antigen-binding activity of a predetermined value or higher. Figure 9A (2') Graph showing confirmation of the antigen-binding activity of the single-domain antibody in each fusion polypeptide. A fusion polypeptide comprising a single-domain antibody that binds to the target antigen or has an antigen-binding activity of a predetermined value or higher in the state of the fusion polypeptide alone is selected. Figure 9A (3') Graph showing the antigen-binding activity of the single-domain antibody in a state where the fusion polypeptide selected in (2') is associated with an association partner. A fusion polypeptide comprising a single-domain antibody is selected, wherein the single-domain antibody does not bind to the target antigen or has an antigen-binding activity of a predetermined value or lower in this associated state.

[0382] [ Figure 9B ] Figure 9B Another specific embodiment of a method for screening fusion polypeptides from a library comprising a plurality of single-domain antibody fusion polypeptides, wherein each single-domain antibody is linked to a first association-maintaining domain, wherein the fusion polypeptide comprises a single-domain antibody whose antigen-binding activity can be inhibited or lost by its association with a specific inhibitory domain. (1) The fusion polypeptides each comprising a single-domain antibody and a first association-maintaining domain are displayed together with an association partner comprising a protease cleavage sequence between the inhibitory domain and the second association-maintaining domain to form a Fab-like structure; (2) From the thus displayed Fab-like structures, a structure that does not bind to an antigen or has an antigen-binding activity of a predetermined value or lower is selected; and (3) The association partner is cleaved by a protease, and a fragment comprising a single-domain antibody that binds to an antigen or has an antigen-binding activity of a predetermined value or higher is selected.

[0383] [ Figure 9C ] Figure 9CAnother specific embodiment of a method for screening fusion polypeptides from a library comprising a plurality of single-domain antibody fusion polypeptides, wherein each single-domain antibody is linked to a first association-maintaining domain, wherein the fusion polypeptide comprises a single-domain antibody whose antigen-binding activity can be inhibited or potentially lost by its association with a specific inhibitory domain. (1) A fusion polypeptide, each comprising a protease cleavage sequence between the single-domain antibody and the first association-maintaining domain, and an association partner of the inhibitory domain linked to the second association-maintaining domain are displayed together to form a Fab-like structure. (2) From the Fab-like structures thus displayed, a structure that does not bind to an antigen or has an antigen-binding activity of a predetermined value or less is selected; and (3) The fusion polypeptide is cleaved by a protease, and a fragment comprising a single-domain antibody that binds to an antigen or has an antigen-binding activity of a predetermined value or greater is selected.

[0384] [ Figure 9D ] Figure 9D An alternative embodiment of a method for screening fusion polypeptides from a library comprising a plurality of single-domain antibody fusion polypeptides, wherein each single-domain antibody is linked to a first association-maintaining domain, wherein the fusion polypeptide comprises a single-domain antibody whose antigen-binding activity can be inhibited or lost by its association with a specific inhibitory domain. (1) The fusion polypeptides each comprising a single-domain antibody and a first association-maintaining domain are displayed together with an association partner of an inhibitory domain linked to a second association-maintaining domain to form Fab-like structures, and from the Fab-like structures thus displayed, structures that do not bind to an antigen or have an antigen-binding activity of a predetermined value or lower are selected; and (2) a portion comprising the single-domain antibody in the Fab-like structure thus selected in (1) is again displayed so as not to co-express an inhibitory domain therewith, and a fragment that binds to an antigen or has an antigen-binding activity of a predetermined value or higher is selected. Figure 9D (2') and 9D(2") each illustrate an alternative embodiment, in which the portion comprising a single domain antibody in (2) is displayed again so as not to express an inhibitory domain together with it. The order of (1) and (2), (2') or (2") can be (2), (2') or (2") then (1). Specifically, the portion comprising a single domain antibody is displayed so as not to express an inhibitory domain together with it, and fragments having an antigen-binding activity of a predetermined value or higher are selected. Next, a fusion polypeptide each comprising a single domain antibody and a first association-maintaining domain and an association partner of an inhibitory domain linked to a second association-maintaining domain are displayed together to form a Fab-like structure, wherein the single domain antibody comprises a fragment having a predetermined or higher binding, and from the Fab-like structures thus displayed, a structure that does not bind to the antigen or has an antigen-binding activity of a predetermined value or lower is selected.

[0385] [ Figure 10 ] Figure 10 The graph shows the results of evaluating the binding of antibody-like molecules to human IL-6R, wherein the antibody-like molecules were prepared by allowing various light chains to associate with IL6R90-G1m, wherein IL6R90-G1m comprises an anti-human IL-6R VHH (IL6R90) fused to a human IgG1 constant region (CH1-hinge-CH2-CH3). The starting time of the action of the antigen-like molecule on the antigen-immobilized sensor is indicated as the starting point on the horizontal axis.

[0386] [ Figure 11 ] Figure 11 (A) Schematic representation of an antibody-like molecular model prepared by inserting a protease cleavage sequence near the boundary between the VHH and constant regions in IL6R90-G1m. Figure 11 (B) The name of each prepared antibody heavy chain, the insertion site of the amino acid sequence, and the inserted amino acid sequence are shown. The insertion site is indicated by [Insert].

[0387] [ Figure 12-1 ] Figure 12-1 The figure shows the results of evaluating the degree of cleavage by reducing SDS-PAGE after protease (MT-SP1) treatment of IL6R90-G1m or an antibody-like molecule (prepared by inserting a protease cleavage sequence near the boundary between the VHH and constant regions of IL6R90-G1m). Of the two new bands generated by protease treatment, the band appearing at 25 kDa or less is derived from the VHH, and the band appearing at 25-50 kDa is derived from the constant region.

[0388] [ Figure 12-2 ] Figure 12-2 Continue Figure 12-1 .

[0389] [ Figure 13 ] Figure 13 The graph shows the results of human IL-6R binding evaluation using IL6R90-G1m, an antibody-like molecule prepared by inserting a protease cleavage sequence near the boundary between the VHH and constant regions in IL6R90-G1m, or samples treated with another protease (MT-SP1). The sensorgram "Protease-" depicts the sensorgram evaluating antigen binding of the protease-untreated antibody-like molecule, while the sensorgram "Protease+" depicts the sensorgram evaluating antigen binding of the protease-treated antibody-like molecule. The starting point of the horizontal axis is 30 seconds before the onset of the antibody-like molecule's interaction with the antigen-immobilized sensor.

[0390] [ Figure 14 ] Figure 14The graph shows the results of evaluating the binding of antibody-like molecules to human IL-6R, wherein the antibody-like molecules were prepared by allowing various light chains to associate with 20A11-G1m, wherein 20A11-G1m contains anti-human IL-6RVHH (20A11) fused to a human IgG1 constant region (CH1-hinge-CH2-CH3). The starting point of the horizontal axis is 30 seconds before the start of the action of the antibody-like molecule on the antigen-immobilized sensor.

[0391] [ Figure 15 ] Figure 15 The graph shows the results of evaluating human IL-6R binding by 20A11-G1m or an antibody-like molecule prepared by introducing mutations into amino acids present at the interface between 20A11 and VL and allowing various light chains to associate with 20A11hu-G1m, wherein 20A11hu-G1m contains the thus prepared 20A11hu fused to a human IgG1 constant region (CH1-hinge-CH2-CH3). The starting point of the horizontal axis is 60 seconds before the start of the action of the antibody-like molecule on the antigen-immobilized sensor.

[0392] [ Figure 16 ] Figure 16 The figure shows the results of evaluating the degree of cleavage by reducing SDS-PAGE after protease (MT-SP1) treatment of 20A11-G1m or four antibody-like molecules (prepared by inserting a protease cleavage sequence near the boundary between 20A11hu and the constant region in 20A11-G1m). Of the two new bands generated by protease treatment, the band appearing at 25 kDa or less is derived from the VHH, and the band appearing at a position of 25-50 kDa is derived from the constant region.

[0393] [ Figure 17 ] Figure 17 The graph shows the results of evaluating binding to human IL-6R by 20A11hu-G1m, antibody-like molecules prepared by inserting a protease cleavage sequence near the boundary between the VHH and constant regions in 20A11hu-G1m, or these samples after protease (MT-SP1) treatment. The sensorgrams labeled "Protease-" evaluate the binding of the antibody-like molecules to the antigen without protease treatment, while the sensorgrams labeled "Protease+" evaluate the binding of the antibody-like molecules to the antigen after protease treatment. The starting point of the horizontal axis is 60 seconds before the onset of antibody action on the antigen-immobilized sensor. Samples labeled "Untested" indicate that the samples were not tested.

[0394] [ Figure 18 ] Figure 18The diagram shows the results of evaluating the extent of cleavage by electrophoresis in reducing SDS-PAGE and CBB detection after protease (MT-SP1) treatment of an antibody-like molecule (having an anti-human CD3 VHH within its heavy chain variable region and prepared by inserting a protease cleavage sequence near the boundary between the VHH and heavy chain constant regions). Of the two new bands generated by protease treatment, the band appearing around 10-15 kDa is derived from the VHH, and the band appearing around 37 kDa is derived from the heavy chain constant region.

[0395] [ Figure 19 ] Figure 19 The diagram shows the results of evaluating binding of samples to human CD3 ed-Fc after protease (MT-SP1) treatment of antibody-like molecules (having an anti-human CD3 VHH within their heavy chain variable region and prepared by inserting a protease cleavage sequence near the boundary between the VHH and heavy chain constant regions). Protease- plots the sensorgram evaluating antigen binding of the protease-untreated antibody-like molecule, while Protease+ plots the sensorgram evaluating antigen binding of the protease-treated antibody-like molecule. The starting point of the horizontal axis is 30 seconds before the onset of interaction of the antibody-like molecule with the antigen-immobilized sensor. Binding is indicated when the response before antigen binding is defined as 0 and the response before antibody interaction is defined as 100. The time starting 30 seconds before antibody interaction is indicated.

[0396] [ Figure 20 ] Figure 20 The diagram shows the results of evaluating the degree of cleavage by electrophoresis in reducing SDS-PAGE and CBB detection after treating a molecule having IL6R90-G1m as the heavy chain and Vk1-39-k0MT as the light chain, or an antibody-like molecule (prepared by inserting a protease cleavage sequence near the boundary between the light chain variable region and the light chain constant region of the molecule having IL6R90-G1m as the heavy chain and Vk1-39-k0MT as the light chain) with protease (MT-SP1). Two bands derived from the light chain are generated by protease treatment, indicating that the light chain is cleaved by the protease.

[0397] [ Figure 21 ] Figure 21The diagram shows the results of evaluating binding of samples to human IL-6R after protease (MT-SP1) treatment of molecules having IL6R90-G1m as the heavy chain and Vk1-39-k0MT as the light chain, or antibody-like molecules (prepared by inserting a protease cleavage sequence near the boundary between the light chain variable region and the light chain constant region of molecules having IL6R90-G1m as the heavy chain and Vk1-39-k0MT as the light chain). Sensorgrams evaluating antigen binding of protease-untreated antibody-like molecules are plotted, while sensorgrams evaluating antigen binding of protease-treated antibody-like molecules are plotted. An antibody (MRA) demonstrating IL-6R binding was used as a positive control. The time at which the antibody-like molecule initiated its action on the antigen-immobilized sensor is indicated as the starting point on the horizontal axis.

[0398] [ Figure 22 ] Figure 22 The graph shows the results of SDS-PAGE evaluating protease cleavage of IgG antibody-like molecules with VHH incorporated into human clusterin A1. The protease (+) lane depicts samples treated by protease cleavage and the protease (-) lane depicts negative control samples without protease cleavage treatment.

[0399] [ Figure 23 ] Figure 23 The figure shows an Octet sensorgram evaluating human plexin A1 binding to VHHs, where the VHHs were released by protease cleavage from IgG antibody-like molecules with incorporated VHHs bound to human plexin A1. Protease+ depicts samples treated with protease cleavage, and Protease- depicts samples not treated with protease cleavage. The concentrations of the IgG antibody-like molecules used are reported on the left side of the figure.

[0400] [ Figure 24 ] Figure 24 The graphic shows the results of SDS-PAGE evaluating protease cleavage of bispecific VHH-VHH containing polypeptides.

[0401] [ Figure 25 ] Figure 25 Luciferase activity before and after protease cleavage is shown. The dashed line depicts the sample without protease treatment and the solid line depicts the sample with protease treatment.

[0402] [ Figure 26 ] Figure 26 Luciferase activity before and after protease cleavage is shown. The dashed line depicts the sample without protease treatment and the solid line depicts the sample with protease treatment.

[0403] [ Figure 27 ] Figure 27The figure shows SDS-PAGE evaluation of protease cleavage of IgG antibody-like molecules containing anti-human IL-6R VHH.

[0404] [ Figure 28 ] Figure 28 The diagram shows the evaluation of protease cleavage of IgG antibody-like molecules comprising a protease cleavage sequence in their light chain.

[0405] [ Figure 29 ] Figure 29 Graphed is the evaluation of the degree of activation of an IgG-like antibody molecule comprising a protease cleavage sequence in its light chain based on the presence or absence of protease treatment.

[0406] [ Figure 30A ] Figure 30A The diagram shows the evaluation of protease cleavage of IgG antibody-like molecules comprising a protease cleavage sequence in their heavy chain.

[0407] [ Figure 30B ] Figure 30B The figure shows the evaluation of protease cleavage of IgG antibody-like molecules containing a protease cleavage sequence in their heavy chain. Protease cleavage was performed using assay buffer (MMP Activity Assay Kit (Fluorometric-Green) (ab112146), component C: assay buffer).

[0408] [ Figure 31 ] Figure 31 The figure shows the evaluation of in vivo cleavage efficiency when an antibody molecule into which a protease cleavage sequence has been inserted is administered to mice.

[0409] [Implementation Plan Description]

[0410] The polypeptide according to the present invention generally refers to a peptide or protein having a length of 4 amino acids or longer. Furthermore, the polypeptide according to the present invention is generally a polypeptide consisting of an artificially designed sequence, but is not particularly limited thereto. For example, a polypeptide from an organism can be used. Alternatively, the polypeptide according to the present invention can be any one of a natural polypeptide, a synthetic polypeptide, a recombinant polypeptide, etc. In addition, fragments of these polypeptides are also included in the polypeptide of the present invention.

[0411] In this specification, each amino acid is represented by a single-letter code or a three-letter code, or both, such as Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I, or Val / V. To express an amino acid at a specific position, a combination of a number representing the specific position and the single-letter code or three-letter code of the amino acid can be appropriately used. For example, amino acid 37V, which is an amino acid contained in a single domain antibody, represents Val at position 37 as defined by Kabat numbering.

[0412] In order to change the amino acids in the amino acid sequence of a polypeptide, methods known in the art, such as site-directed mutagenesis (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) or overlap extension PCR can be appropriately used. Various methods known in the art can also be used as a method for changing amino acids by replacing amino acids other than natural amino acids (Annu. Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; and Proc. Natl. Acad. Sci. USA (2003) 100 (11), 6353-6357). For example, a cell-free translation system (Clover Direct (Protein Express)) containing tRNA with a non-natural amino acid bound to an amber suppressor tRNA is also preferably used, and the amber suppressor tRNA is complementary to the UAG codon (amber codon) serving as a stop codon. In the present specification, examples of changes include, but are not limited to, substitution.

[0413] In this specification, the term "and / or" used to indicate an amino acid change site is intended to include each combination appropriately represented by "and" and "or". Specifically, for example, the phrase "amino acids at positions 37, 45 and / or 47 are substituted" includes variants in which the following amino acids are changed: (a) position 37, (b) position 45, (c) position 47, (d) positions 37 and 45, (e) positions 37 and 47, (f) positions 45 and 47, and (g) positions 37, 45 and 47.

[0414] In this specification, the following method can be used to express amino acid changes: the single-letter or three-letter code of the amino acid before or after the change is used before or after the number representing the specific position. For example, the change F37V or Phe37Val, which is used to replace an amino acid contained in an antibody variable region or single-domain antibody, indicates that Phe at position 37 defined by Kabat numbering is replaced by Val. Specifically, the number represents the amino acid position defined by Kabat numbering; the single-letter or three-letter code of the amino acid before the number represents the amino acid before the replacement; and the single-letter or three-letter code of the amino acid after the number represents the amino acid after the replacement. Similarly, the change P238A or Pro238Ala, which is used to replace an amino acid in the Fc region contained in an antibody constant region, indicates that Pro at position 238 defined by EU numbering is replaced by Ala. Specifically, the number represents the amino acid position defined by EU numbering; the single-letter or three-letter code of the amino acid before the number represents the amino acid before the replacement; and the single-letter or three-letter code of the amino acid after the number represents the amino acid after the replacement.

[0415] In this specification, the term "antibody" is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single domain antibodies and antibody fragments, so long as the antibody exhibits the desired antigen-binding activity.

[0416] An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0417] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure, or an antibody having heavy chains that include an Fc region as defined herein.

[0418] The term "variable region" or "variable domain" refers to the region or domain of an antibody heavy chain or light chain that is involved in binding an antibody to an antigen. Typically, antibody heavy and light chain variable domains (VH and VL, respectively) are structurally similar and each contain four conserved framework regions (FRs) and three complementarity determining regions (CDRs) (see, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). One VH or VL domain may be sufficient to confer antigen binding specificity.

[0419] As used herein, the term "complementarity determining region" or "CDR" is hypervariable in sequence and / or forms a structurally defined loop ("hypervariable loop") and / or refers to each region of an antibody variable region that contacts the antigen ("antigen contact"). Typically, antibodies contain six CDRs: three (H1, H2, and H3) in VH and three (L1, L2, and L3) in VL. In this specification, exemplary CDRs include the following:

[0420] (a) hypervariable loops present at amino acid residues 26 to 32 (L1), 50 to 52 (L2), 91 to 96 (L3), 26 to 32 (H1), 53 to 55 (H2), and 96 to 101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0421] (b) CDRs present at amino acid residues 24 to 34 (L1), 50 to 56 (L2), 89 to 97 (L3), 31 to 35b (H1), 50 to 65 (H2), and 95 to 102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0422] (c) antigenic contacts present at amino acid residues 27c to 36 (L1), 46 to 55 (L2), 89 to 96 (L3), 30 to 35b (H1), 47 to 58 (H2), and 93 to 101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)); and

[0423] (d) a combination of (a), (b) and / or (c) containing HVR amino acid residues 46 to 56 (L2), 47 to 56 (L2), 48 to 56 (L2), 49 to 56 (L2), 26 to 35 (H1), 26 to 35b (H1), 49 to 65 (H2), 93 to 102 (H3) and 94 to 102 (H3).

[0424] Typically, a single domain antibody comprises three CDRs: CDR1, CDR2, and CDR3. When a single domain antibody is a single domain antibody prepared from VHH or antibody VH, the single domain antibody CDRs exemplarily include the following:

[0425] (a) hypervariable loops present at amino acid residues 26 to 32 (CDR1), 53 to 55 (CDR2), and 91 to 101 (CDR3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0426] (b) CDRs present at amino acid residues 31 to 35b (CDR1), 50 to 65 (CDR2), and 95 to 102 (CDR3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0427] (c) antigenic contacts present at amino acid residues 30 to 35b (CDR1), 47 to 58 (CDR2), and 93 to 101 (CDR3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)); and

[0428] (d) a combination of (a), (b) and / or (c) comprising CDR amino acid residues 26 to 35 (CDR1), 26 to 35b (CDR1), 49 to 65 (CDR2), 93 to 102 (CDR3) or 94 to 102 (CDR3).

[0429] When the single-domain antibody is a single-domain antibody prepared from VL, the single-domain antibody CDR exemplarily includes the following:

[0430] (a) hypervariable loops present at amino acid residues 26 to 32 (CDR1), 50 to 52 (CDR2), and 91 to 96 (CDR3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0431] (b) CDRs present at amino acid residues 24 to 34 (CDR1), 50 to 56 (CDR2), and 89 to 97 (CDR3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0432] (c) antigenic contacts present at amino acid residues 27c to 36 (CDR1), 46 to 55 (CDR2), and 89 to 96 (CDR3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)); and

[0433] (d) a combination of (a), (b) and / or (c) comprising CDR amino acid residues 46 to 56 (CDR2), 47 to 56 (CDR2), 48 to 56 (CDR2) or 49 to 56 (CDR2).

[0434] In this specification, unless otherwise indicated, CDR residues and other residues in the variable domain (eg, FR residues) are numbered according to Kabat et al. (supra).

[0435] The term "framework" or "FR" refers to the variable domain residues excluding the complementarity determining region (CDR) residues. The FR in the variable domain is generally composed of four FR domains: FR1, FR2, FR3, and FR4. Thus, the sequences of CDRs and FRs generally appear in the following order in VH (or VL): FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4. In single-domain antibodies, the sequences of CDRs and FRs generally appear in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0436] Generally, a single domain antibody of the present invention can be defined as a polypeptide comprising:

[0437] a) an amino acid sequence consisting of four framework regions / sequences with three complementarity determining regions / sequences inserted therebetween (the amino acid residue at position 11 according to Kabat numbering is chosen from the group consisting of L, M, S, V and W, and is preferably L); and / or

[0438] b) an amino acid sequence consisting of four framework regions / sequences with three complementarity determining regions / sequences inserted therebetween (the amino acid residue at position 37 according to Kabat numbering is chosen from the group consisting of F, Y, H, I, L and V, and is preferably F or Y); and / or

[0439] c) an amino acid sequence consisting of four framework regions / sequences with three complementarity determining regions / sequences inserted therebetween (the amino acid residue at position 44 according to Kabat numbering is chosen from the group consisting of G, E, A, D, Q, R, S and L, preferably G, E or Q, and more preferably G or E); and / or

[0440] d) an amino acid sequence consisting of four framework regions / sequences with three complementarity determining regions / sequences inserted therebetween (the amino acid residue at position 45 according to Kabat numbering is chosen from the group consisting of L, R, C, I, L, P, Q and V, and is preferably L or R); and / or

[0441] e) an amino acid sequence consisting of four framework regions / sequences with three complementarity determining regions / sequences inserted therebetween (the amino acid residue at position 47 according to Kabat numbering is chosen from the group consisting of W, L, F, A, G, I, M, R, S, V and Y, and is preferably W, L, F or R); and / or

[0442] f) an amino acid sequence consisting of four framework regions / sequences with three complementarity determining regions / sequences inserted therebetween (the amino acid residue at position 83 according to Kabat numbering is chosen from the group consisting of R, K, N, E, G, I, M, Q and T, preferably K or R, and more preferably K); and / or

[0443] g) an amino acid sequence consisting of four framework regions / sequences with three complementarity determining regions / sequences inserted therebetween (the amino acid residue at position 84 according to Kabat numbering is chosen from the group consisting of P, A, L, R, S, T, D and V, and is preferably P); and / or

[0444] h) an amino acid sequence consisting of four framework regions / sequences with three complementarity determining regions / sequences inserted therebetween (the amino acid residue at position 103 according to Kabat numbering is chosen from the group consisting of W, P, R and S, and is preferably W); and / or

[0445] i) an amino acid sequence consisting of four framework regions / sequences with three complementarity determining regions / sequences interposed therebetween (the amino acid residue at position 104 according to Kabat numbering is G or D, and preferably is G); and / or

[0446] j) an amino acid sequence consisting of four framework regions / sequences with three complementarity determining regions / sequences interposed therebetween (the amino acid residue at position 108 according to Kabat numbering is chosen from the group consisting of Q, L and R, and is preferably Q or L).

[0447] More specifically, but not exclusively, a single domain antibody of the invention may be defined as a polypeptide comprising any one of the amino acid sequences consisting of four framework regions / sequences with three complementarity determining regions / sequences interposed therebetween:

[0448] k) an amino acid sequence in which the amino acid residues at positions 43 to 46 according to Kabat numbering are KERE or KQRE;

[0449] 1) an amino acid sequence in which the amino acid residues at positions 44 to 47 according to Kabat numbering are GLEW; and

[0450] m) an amino acid sequence in which the amino acid residues at positions 83 to 84 according to the Kabat numbering are KP or EP.

[0451] In this specification, the term "constant region" or "constant domain" refers to the region or domain other than the variable region in an antibody. For example, an IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 Da, composed of two identical light chains and two identical heavy chains connected by a disulfide bond. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also referred to as a variable heavy chain domain or a heavy chain variable domain, followed by a heavy chain constant region (CH) containing a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also referred to as a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain. Based on the amino acid sequence of its constant domain, the light chain of a natural antibody can be classified as one of two types, referred to as κ and λ.

[0452] In this specification, the term "Fc region" herein is used to define the C-terminal region of the immunoglobulin heavy chain comprising at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG1 Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) in the Fc region may or may not exist. In this specification, unless otherwise indicated, the encoding of the amino acid residues in the Fc region or constant region is in accordance with the EU numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition Public Health Service, National Institutes of Health, Bethesda, MD 1991, also referred to as the EU index.

[0453] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. Antibodies have five major classes: IgA, IgD, IgE, IgG, and IgM. Some of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0454] In this specification, "antigen binding domain" is limited to binding to the target antigen.Antigen binding domain can be a domain with any structure, as long as the domain used is used to bind to the target antigen. Examples of such domains include, but are not limited to, antibody heavy chain variable region (VH), antibody light chain variable region (VL), single domain antibodies (sdAb), a module of about 35 amino acids contained in the cell membrane protein avimer referred to as the A domain (WO2004 / 044011 and WO2005 / 040229), an adnectin containing 10Fn3 domains, the 10Fn3 domains being derived from the protein binding domain of the glycoprotein fibronectin expressed on the cell membrane (WO2002 / 032925), an affibody containing an IgG binding domain scaffold, the IgG binding domain scaffold constituting a three-helix bundle consisting of 58 amino acids of protein A (WO1995 / 001937), DARPins (designed ankyrin repeat proteins), which are anchor proteins. The molecular surface-exposed regions of the white repeat sequence (AR), each having a 33-amino acid residue structure folded into a turn subunit, two antiparallel helices and a loop (WO2002 / 020565), anticalin having four loop regions connecting eight antiparallel chains, which bends towards the central axis at one end of the barrel-like structure, the barrel structure being highly conserved in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) (WO2003 / 029462), and a concave region in a horseshoe-folded internal parallel plate structure, which is composed of repeated leucine-rich repeat (LRR) modules of variable lymphocyte receptors (VLRs) that do not have an immunoglobulin structure, as seen in the acquired immune system of jawless vertebrates such as lampreys or hagfishes (WO2008 / 016854).

[0455] Preferred embodiments of the antigen-binding domains of the present invention include those that can exert antigen-binding function via a molecule consisting solely of the antigen-binding domain, as well as those that can exert antigen-binding function on their own after being released from another peptide to which they are attached. Examples of such antigen-binding domains include, but are not limited to, single-domain antibodies, scFv, Fv, Fab, Fab', and F(ab')2.

[0456] A preferred embodiment of the antigen-binding domains of the present invention includes an antigen-binding domain with a molecular weight of 60 kDa or less. Examples of such antigen-binding domains include, but are not limited to, single-domain antibodies, scFv, Fab, and Fab'. When present as monomers in the blood, antigen-binding domains with a molecular weight of 60 kDa or less may generally undergo clearance through the kidneys (see J Biol Chem. 1988 Oct 15; 263(29): 15064-70).

[0457] From another perspective, a preferred embodiment of the antigen-binding domain of the present invention includes an antigen-binding domain with a half-life in the blood of 12 hours or less. Examples of such antigen-binding domains include, but are not limited to, single-domain antibodies, scFv, Fab, and Fab'.

[0458] A preferred embodiment of the antigen binding domain of the present invention comprises a single domain antibody (sdAb).

[0459] In this specification, the term "single-domain antibody" is not limited by its structure, as long as the domain itself can exert antigen-binding activity. It is known that general antibodies, such as IgG antibodies, exhibit antigen-binding activity in a state where the variable region is formed by pairing of VH and VL, while the self-domain structure of a single-domain antibody can exert antigen-binding activity by itself without pairing with another domain. Generally, single-domain antibodies have a relatively low molecular weight and exist in the form of monomers.

[0460] Examples of single domain antibodies include, but are not limited to, antigen binding molecules that inherently lack light chains, such as camelid VHHs and shark VHHs. NAR , and antibody fragments containing all or part of an antibody VH domain, or containing all or part of an antibody VL domain. Examples of single-domain antibodies that are antibody fragments containing all or part of an antibody VH or VL domain include, but are not limited to, artificially prepared single-domain antibodies derived from human antibody VH or human antibody VL as described in U.S. Patent No. 6,248,516B1, etc. In some embodiments of the present invention, a single-domain antibody has three CDRs (CDR1, CDR2, and CDR3).

[0461] Single-domain antibodies can be obtained from animals capable of producing single-domain antibodies, or obtained by immunizing animals capable of producing single-domain antibodies. Examples of animals capable of producing single-domain antibodies include, but are not limited to, camelids, and transgenic animals comprising genes capable of producing single-domain antibodies. Animals of the Camelidae family include camels, llamas, alpacas, dromedaries, and guanacos. Examples of transgenic animals comprising genes capable of producing single-domain antibodies include, but are not limited to, the transgenic animals described in WO2015 / 143414 and U.S. Patent Publication No. US2011 / 0123527A1. The framework sequence of the single-domain antibody obtained from the animal can be converted into a human germline sequence or a sequence similar thereto to obtain a humanized single-domain antibody. Humanized single-domain antibodies (e.g., humanized VHH) are also an embodiment of the single-domain antibodies of the present invention. "Humanized single domain antibody" refers to a chimeric single domain antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, in a humanized single domain antibody, all or substantially all CDRs correspond to those of a non-human antibody, and all or substantially all FRs correspond to those of a human antibody. In a humanized antibody, even when a portion of the residues in the FR do not correspond to residues in a human antibody, substantially all FRs are considered to correspond, for example, to those in a human antibody. For example, when humanizing VHH, which is an embodiment of a single domain antibody, a portion of the residues in the FR need to be residues that do not correspond to residues in a human antibody (C Vincke et al., The Journal of Biological Chemistry 284, 3273-3284).

[0462] Alternatively, single domain antibodies can be obtained from a polypeptide library containing single domain antibodies by ELISA, panning, or the like. Examples of polypeptide libraries containing single domain antibodies include, but are not limited to, naive antibody libraries obtained from various animals or humans (e.g., Methods in Molecular Biology 2012 911(65-78); and Biochimica et Biophysica Acta-Proteins and Proteomics 2006 1764:8(1307-1319)), antibody libraries obtained by immunization of various animals (e.g., Journal of Applied Microbiology 2014 117:2(528-536)), and synthetic antibody libraries prepared from antibody genes of various animals or humans (e.g., Journal of Biomolecular Screening 2016 21:1(35-43); Journal of Biological Chemistry 2016 291:24(12641-12657); and AIDS 2016 117:1264-12657). 30:11(1691-1701)).

[0463] In this specification, "antigen" is limited to containing epitopes that bind to antigen binding domains. Preferred examples of antigens include, but are not limited to, peptides, polypeptides, and proteins of animal or human origin. Preferred examples of antigens for treating diseases caused by target tissues include, but are not limited to, molecules expressed on the surface of target cells (e.g., cancer cells and inflammatory cells), molecules expressed on the surface of other cells in tissues containing target cells, molecules expressed on the surface of cells that have immunological effects on target cells and tissues containing target cells, and macromolecules present in the matrix of tissues containing target cells.

[0464] Examples of antigens may include the following molecules: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic factor , av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2BMP-2a, BMP-3 osteogenic factor, BMP-4BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 ( OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5,CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6 , CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L , CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, PD-1, PD-L1, LAG3, TIM3, galectin-9, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor phase Related antigens, DAN, DCC, DcR3, DC-SIGN, decay accelerating factor, des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, deoxyribonuclease, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, neprilysin, eNOS, Eot, eotaxin 1, EpCAM, ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, factor IIa, factor VII, factor VIIIc, factor IX,Fibroblast activation protein (FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle-stimulating hormone, CXXXC chemotactic molecule (fractalkine), FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP -14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-α1, GFR-α2, GFR-α3, 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, HIVIIIB gp 120V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human myocardial globulin, 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)-α, INF-β, INF-γ, inhibin, iNOS, insulin chain A, insulin chain B,Insulin-like growth factor 1, integrin α2, integrin α3, integrin α4, integrin α4 / β1, integrin α4 / β7, integrin α5 (αV), integrin α5 / β1, integrin α5 / β3, integrin α6, integrin β1, integrin β2, interferon γ, 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, left heparin, 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, metalloproteinases, MGDF receptor, MGMT, MHC (HLA-DR), M IF, MIG, MIP, MIP-1-α, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-2 4. MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Müllerian inhibitory substance, Mug, MuSK, NAIP, NAP, NCAD, NC adherin,NCA 90, NCAM, NCAM, neprilysin, neurotrophin-3, -4 or -6, neurturin, nerve growth factor (NGF), NGFR, NGF-β, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA,Prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSV Fgp, 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 receptors (such as T cell receptor α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP Like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-βPan specific, TGF-βRI (ALK-5), TGF-betaRII, TGF-βRIIb, TGF-βRIII, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-α, TNF-α / β, TNF-β2, 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 R4DcR2, 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 (TNFRRH3), TNFRSF3 (LTbR TNF RIII, TNFCR), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA)), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-aConectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF, Trk, TROP-2, TLR (toll-like receptor) 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 antigens, 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, WNT 9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, HMGB1, IgA, Aβ, CD81, CD97, CD98, DDR1, DKK1, EREG, Hsp90, IL-17 / IL-17R, IL-20 / IL-20R, oxidized LDL, PCSK9, prekallikrein, RON, TMEM16F, SOD1, chromogranin A, chromogranin B, tau, VAP1, high-molecular-weight kininogen, IL-31, IL-31R 、Nav1.1、Nav1.2、Nav1.3、Nav1.4、Nav1.5、Nav1.6、Nav1.7、Nav1.8、Nav1.9、EPCR、C1、C1q、C1r、C1s、C2、C2a、C2b、C3、C3a、C3b、C4、C4a、C4b、C5、C5a、C5b、C6、C7、C8、C9、Factor B、Factor D、Factor H、Properdin、Sclerostin、Fibrinogen、Fibrin、Prothrombin、Thrombin、Tissue Factor、Factor V、Factor Va、Factor VII、Factor VIIa、Factor VIIiI、Factor VIIIa、Factor IX, factor IXa, factor X, factor Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA, plasminogen, plasmin, PAI-1, PAI-2, GPC3, syndecan-1, syndecan-2, syndecan-3, syndecan-4, LPA, S1P, and receptors for hormones or growth factors.

[0465] Although the examples of antigens listed above also include receptors, these receptors can still be used as antigens bound by the antigen-binding domains of the present invention even if they are present in soluble form in body fluids. A non-limiting example of such a soluble receptor can include the protein represented by SEQ ID NO: 35, which is a soluble IL-6R described by Mullberg et al. (J. Immunol. (1994) 152(10), 4958-4968).

[0466] Examples of antigens listed above include membrane molecules expressed on cell membranes and soluble molecules secreted from cells. When the antigen-binding domains of the present invention bind to soluble molecules secreted from cells, the antigen-binding domains preferably have neutralizing activity.

[0467] The solution containing soluble molecules is not limited, and the soluble molecules can be present in body fluids (i.e., each vascular fluid or each liquid filled between tissues or cells in an organism). In non-limiting aspects, the soluble molecules combined by the antigen-binding domains of the present invention can be present in extracellular fluid. Extracellular fluid refers to such components in plasma, intercellular fluid, lymph, tight connective tissue, cerebrospinal fluid, spinal fluid, puncture fluid (aspirate), synovial fluid or bone and cartilage in vertebrates, alveolar fluid (bronchoalveolar lavage fluid), ascites, pleural effusion, pericardial effusion, cystic fluid, aqueous humor (watery) or the common name of such transcellular fluids (various liquids in the glandular cavity caused by the active transport or secretory activity of cells, as well as the liquid in the intestinal cavity and other body cavities).

[0468] An epitope (meaning an antigenic determinant) present in an antigen refers to a site on the antigen that binds to an antigen-binding domain disclosed in this specification. Thus, for example, an epitope can be defined by its structure. Alternatively, an epitope can be defined by the antigen-binding activity of an antigen-binding domain that recognizes the epitope. When the antigen is a peptide or polypeptide, the epitope can be identified by the amino acid residues that constitute the epitope. When the epitope is a sugar chain, the epitope can be identified by the specific sugar chain structure.

[0469] A linear epitope is an epitope comprising an epitope recognized by its primary amino acid sequence. A linear epitope typically contains at least 3, most commonly at least 5, such as about 8 to about 10 or 6 to 20 amino acids in its unique sequence.

[0470] In contrast to linear epitopes, conformational epitopes refer to epitopes contained in the primary sequence of amino acids, wherein the primary sequence contains components other than the single limiting component of the epitope to be identified (e.g., an epitope whose primary sequence of amino acids may not be recognized by an antibody that determines the epitope). Compared to linear epitopes, conformational epitopes may contain an increased number of amino acids. As for the recognition of conformational epitopes, the three-dimensional structure of the antigen-binding domain recognition peptide or protein. For example, when the protein molecule is folded to form a three-dimensional structure, certain amino acids and / or polypeptide main chains constituting the conformational epitope are arranged in parallel to allow antibodies to recognize the epitope. Examples of methods for determining epitope conformation include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance spectroscopy, and site-specific spin labeling and electron paramagnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology (1996), Vol. 66, edited by Morris.

[0471] The structure of the antigen binding domain that binds to the epitope is called the paratope. The paratope stably binds to the epitope through hydrogen bonds, electrostatic forces, van der Waals forces, hydrophobic bonds, etc. that work between the epitope and the paratope. This binding force between the epitope and the paratope is called affinity. The total binding force when multiple antigen binding domains bind to multiple antigens is called avidity. When, for example, an antibody comprising multiple antigen binding domains (i.e., a multivalent antibody) binds to multiple epitopes, affinity acts synergistically. Therefore, avidity is higher than avidity.

[0472] In specific embodiments, the dissociation constant (Kd) of the antigen binding domain provided herein is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, such as 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 M).

[0473] Hereinafter, exemplary methods for confirming that an antigen-binding domain or a polypeptide comprising such an antigen-binding domain binds to an epitope will be described. However, according to the examples given below, methods for confirming that an antigen-binding domain or a polypeptide comprising such an antigen-binding domain binds to an epitope other than IL-6R can also be appropriately performed.

[0474] For example, it can be confirmed whether the antigen-binding domain for IL-6R recognizes a linear epitope present in the IL-6R molecule, for example, as follows: For the above purpose, a linear peptide comprising an amino acid sequence constituting the extracellular domain of IL-6R is synthesized. The peptide can be chemically synthesized. Alternatively, the peptide can be obtained by genetic engineering using a region encoding an amino acid sequence corresponding to the extracellular domain in the IL-6R cDNA. Next, the binding activity of the antigen-binding domain for IL-6R to a linear peptide comprising an amino acid sequence constituting the extracellular domain is evaluated. For example, the binding activity of the antigen-binding domain to the peptide can be evaluated by ELISA using an immobilized linear peptide as an antigen. Alternatively, the binding activity to the linear peptide can be determined based on the level at which the linear peptide inhibits the binding of the antigen-binding domain to IL-6R-expressing cells. These tests can determine the binding activity of the antigen-binding domain to the linear peptide.

[0475] Furthermore, whether an antigen-binding domain against IL-6R recognizes a conformational epitope can be confirmed by preparing IL-6R-expressing cells for the above purpose. Recognition of a conformational epitope by an antigen-binding domain against IL-6R can be confirmed, for example, by showing that the antigen-binding domain against IL-6R strongly binds to IL-6R-expressing cells upon contact with the cells, while the antigen-binding domain does not substantially bind to an immobilized linear peptide comprising the amino acid sequence constituting the extracellular domain of IL-6R, or to a denatured (using a common denaturing agent, such as guanidine) linear peptide comprising the amino acid sequence constituting the extracellular domain of IL-6R. As used herein, the term "does not substantially bind" means that the binding activity is 80% or less, typically 50% or less, preferably 30% or less, and particularly preferably 15% or less, of the binding activity against human IL-6R-expressing cells.

[0476] Methods for confirming the antigen-binding activity of antigen-binding domains also include methods for measuring Kd values ​​by, for example, radiolabeled antigen binding assays (RIA). In one embodiment, RIA is performed using the antigen-binding domains of interest and their antigens. For example, the binding affinity of an antigen-binding domain to an antigen in solution can be measured by balancing the antigen-binding domains with a minimum concentration of (125I)-labeled antigen in the presence of a titration series of unlabeled antigen, and subsequently capturing the bound antigen (see, for example, Chen et al., J.Mol.Biol.293:865-881 (1999)) by a flat plate coated with the antigen-binding domains.

[0477] According to alternative embodiment, Kd is measured by the surface plasmon resonance method using BIACORE (registered trademark).For example, using the antigen with about 10 response units (RU) fixed thereon CMAC chip, at 25 DEG C, carry out using BIACORE (registered trademark)-2000 or BIACORE (registered trademark)-3000 (BIAcore, Inc., Piscataway, NJ) mensuration.In one embodiment, according to the explanation of supplier, N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are used to activate carboxymethylated dextran biosensor chip (CM5, BIAcore, Inc.).With 10mM sodium acetate (pH 4.8), antigen is diluted to 5 μ g / mL (about 0.2 μ m), then injected wherein with the flow velocity of 5 μ l / min, to obtain the protein binding of about 10 response units (RU). After antigen injection, 1 M ethanolamine was injected into it to block unreacted groups. For kinetic measurements, a 2-fold dilution (0.78 nM to 500 nM) of the antigen-binding domain in PBS containing 0.05% polysorbate 20 (TWEEN-20 (trademark)) as a surfactant (PBST) was injected into it at a flow rate of approximately 25 μl / min at 25 ° C. The association rate (kon) and dissociation rate (koff) were calculated by simultaneously fitting the sensorgrams of association and dissociation using a simple 1:1 Langmuir binding model (BIACORE (registered trademark) evaluation software version 3.2). The equilibrium dissociation constant (Kd) was calculated as the ratio of koff / kon. In addition, the apparent dissociation constant (Kd) can be determined using equilibrium analysis. For these steps, please refer to the protocol included with BIACORE (registered trademark). See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999) and Methods Enzymol. 2000; 323:325-40. In surface plasmon resonance assays, one skilled in the art can vary the amount of immobilized protein, the amount of protein used in the reaction, the temperature, and the solution composition. When the on-rate in the surface plasmon resonance assay exceeds 10 6 M -1 s -1When the binding rate is determined, the binding rate can be determined by using a spectrometer's fluorescence quenching technique (e.g., a stopped-flow spectrophotometer (Aviv Instruments, Inc.) or a SLM-AMINCO (trademark) spectrophotometer 8000 series (Thermo Spectronic / Thermo Fisher Scientific Inc.) using a stirred cuvette) to measure the increase or decrease in fluorescence intensity (excitation = 295 nM; emission = 340 nM, band gap: 16 nM)) in the presence of increasing concentrations of antigen at 25°C for 20 nM antigen binding domain in PBS (pH 7.2).

[0478] Furthermore, the antigen-binding activity of the antigen-binding domain can also be measured by known molecule-molecule interaction measurement methods such as electrochemiluminescence.

[0479] Examples of methods for measuring the binding activity of an antigen-binding domain against IL-6R to IL-6R-expressing cells include the methods described in Antibodies: A Laboratory Manual (Ed Harlow, David Lane, Cold Spring Harbor Laboratory (1988) 359-420). Specifically, binding activity can be evaluated based on the principles of ELISA or FACS (fluorescence-activated cell sorting) using IL-6R-expressing cells as antigens.

[0480] In the ELISA format, the binding activity of an antigen-binding domain against IL-6R to IL-6R-expressing cells is quantitatively evaluated by comparing the signal levels generated by the enzymatic reaction. Specifically, a test antigen-binding domain is added to an ELISA plate on which IL-6R-expressing cells are immobilized. Then, the test antigen-binding domain bound to the cells is detected by using an enzyme-labeled antibody that recognizes the test antigen-binding domain. Alternatively, in FACS, a dilution series of the test antigen-binding domain is prepared, and the antibody binding titer to IL-6R-expressing cells can be determined to compare the binding activity of the test antigen-binding domain to IL-6R-expressing cells.

[0481] Flow cytometry can be used to detect the binding of a test antigen-binding domain to an antigen expressed on the surface of cells suspended in a buffer solution, etc. Flow cytometry is known as, for example, the following device:

[0482] FACSCanto(TM)II

[0483] FACSAria(TM)

[0484] FACSArray(TM)

[0485] FACSVantage(TM)SE

[0486] FACSCalibur(TM) (both are trade names of BD Biosciences)

[0487] EPICS ALTRA HyPerSort

[0488] Cytomics FC 500

[0489] EPICS XL-MCL ADC EPICS XL ADC

[0490] Cell Lab Quanta / Cell Lab Quanta SC (both trade names of Beckman Coulter, Inc.)

[0491] A preferred example of a method for measuring the antigen binding activity of an antigen binding domain for IL-6R includes the following method: First, IL-6R expressing cells reacting with the test antigen binding domain are stained with a FITC-labeled secondary antibody that recognizes the test antigen binding domain. The test antigen binding domain is appropriately diluted with a suitable buffer solution to prepare the desired concentration of the antigen binding domain for use. The antigen binding domain can be used at any concentration of, for example, 10 μg / mL to 10 ng / mL. Next, fluorescence intensity and cell number are measured using a FACSCalibur (Becton, Dickinson and Company). The amount of antigen binding domain bound to the cells is reflected in the fluorescence intensity (i.e., geometric mean) obtained by analyzing using CELL QUEST software (Becton, Dickinson and Company). In short, the binding activity of the test antigen binding domain, represented by the binding amount of the test antigen binding domain, can be determined by obtaining a geometric mean.

[0492] Whether an antigen-binding domain targeting IL-6R shares an epitope with another antigen-binding domain can be determined by competition between these antigen-binding domains for the same epitope. Competition between antigen-binding domains can be detected by, for example, a cross-blocking assay. A preferred cross-blocking assay is, for example, a competitive ELISA assay.

[0493] Specifically, in a cross-blocking assay, IL-6R protein-coated wells of a microtiter plate are pre-incubated in the presence or absence of a candidate competing antigen-binding domain. A test antigen-binding domain is then added. The amount of test antigen-binding domain bound to the IL-6R protein in the wells indirectly correlates with the binding ability of the candidate competing antigen-binding domain competing for binding to the same epitope. In short, a greater affinity of the competing antigen-binding domain for the same epitope means a lower binding activity of the test antigen-binding domain to the IL-6R protein-coated wells.

[0494] The amount of test antigen binding domain bound to the wells via the IL-6R protein can be readily measured by pre-labeling the antigen binding domain. For example, a biotinylated antigen binding domain can be assayed using an avidin-peroxidase conjugate and a suitable substrate. In particular, cross-blocking assays utilizing enzyme labels such as peroxidase are referred to as competitive ELISA assays. The antigen binding domain can be labeled with alternative detectable or measurable labeling materials. Specifically, radioactive labels, fluorescent labels, and the like are known in the art.

[0495] If the competing antigen-binding domain blocks the binding of the antigen-binding domain to IL-6R by at least 20%, preferably at least 20 to 50%, and more preferably at least 50%, as compared to the binding activity obtained in a control assay performed in the absence of the candidate competing antigen-binding domain, then the test antigen-binding domain can be determined to be an antigen-binding domain that binds to substantially the same epitope as the competing antigen-binding domain, or that competes for binding to the same epitope.

[0496] When the epitope bound by an antigen-binding domain against IL-6R has an identified structure, whether the test antigen-binding domain and the control antigen-binding domain share the same epitope can be evaluated by comparing the binding activity of these antigen-binding domains to peptides or polypeptides prepared by introducing amino acid mutations into the peptide constituting the epitope.

[0497] In this method of measuring binding activity, for example, the binding activity of the test antigen-binding domain and the control antigen-binding domain to a linear peptide containing an introduced mutation can be compared in the above-mentioned ELISA format. In methods other than ELISA, the binding activity of the mutant peptide bound to the column can be measured by flowing the test antigen-binding domain and the control antigen-binding domain in a column and then quantifying the eluted antigen-binding domain in the eluent. Methods for adsorbing mutant peptides (e.g., fusion peptides with GST) onto columns are known in the art.

[0498] When the identified epitope is a conformational epitope, it is possible to evaluate whether the test antigen-binding domain and the control antigen-binding domain share the epitope by the following method: First, prepare IL-6R expressing cells and cells expressing IL-6R in which a mutation is introduced into the epitope. The test antigen-binding domain and the control antigen-binding domain are added to a cell suspension containing these cells suspended in an appropriate buffer solution (e.g., PBS). Subsequently, the cell suspension is appropriately washed with a buffer solution, and then a FITC-labeled antibody capable of recognizing the test antigen-binding domain and the control antigen-binding domain is added thereto. The fluorescence intensity and number of cells stained with the labeled antibody are measured using a FACSCalibur (Becton, Dickinson and Company). The test antigen-binding domain and the control antigen-binding domain are appropriately diluted with an appropriate buffer solution and used at a certain concentration to adjust to the desired concentration. These antigen-binding domains are used, for example, at any concentration from 10 μg / mL to 10 ng / mL. The amount of labeled antibody bound to the cells is reflected in the fluorescence intensity (i.e., geometric mean) obtained by analysis using CELL QUEST software (Becton, Dickinson and Company). In short, the binding activity of the test antigen-binding domain and the control antigen-binding domain, represented by the amount of labeled antibody bound, can be determined by obtaining the geometric mean.

[0499] In addition to the above-mentioned ELISA or FACS, competition between an antigen-binding domain and another antigen-binding domain for the same epitope can also be confirmed by using a radiolabeled antigen binding assay (RIA), BIACORE (registered trademark) surface plasmon resonance assay, electrochemiluminescence, etc.

[0500] In the present method, whether or not "substantially does not bind to cells expressing mutant IL-6R" can be determined by, for example, the following method: First, a test antigen-binding domain and a control antigen-binding domain that bind to cells expressing mutant IL-6R are stained with a labeled antibody. Subsequently, the fluorescence intensity of the cells is measured. When a FACSCalibur is used for fluorescence detection by flow cytometry, the obtained fluorescence intensity can be analyzed using CELL QUEST software. The geometric means obtained in the presence and absence of the polypeptide association product can be compared to calculate their value (ΔGeo-Mean) according to Expression 1 given below to determine the rate of increase in fluorescence intensity due to binding of the antigen-binding domain.

[0501] (Expression 1)

[0502] ΔGeo-Mean = Geo-Mean (in the presence of polypeptide association products) / Geo-Mean (in the absence of polypeptide association products)

[0503] The geometric mean comparative value (ΔGeo-Mean value of the mutant IL-6R molecule) obtained by the analysis is compared with the ΔGeo-Mean comparative value, wherein the geometric mean comparative value reflects the amount of the test antigen-binding domain bound to cells expressing the mutant IL-6R, and wherein the ΔGeo-Mean comparative value reflects the amount of the test antigen-binding domain bound to IL-6R-expressing cells. In this case, it is particularly preferred to adjust the concentrations of the test antigen-binding domain used to determine the ΔGeo-Mean comparative values ​​for cells expressing the mutant IL-6R and cells expressing IL-6R to be equal or substantially equal. An antigen-binding domain that has been shown to recognize an epitope in IL-6R is used as a control antigen-binding domain.

[0504] If the ΔGeo-Mean comparison value of the test antigen-binding domain against cells expressing mutant IL-6R 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 against cells expressing IL-6R, then the test antigen-binding domain "does not substantially bind to cells expressing mutant IL-6R." The calculation expression for determining the Geo-Mean value (geometric mean) is described in the CELL QUEST software user guide (BD biosciences). As a result of the comparison, when their comparison values ​​are considered to be substantially the same, then the epitope of the test antigen-binding domain and the epitope of the control antigen-binding domain can be evaluated as being identical.

[0505] As used herein, the term "delivery moiety" refers to the portion of a polypeptide other than the antigen-binding domain. The delivery moiety of the present invention is typically a peptide or polypeptide composed of amino acids. In one embodiment, the delivery moiety of the polypeptide is linked to the antigen-binding domain via a cleavage site. The delivery moiety of the present invention can be a series of peptides or polypeptides linked by amide bonds, or can be a complex formed by multiple peptides or polypeptides through covalent bonds such as disulfide bonds or non-covalent bonds such as hydrogen bonds or hydrophobic interactions.

[0506] The transport portion of the present invention has an inhibitory domain that inhibits the antigen-binding activity of the antigen-binding domain. In this specification, the term "inhibitory domain" is limited only to the antigen-binding activity of the antigen-binding domain. The inhibitory domain can be a domain with any structure, as long as the domain used can inhibit the antigen-binding activity of the antigen-binding domain. Examples of such an inhibitory domain include, but are not limited to, antibody heavy chain variable region (VH), antibody light chain variable region (VL), pre-B cell receptor and single domain antibody. The inhibitory domain can constitute the entire transport portion or can constitute a portion of the transport portion.

[0507] In some embodiments of the present invention, the antigen-binding domain released from the polypeptide has a higher antigen-binding activity than before release. In other words, the antigen-binding activity of the antigen-binding domain is inhibited by the inhibitory domain when the antigen-binding domain is not released from the polypeptide. Whether the antigen-binding activity of the antigen-binding domain is inhibited by the inhibitory domain is confirmed by methods such as FACS (fluorescence-activated cell sorting), ELISA (enzyme-linked immunosorbent assay), ECL (electrochemiluminescence), SPR (surface plasmon resonance) method (Biacore), BLI (biolayer interferometry) (Octet). In some embodiments of the invention, the antigen-binding activity of the antigen-binding domain released from the polypeptide is equal to or greater than 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times, 2000 times or 3000 times the binding activity of the antigen-binding domain not released from the polypeptide. In some more specific embodiments of the invention, when the antigen-binding activity of the antigen-binding domain is measured by a method selected from the above methods, no binding of the antigen-binding domain to the antigen before release is observed.

[0508] In some aspects of the invention, the cleavage site is cut so that the antigen-binding domain becomes able to be released from the polypeptide. Therefore, in these aspects, the antigen-binding activity can be compared before and after the polypeptide is cut. Specifically, the antigen-binding activity measured using the cut polypeptide is equal to or greater than 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times, 2000 times or 3000 times the value of the antigen-binding activity measured using the uncut polypeptide. In some more specific embodiments, when the antigen-binding activity is measured by a method selected from the above methods, no binding of the antigen-binding domain of the uncut polypeptide to the antigen is observed.

[0509] In some aspects of the present invention, the cleavage site is cut by protease.Therefore, in these aspects, can be before and after the protease treatment polypeptide relatively antigen-binding activity.Particularly, the antigen-binding activity measured by the polypeptide after using the protease treatment is equal to or greater than 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times, 2000 times or 3000 times the value of the antigen-binding activity measured by the polypeptide without protease treatment.In some more specific embodiments, when measuring antigen-binding activity by a method selected from the above-mentioned method, the antigen-binding domains of the untreated polypeptide of protease are not observed to be combined with the antigen.

[0510] In the present invention, a polypeptide comprising an antigen binding domain and a transport moiety has a half-life in the blood that is longer than the half-life of the antigen binding domain alone. In some embodiments of the present invention, for a longer half-life of the polypeptide, the transport moiety is designed so as to have a longer half-life in the blood. In such embodiments, examples of methods for extending the half-life of the transport moiety in the blood include, but are not limited to, a large molecular weight of the transport moiety, FcRn binding activity of the transport moiety, albumin binding activity of the transport moiety, and PEGylation of the transport moiety. In some embodiments of the present invention, the transport moiety has a longer half-life in the blood than the antigen binding domain (in other words, the antigen binding domain has a shorter half-life in the blood than the transport moiety).

[0511] In the present invention, the half-life of the antigen-binding domain and polypeptide alone, or the half-life of the antigen-binding domain and the transport moiety in blood, is preferably compared in terms of half-life in human blood. If the half-life in blood is difficult to measure in humans, the half-life in human blood can be predicted based on their half-life in the blood of mice (e.g., normal mice, transgenic mice expressing human antigens, transgenic mice expressing human FcRn) or monkeys (e.g., cynomolgus monkeys).

[0512] In one embodiment, a method for extending the half-life of a transport moiety in the blood comprises a large molecular weight of the transport moiety. In one embodiment, a method for making the half-life of a transport moiety in the blood longer than the half-life of an antigen binding domain in the blood comprises making the molecular weight of the transport moiety greater than the molecular weight of the antigen binding domain.

[0513] In one embodiment, the method for extending the half-life of a transport moiety in the blood comprises conferring FcRn binding activity to the transport moiety. The transport moiety can generally be conferred with FcRn binding activity by establishing an FcRn binding region in the transport moiety. The FcRn binding region refers to a region that has binding activity for FcRn and can have any structure as long as the region has binding activity for FcRn.

[0514] The transport portion containing the FcRn binding domain can be absorbed into the cell and then returned to the plasma through the salvage pathway of FcRn. For example, IgG molecules have a relatively long circulation time (slow disappearance) in plasma because FcRn, which is known to act as a salvage receptor for IgG molecules, works. The IgG molecules taken into the endosome by pinocytosis bind to the FcRn expressed in the endosome under acidic conditions in the endosome. The IgG molecules that fail to bind to FcRn move to the lysosome and degrade therein, while the IgG molecules that bind to FcRn are transferred to the cell surface and then dissociate with FcRn under neutral conditions in the plasma, thereby returning to the plasma.

[0515] The FcRn binding region is preferably a region that directly binds to FcRn. Preferred examples of FcRn binding regions may include antibody Fc regions. However, regions capable of binding to polypeptides (e.g., such as albumin or IgG) (which have FcRn binding ability) may indirectly bind to FcRn via albumin, IgG, etc. Therefore, the FcRn binding region according to the present invention may be a region that binds to such a polypeptide having FcRn binding ability.

[0516] The binding activity of the FcRn binding domain according to the present invention for FcRn, particularly for human FcRn, can be measured by methods known to those skilled in the art, as described above in the section on binding activity. The conditions can be appropriately determined by those skilled in the art. The binding activity for human FcRn can be evaluated as KD (dissociation constant), apparent KD (apparent dissociation constant), kD (dissociation rate), or apparent kD (apparent dissociation rate), etc. These values ​​can be measured by methods known to those skilled in the art. For example, Biacore (GE Healthcare Japan Corp.), Scatchard plot, flow cytometer, etc. can be used.

[0517] The conditions for measuring the binding activity of the FcRn binding region to FcRn are not particularly limited and can be appropriately selected by those skilled in the art. The binding activity can be measured under conditions including, for example, MES buffer and at 37°C as described in WO2009 / 125825. In addition, the binding activity of the FcRn binding region of the present invention to FcRn can be measured by methods known to those skilled in the art and can be measured using, for example, Biacore (GE Healthcare Japan Corp.). When measuring the binding activity of the FcRn binding region to FcRn, FcRn and the FcRn binding region or a transport portion containing the FcRn binding region can be injected as analytes onto a chip, wherein the FcRn binding region or a transport portion containing the FcRn binding region and FcRn are respectively fixed on the chip, and then evaluated.

[0518] Regarding the pH used as measurement conditions, the binding affinity of the FcRn binding region for FcRn can be evaluated at any pH between 4.0 and 6.5. Preferably, pH 5.8-6.0, which approximates the pH in early endosomes in vivo, is used to determine the binding affinity of the FcRn binding region for human FcRn. Regarding the temperature used as measurement conditions, the binding affinity of the FcRn binding region for FcRn can be evaluated at any temperature between 10°C and 50°C. Preferably, a temperature between 15°C and 40°C is used to determine the binding affinity of the FcRn binding region for human FcRn. More preferably, any temperature between 20°C and 35°C, such as any of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34, is also used to determine the binding affinity of the FcRn binding region for FcRn. A temperature of 25°C is a non-limiting example of an embodiment of the present invention.

[0519] An example of an FcRn-binding region includes, but is not limited to, an IgG antibody Fc region. When an IgG antibody Fc region is used, its type is not limited; for example, an IgG1, IgG2, IgG3, or IgG4 Fc region can be used. For example, an Fc region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 23, and 24 can be used.

[0520] A native IgG antibody Fc region as well as Fc region variants having one or more amino acid substitutions can be used as long as the Fc region has FcRn-binding activity.

[0521] For example, an Fc region variant containing an amino acid sequence derived from an IgG antibody Fc region by replacing at least one amino acid selected from the group consisting of positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 by EU numbering with another amino acid may be used.

[0522] More specifically, Fc region variants containing at least one amino acid substitution in the Fc region of an IgG antibody selected from the group consisting of:

[0523] The amino acid substituted with Met at position 237 is substituted with Gly.

[0524] The amino acid substituted with Ala at position 238 Pro was substituted.

[0525] The amino acid at position 239 Ser was replaced with Lys,

[0526] The Lys at position 248 is replaced with an Ile-substituted amino acid.

[0527] The Thr at position 250 is replaced with an amino acid selected from the group consisting of Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr.

[0528] The Met at position 252 is replaced with an amino acid substituted with Phe, Trp or Tyr,

[0529] The amino acid at position 254 Ser was replaced with Thr,

[0530] The amino acid in position 255 Arg is replaced with Glu,

[0531] The Thr at position 256 is replaced with an amino acid substituted with Asp, Glu or Gln,

[0532] The Pro at position 257 is replaced with an amino acid substituted with Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr or Val,

[0533] The amino acid substituted by His at position 258 Glu,

[0534] The Asp at position 265 was replaced with an Ala-substituted amino acid,

[0535] The Asp at position 270 was replaced with a Phe-substituted amino acid.

[0536] The amino acid substituted by Ala or Glu at position 286 Asn is replaced.

[0537] Replace the Thr at position 289 with a His-substituted amino acid.

[0538] The Asn at position 297 is replaced with an Ala-substituted amino acid,

[0539] Ser at position 298 was replaced with a Gly-substituted amino acid.

[0540] The Val at position 303 is replaced with an Ala-substituted amino acid,

[0541] The Val at position 305 is replaced with an Ala-substituted amino acid,

[0542] The Thr at position 307 is replaced with an amino acid selected from the group consisting of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr.

[0543] The Val at position 308 is replaced with an amino acid substituted with Ala, Phe, Ile, Leu, Met, Pro, Gln or Thr,

[0544] Leu or Val at position 309 is replaced with an amino acid substituted with Ala, Asp, Glu, Pro or Arg,

[0545] The Gln at position 311 is replaced with an amino acid substituted with Ala, His or Ile,

[0546] The Asp at position 312 is replaced with an amino acid substituted with Ala or His,

[0547] Leu at position 314 is replaced with an amino acid substituted with Lys or Arg,

[0548] The Asn at position 315 is replaced with an amino acid such as Ala or His,

[0549] The Lys at position 317 was replaced with an Ala-substituted amino acid.

[0550] The Asn at position 325 was replaced with a Gly-substituted amino acid,

[0551] The amino acid substituted with Val at position 332 is substituted with Ile.

[0552] Lys at position 334 was replaced with a Leu-substituted amino acid,

[0553] The Lys at position 360 was replaced with a His-substituted amino acid.

[0554] Asp at position 376 was replaced with an Ala-substituted amino acid,

[0555] The Glu at position 380 is replaced with an Ala-substituted amino acid.

[0556] The Glu at position 382 is replaced with an Ala-substituted amino acid.

[0557] The Asn or Ser at position 384 is replaced with the amino acid Ala.

[0558] The Gly at position 385 is replaced with an amino acid substituted with Asp or His,

[0559] The Gln at position 386 was replaced with a Pro-substituted amino acid,

[0560] The Pro at position 387 was replaced with a Glu-substituted amino acid.

[0561] The Asn at position 389 is replaced with an amino acid substituted with Ala or Ser,

[0562] The Ser at position 424 was replaced with an Ala-substituted amino acid.

[0563] Met at position 428 is replaced with an amino acid selected from the group consisting of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, and Tyr.

[0564] The His at position 433 was replaced with a Lys-substituted amino acid.

[0565] Asn at position 434 is replaced with an amino acid substituted with Ala, Phe, His, Ser, Trp or Tyr, and

[0566] Tyr or Phe at position 436 was substituted with a His-substituted amino acid (all according to EU numbering).

[0567] From another perspective, an Fc region containing at least one of the following amino acids selected from the group consisting of IgG antibody Fc regions can be used:

[0568] Met is the 237th amino acid.

[0569] Ala is the amino acid at position 238,

[0570] Lys is the amino acid at position 239,

[0571] Ile is the amino acid at position 248,

[0572] Ala, Phe, Ile, Met, Gln, Ser, Val, Trp or Tyr as the amino acid at position 250,

[0573] Phe, Trp or Tyr as the amino acid at position 252,

[0574] Thr as the amino acid at position 254,

[0575] Glu is the amino acid at position 255.

[0576] Asp, Glu or Gln as the amino acid at position 256,

[0577] Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr or Val as the amino acid at position 257,

[0578] His is the amino acid at position 258,

[0579] Ala is the amino acid at position 265,

[0580] Phe as amino acid at position 270,

[0581] Ala or Glu as the amino acid at position 286,

[0582] His is the amino acid at position 289,

[0583] Ala is the amino acid at position 297,

[0584] Gly as amino acid at position 298,

[0585] Ala is the amino acid at position 303,

[0586] Ala is the amino acid at position 305,

[0587] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp or Tyr as the amino acid at position 307,

[0588] Ala, Phe, Ile, Leu, Met, Pro, Gln or Thr as the amino acid at position 308,

[0589] Ala, Asp, Glu, Pro or Arg as the amino acid at position 309,

[0590] Ala, His or Ile as the amino acid at position 311,

[0591] Ala or His as amino acid at position 312,

[0592] Lys or Arg as amino acid at position 314,

[0593] Ala or His as amino acid at position 315

[0594] Ala is the amino acid at position 317,

[0595] Gly as amino acid at position 325,

[0596] Val is the amino acid at position 332,

[0597] Leu as amino acid at position 334,

[0598] His is the amino acid at position 360,

[0599] Ala is the amino acid at position 376,

[0600] Ala is the amino acid at position 380,

[0601] Ala is the amino acid at position 382,

[0602] Ala is the amino acid at position 384,

[0603] Asp or His as amino acid at position 385,

[0604] Pro is the amino acid at position 386,

[0605] Glu is the amino acid at position 387.

[0606] Ala or Ser as the amino acid at position 389,

[0607] Ala is the amino acid at position 424,

[0608] Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp or Tyr as the amino acid at position 428,

[0609] Lys is the amino acid at position 433,

[0610] Ala, Phe, His, Ser, Trp or Tyr as the amino acid at position 434, and

[0611] His is used as the amino acid at position 436 (all according to EU numbering).

[0612] The fact that the transport moiety has FcRn-binding activity does not mean that the antigen-binding domain does not have FcRn-binding activity. In embodiments where the transport moiety has a longer half-life in the blood than the antigen-binding domain, the antigen-binding domain may of course not have FcRn-binding activity, or the antigen-binding domain may have FcRn-binding activity as long as its FcRn-binding activity is weaker than that of the transport moiety.

[0613] In one embodiment, the method for extending the half-life of the transport portion in the blood includes binding the transport portion to albumin. Since albumin does not undergo renal excretion and has FcRn binding ability, its half-life in the blood is as long as 17 to 19 days (J Clin Invest. 1953 August; 32 (8): 746-768). Therefore, it is reported that the protein bound to albumin becomes bulky and can indirectly bind to FcRn, thereby increasing the half-life in the blood (Antibodies 2015, 4 (3), 141-156).

[0614] In one embodiment, an alternative method for extending the half-life of the transport moiety in the blood comprises PEGylating the transport moiety. PEGylation of proteins is believed to make the protein bulky and also inhibit its degradation by proteases in the blood, thereby extending the half-life of the protein in the blood (JPharm Sci. 2008 Oct; 97(10):4167-83).

[0615] In some embodiments of the present invention, the transport portion comprises an antibody Fc region. In one embodiment, the transport portion comprises the CH2 domain and CH3 domain of a human IgG antibody. In one embodiment, the transport portion comprises a portion extending from Cys226 or Pro230 of the heavy chain of a human IgG1 antibody to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region may or may not be present.

[0616] In some embodiments of the invention, the transport moiety comprises an antibody constant region. In more preferred embodiments, the transport moiety comprises an IgG antibody constant region. In further preferred embodiments, the transport moiety comprises a human IgG antibody constant region.

[0617] In some embodiments of the present invention, the transport portion comprises: a region having a structure substantially similar to the constant region of an antibody heavy chain; and a region having a structure substantially similar to the light chain of an antibody, which is connected to the region by a covalent bond (e.g., a disulfide bond) or a non-covalent bond (e.g., a hydrogen bond or hydrophobic interaction).

[0618] In this specification, a "polypeptide comprising an antigen-binding domain and a transport moiety" is generally a series of polypeptides linked by amide bonds, or a protein containing multiple polypeptides linked by amide bonds.

[0619] In some embodiments of the present invention, the antigen-binding domain can be released from the polypeptide, and the antigen-binding domain released from the polypeptide has a higher antigen-binding activity. In this specification, the term "release" refers to the mutual separation of the two parts of the polypeptide. The release of the antigen-binding domain from the polypeptide can be attributed to the elimination of the interaction between the antigen-binding domain and the transport portion. The antigen-binding activity of the antigen-binding domain incorporated into the polypeptide is suppressed. Therefore, it is possible to confirm the antigen-binding domain released from the polypeptide by measuring the antigen-binding activity of the subject and comparing it with the antigen-binding activity of the antigen-binding domain incorporated into the polypeptide.

[0620] In some embodiments, the polypeptide comprises a cleavage site, and the cleavage site is cut so that the antigen-binding domain is released from the polypeptide. The cleavage site can be cut by, for example, an enzyme, can be reduced with a reducing agent, or can be photodegraded. The cleavage site can be placed at any position in the polypeptide, as long as the antigen-binding domain can be released and does not lose its antigen-binding activity after release. The polypeptide can further contain an additional cleavage site in addition to the cleavage site for releasing the antigen-binding domain. In one embodiment of the invention, the cleavage site comprises a protease cleavage sequence and can be cut by a protease.

[0621] In this specification, the term "cleaved" refers to a state in which the antigen-binding domain and the transport moiety are separated from each other after modification of the cleavage site by a protease, 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 the transport moiety are linked in the absence of protease cleavage at the cleavage site, reduction of the cysteine-cysteine ​​disulfide bond at the cleavage site, and / or in the absence of light.

[0622] Cleavage of the cleavage site can be detected by subjecting a solution containing the polypeptide containing the cleavage site to SDS-PAGE (polyacrylamide gel electrophoresis) and measuring the molecular weight of the fragments or detecting a change in molecular weight before and after cleavage.

[0623] The cleavage site can be cleaved by an agent (i.e., a protease, a reducing agent, or light) at a rate of about 0.001 to 1500×10 4 M -1 S -1 , or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500×10 4 M -1 S -1 The ratio of isocyanate to phosphodiesterase is specifically modified (cleaved, reduced or photodegraded).

[0624] The specific cleavage of the protease is carried out by contact between the protease and the cleavage site or the molecule containing the cleavage site. The cleavage site can be cleaved in the presence of sufficient enzymatic activity. Sufficient enzymatic activity can refer to the ability of the enzyme to cleave when in contact with the cleavage site.

[0625] In this specification, the term "protease" refers to an enzyme, such as an endopeptidase or exopeptidase that hydrolyzes peptide bonds, typically an endopeptidase. The protease used in the present invention is only limited by the ability to cleave the protease cleavage sequence and is not particularly limited by its type. In some embodiments, a target tissue-specific protease is used. The target tissue-specific protease can refer to, for example, any of the following:

[0626] (1) Proteases that are expressed at higher levels in target tissues than in normal tissues,

[0627] (2) Proteases that are more active in target tissues than in normal tissues,

[0628] (3) a protease that is expressed at a higher level in target cells than in normal cells, and

[0629] (4) A protease that has higher activity in target cells than in normal cells.

[0630] In more specific embodiments, a cancer tissue-specific protease or an inflammatory tissue-specific protease is used.

[0631] In this specification, the term "target tissue" means a tissue containing at least one target cell. In some embodiments of the present invention, the target tissue is a cancerous tissue. In some embodiments of the present invention, the target tissue is an inflammatory tissue.

[0632] The term "cancerous tissue" refers to tissue containing at least one cancer cell. Thus, considering, for example, that the cancerous tissue contains cancer cells and blood vessels, any cell type that contributes to the formation of a tumor mass containing cancer cells and endothelial cells is included within the scope of the present invention. Throughout this specification, a tumor mass refers to a lesion of tumor tissue. The term "tumor" is generally used to refer to either a benign or malignant tumor.

[0633] In this specification, examples of “inflammatory tissue” include the following:

[0634] joint tissue in rheumatoid arthritis or osteoarthritis,

[0635] Lungs (alveoli) in bronchial asthma or COPD,

[0636] Digestive tissue in inflammatory bowel disease, Crohn's disease, or ulcerative colitis,

[0637] fibrotic tissue in liver, kidney, or lung fibrosis,

[0638] Tissues that cause organ transplant rejection,

[0639] Blood vessels or the heart (myocardium) in arteriosclerosis or heart failure,

[0640] Visceral adipose tissue in metabolic syndrome,

[0641] skin tissue in atopic dermatitis and other dermatitis, and

[0642] Spinal nerves for herniated disc or chronic low back pain.

[0643] For certain types of target tissues, proteases that are specifically expressed or specifically activated, or proteases that are believed to be associated with the disease state of the target tissue (target tissue-specific proteases) are known. For example, WO2013 / 128194, WO2010 / 081173 and WO2009 / 025846 disclose proteases that are specifically expressed in cancer tissues. In addition, J Inflamm (Lond). 2010; 7: 45, Nat Rev Immunol. 2006 July; 6 (7): 541-50, Nat Rev Drug Discov. 2014 December; 13 (12): 904-27, Respir Res. 2016 March 4; 17: 23, Dis Model Mech. 2014 February; 7 (2): 193-203 and Biochim Biophys Acta. 2012 January; 1824 (1): 133-45 disclose proteases that are believed to be associated with inflammation.

[0644] In addition to proteases specifically expressed in target tissues, there are also proteases that are specifically activated in target tissues. For example, proteases can be expressed in an inactive form and then converted to an activated form. Many tissues contain substances that inhibit active proteases, and their activity is controlled by the activation process and the presence of inhibitors (Nat Rev Cancer. 2003 July; 3(7): 489-501). In target tissues, active proteases can be specifically activated by escaping inhibition.

[0645] The active protease can be measured by a method using an antibody that recognizes the active protease (PNAS 2013 Jan 2;110(1):93-98), or a method in which a peptide that can be recognized by the protease is fluorescently labeled so that the fluorescence is quenched before cleavage but emits light after cleavage (Nat Rev Drug Discov. 2010 Sep;9(9):690-701. doi:10.1038 / nrd3053).

[0646] From one perspective, the term "target tissue-specific protease" may refer to any of the following:

[0647] (i) a protease that is expressed at higher levels in target tissues than in normal tissues,

[0648] (ii) a protease that is more active in the target tissue than in normal tissue,

[0649] (iii) a protease that is expressed at a higher level in target cells than in normal cells, and

[0650] (iv) A protease that has higher activity in target cells than in normal cells.

[0651] Specific examples of proteases include, but are not limited to, cysteine ​​proteases (including cathepsin family B, L, S, etc.), aspartyl proteases (cathepsins D, E, K, O, etc.), serine proteases (including matriptase (including MT-SP1), cathepsins A and G, thrombin, plasmin, urokinase (uPA), tissue plasminogen activator (tPA), elastase, proteinase 3, thrombin, kallikrein, tryptase, and chymotrypsin), metalloproteases (metalloproteases (MMP1-28) including membrane-bound forms (MMP14-17 and MMP24-25) and secreted forms (MMP1-13, MMP18-23, and MMP26-28)), disintegrin and metalloproteases (ADAM ), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), transmembrane peptidases (transmembrane peptidase α and transmembrane peptidase β), CD10 (CALLA), prostate-specific antigen (PSA), legumain, TMPRSS3, TMPRSS4, neutrophil elastase (HNE), beta-secretase (BACE), fibroblast activation protein α (FAP), granzyme B, guanidinobenzoic acidase (GB), hepsin, neprilysin, NS3 / 4A, HCV-NS3 / 4, calpain, ADAMDEC1, renin, cathepsin C, cathepsin V / L2, cathepsin X / Z / P, cruzipain, otubain 2. Kallikrein-related peptidases (KLKs (KLK3, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and KLK14)), bone morphogenetic protein 1 (BMP-1), activated protein C, coagulation-related proteases (factor VIIa, factor IXa, factor Xa, factor XIa, and factor XIIa), HtrA1, lactoferrin, marapsin, PACE4, DESC1, dipeptidyl peptidase 4 (DPP-4), TMPRSS2, cathepsin F, cathepsin H, cathepsin L2, cathepsin O, cathepsin S, granzyme A, protease calpain 2, glutamate carboxypeptidase 2, AMSH-like protease, AMSH, γ-secretase, antiplasmin-cleaving enzyme (APCE), decysin1, N-acetylated α-linked acid dipeptidase-like 1 (NAALADL1), and furin.

[0652] From another perspective, the target tissue-specific protease may refer to a cancer tissue-specific protease or an inflammatory tissue-specific protease.

[0653] Examples of cancer tissue-specific proteases include proteases specifically expressed in cancer tissues disclosed in WO2013 / 128194, WO2010 / 081173, and WO2009 / 025846.

[0654] As for the type of cancer tissue-specific protease, a protease with a higher expression specificity in the cancer tissue to be treated is more effective for reducing side effects. The concentration of the preferred cancer tissue-specific protease in the cancer tissue is 5 times or more of its concentration in normal tissue, more preferably 10 times or more, further preferably 100 times or more, particularly preferably 500 times or more, and most preferably 1000 times or more. In addition, the activity of the preferred cancer tissue-specific protease in the cancer tissue is 2 times or more of its activity in normal tissue, more preferably 3 times or more, 4 times or more, 5 times or more or 10 times or more, further preferably 100 times or more, particularly preferably 500 times or more, and most preferably 1000 times or more.

[0655] Cancer tissue specific protease can be the form that is combined with cancer cell membrane, or can be the form that extracellular secretion is not combined with cell membrane.When cancer tissue specific protease is not combined with cancer cell membrane, for the cytotoxicity of the immune cell mediation of cancer cell specificity, preferred cancer tissue specific protease should be present in the cancerous tissue or near the cancerous tissue.In this manual, " near the cancerous tissue " refers to fall into the position range that the cancer tissue specific protease cleavage sequence is cut, so that the antigen-binding domains are brought into play antigen-binding activity.But preferably, should make the infringement to normal cell minimized within this position range.

[0656] Alternatively, the cancer tissue-specific protease is any of the following:

[0657] (i) a protease that is expressed at higher levels in cancer tissue than in normal tissue,

[0658] (ii) a protease that is more active in cancerous tissue than in normal tissue,

[0659] (iii) proteases that are expressed at higher levels in cancer cells than in normal cells, and

[0660] (iv) Proteases that are more active in cancer cells than in normal cells.

[0661] One cancer tissue-specific protease may be used alone, or two or more cancer tissue-specific proteases may be used in combination. Those skilled in the art can appropriately set the number of types of cancer tissue-specific proteases in consideration of the type of cancer to be treated.

[0662] From these viewpoints, the cancer tissue-specific protease is preferably a serine protease or metalloprotease among the proteases listed above, more preferably a matrices (including MT-SP1), urokinase (uPA) or a metalloprotease, and further preferably MT-SP1, uPA, MMP-2 or MMP-9.

[0663] As for the type of inflammatory tissue-specific protease, proteases with higher expression specificity in the inflammatory tissue to be treated are more effective for reducing side effects. The concentration of the preferred inflammatory tissue-specific protease in the inflammatory tissue is 5 times or more of its concentration in normal tissue, more preferably 10 times or more, further preferably 100 times or more, particularly preferably 500 times or more, most preferably 1000 times or more. In addition, the activity of the preferred inflammatory tissue-specific protease in the inflammatory tissue is 2 times or more of its activity in normal tissue, more preferably 3 times or more, 4 times or more, 5 times or more, or 10 times or more, further preferably 100 times or more, particularly preferably 500 times or more, and most preferably 1000 times or more.

[0664] The inflammatory tissue specific protease can be a form combined with the inflammatory cell membrane, or can be a form of extracellular secretion and not combined with the cell membrane. When the inflammatory tissue specific protease is not combined with the inflammatory cell membrane, for the cytotoxicity of the immune cell-mediated specificity of inflammatory cells, preferably the inflammatory tissue specific protease should be present in the inflammatory tissue or near the inflammatory tissue. In this manual, "near the inflammatory tissue" refers to fall within the position range where the protease cleavage sequence specific to the inflammatory tissue is cut, so that the antigen-binding domains are brought into play antigen-binding activity. However, preferably, the damage to normal cells should be minimized within this position range.

[0665] From an alternative perspective, an inflammatory tissue-specific protease is any of the following: (i) a protease that is expressed at higher levels in inflamed tissue than in normal tissue,

[0666] (ii) proteases that are more active in inflamed tissues than in normal tissues,

[0667] (iii) a protease that is expressed at a higher level in inflammatory cells than in normal cells, and (iv) a protease that has a higher activity in inflammatory cells than in normal cells.

[0668] One type of inflammatory tissue-specific protease may be used alone, or two or more types of inflammatory tissue-specific proteases may be used in combination. Those skilled in the art may appropriately set the number of types of inflammatory tissue-specific proteases in consideration of the pathological condition to be treated.

[0669] From these viewpoints, the inflammatory tissue-specific protease is preferably a metalloprotease among the proteases listed above. The metalloprotease is more preferably ADAMTS5, MMP-2, MMP-7, MMP-9 or MMP-13.

[0670] The protease cleavage sequence is a specific amino acid sequence that is specifically recognized by the target tissue-specific protease when the polypeptide is hydrolyzed by the target tissue-specific protease in an aqueous solution.

[0671] From the perspective 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, wherein the target tissue-specific protease is specifically expressed in the target tissue or cell to be treated, or is specifically activated in the target tissue / cell to be treated.

[0672] The specific example of protease cleavage sequence includes the target sequence by the following protease specific hydrolysis: the above-mentioned protease specifically expressed in cancer tissue disclosed in WO2013 / 128194, WO2010 / 081173 and WO2009 / 025846, the protease with specificity to inflammatory tissue, etc. It is also possible to use artificially altered sequences, for example, by introducing appropriate amino acid mutations into the target sequence by known protease specific hydrolysis. Alternatively, it is possible to use protease cleavage sequences identified by methods known to those skilled in the art as described in Nature Biotechnology 19,661-667 (2001).

[0673] In addition, naturally occurring protease cleavage sequences can be used. For example, TGFβ is converted to a latent form by protease cleavage. Similarly, protease cleavage sequences in proteins that change their molecular form by protease cleavage can also be used.

[0674] Examples of protease cleavage sequences that can be used include, but are not limited to, sequences disclosed in WO2015 / 116933, WO2015 / 048329, WO2016 / 118629, WO2016 / 179257, WO2016 / 179285, WO2016 / 179335, WO2016 / 179003, WO2016 / 046778, WO2016 / 014974, U.S. Patent Publication No. US2016 / 0289324, U.S. Patent Publication No. US2016 / 0311903, PNAS (2000) 97:7754-7759, Biochemical Journal (2010) 426:219-228, and Beilstein J Nanotechnol. (2016) 7:364-373.

[0675] The protease cleavage sequence is more preferably an amino acid sequence that is specifically hydrolyzed by a suitable target tissue-specific protease as described above. The amino acid sequence that is specifically hydrolyzed by the target tissue-specific protease is preferably a sequence comprising any of the following amino acid sequences:

[0676] LSGRSDNH (SEQ ID NO: 12, cleavable by MT-SP1 or uPA),

[0677] PLALAG (SEQ ID NO: 25, cleavable by MMP-2 or MMP-9), and

[0678] VPLSLTMG (SEQ ID NO: 26, cleavable by MMP-7).

[0679] Any of the following sequences may also be used as protease cleavage sequences:

[0680] TSTSGRSANPRG (SEQ ID NO: 74, cleavable by MT-SP1 or uPA),

[0681] ISSGLLSGRSDNH (SEQ ID NO: 75, cleavable by MT-SP1 or uPA),

[0682] AVGLLAPPGGLSGRSDNH (SEQ ID NO: 76, cleavable by MT-SP1 or uPA),

[0683] GAGVPMSMRGGAG (SEQ ID NO: 77, cleavable by MMP1),

[0684] GAGIPVSLRSGAG ​​(SEQ ID NO: 78, cleavable by MMP2),

[0685] GPLGIAGQ (SEQ ID NO: 79, cleavable by MMP-2),

[0686] GGPLG MLSQS (SEQ ID NO: 80, cleavable by MMP-2),

[0687] PLGLWA (SEQ ID NO: 81, cleavable by MMP-2),

[0688] GAGRPFSMIMGAG (SEQ ID NO: 82, cleavable by MMP-3),

[0689] GAGVPLSLTMGAG (SEQ ID NO: 83, cleavable by MMP-7),

[0690] GAGVPLSLYSGAG (SEQ ID NO: 84, cleavable by MMP-9),

[0691] AANLRN (SEQ ID NO: 85, cleavable by MMP-11),

[0692] AQAYVK (SEQ ID NO: 86, cleavable by MMP-11),

[0693] AANYMR (SEQ ID NO: 87, cleavable by MMP-11),

[0694] AAALTR (SEQ ID NO: 88, cleavable by MMP-11),

[0695] AQNLMR (SEQ ID NO: 89, cleavable by MMP-11),

[0696] AANYTK (SEQ ID NO: 90, cleavable by MMP-11),

[0697] GAGPQGLAGQRGIVAG (SEQ ID NO: 91, cleavable by MMP-13),

[0698] PRFKIIGG (SEQ ID NO: 92, cleavable by prourokinase),

[0699] PRFRIIGG (SEQ ID NO: 93, cleavable by prourokinase),

[0700] GAGSGRSAG (SEQ ID NO: 94, cleavable by uPA),

[0701] SGRSA (SEQ ID NO: 95, cleavable by uPA),

[0702] GSGRSA (SEQ ID NO: 96, cleavable by uPA),

[0703] SGKSA (SEQ ID NO: 97, cleavable by uPA),

[0704] SGRSS (SEQ ID NO: 98, cleavable by uPA),

[0705] SGRRA (SEQ ID NO: 99, cleavable by uPA),

[0706] SGRNA (SEQ ID NO: 100, cleavable by uPA),

[0707] SGRKA (SEQ ID NO: 101, cleavable by uPA),

[0708] QRGRSA (SEQ ID NO: 102, cleavable by tPA),

[0709] GAGSLLKSRMVPNFNAG (SEQ ID NO: 103, cleavable by cathepsin B)

[0710] TQGAAA (SEQ ID NO: 104, cleavable by cathepsin B),

[0711] GAAAAA (SEQ ID NO: 105, cleavable by cathepsin B),

[0712] GAGAAG (SEQ ID NO: 106, cleavable by cathepsin B),

[0713] AAAAAG (SEQ ID NO: 107, cleavable by cathepsin B),

[0714] LCGAAI (SEQ ID NO: 108, cleavable by cathepsin B),

[0715] FAQALG (SEQ ID NO: 109, cleavable by cathepsin B),

[0716] LLQANP (SEQ ID NO: 110, cleavable by cathepsin B),

[0717] LAAANP (SEQ ID NO: 111, cleavable by cathepsin B),

[0718] LYGAQF (SEQ ID NO: 112, cleavable by cathepsin B),

[0719] LSQAQG (SEQ ID NO: 113, cleavable by cathepsin B),

[0720] ASAASG (SEQ ID NO: 114, cleavable by cathepsin B),

[0721] FLGASL (SEQ ID NO: 115, cleavable by cathepsin B),

[0722] AYGATG (SEQ ID NO: 116, cleavable by cathepsin B),

[0723] LAQATG (SEQ ID NO: 117, cleavable by cathepsin B),

[0724] GAGSGVVIATVIVITAG (SEQ ID NO: 118, cleavable by cathepsin L),

[0725] APMAEGGG (SEQ ID NO: 119, cleavable by transmembrane peptidase α or transmembrane peptidase β),

[0726] EAQGDKII (SEQ ID NO: 120, cleavable by transmembrane peptidase α or transmembrane peptidase β),

[0727] LAFSDAGP (SEQ ID NO: 121, cleavable by transmembrane peptidase α or transmembrane peptidase β),

[0728] YVADAPK (SEQ ID NO: 122, cleavable by transmembrane peptidase α or transmembrane peptidase β),

[0729] RRRRR (SEQ ID NO: 123, cleavable by furin),

[0730] RRRRRR (SEQ ID NO: 124, cleavable by furin),

[0731] GQSSRHRRAL (SEQ ID NO: 125, cleavable by furin),

[0732] SSRHRRALD (SEQ ID NO: 126),

[0733] RKSSIIIRMRDVVL (SEQ ID NO: 127, cleavable by plasminogen),

[0734] SSSFDKGKYKKGDDA (SEQ ID NO: 128, cleavable by staphylokinase),

[0735] SSSFDKGKYKRGDDA (SEQ ID NO: 129, cleavable by staphylokinase),

[0736] IEGR (SEQ ID NO: 130, cleavable by Factor Xa),

[0737] IDGR (SEQ ID NO: 131, cleavable by Factor Xa),

[0738] GGSIDGR (SEQ ID NO: 132, cleavable by Factor Xa),

[0739] GPQGIAGQ (SEQ ID NO: 133, cleavable by collagenase),

[0740] GPQGLLGA (SEQ ID NO: 134, cleavable by collagenase),

[0741] GIAGQ (SEQ ID NO: 135, cleavable by collagenase),

[0742] GPLGIAG (SEQ ID NO: 136, cleavable by collagenase),

[0743] GPEGLRVG (SEQ ID NO: 137, cleavable by collagenase),

[0744] YGAGLGVV (SEQ ID NO: 138, cleavable by collagenase),

[0745] AGLGVVER (SEQ ID NO: 139, cleavable by collagenase),

[0746] AGLGISST (SEQ ID NO: 140, cleavable by collagenase),

[0747] EPQALAMS (SEQ ID NO: 141, cleavable by collagenase),

[0748] QALAMSAI (SEQ ID NO: 142, cleavable by collagenase),

[0749] AAYHLVSQ (SEQ ID NO: 143, cleavable by collagenase),

[0750] MDAFLESS (SEQ ID NO: 144, cleavable by collagenase),

[0751] ESLPVVAV (SEQ ID NO: 145, cleavable by collagenase),

[0752] SAPAVESE (SEQ ID NO: 146, cleavable by collagenase),

[0753] DVAQFVLT (SEQ ID NO: 147, cleavable by collagenase),

[0754] VAQFVLTE (SEQ ID NO: 148, cleavable by collagenase),

[0755] AQFVLTEG (SEQ ID NO: 149, cleavable by collagenase),

[0756] PVQPIGPQ (SEQ ID NO: 150, cleavable by collagenase),

[0757] LVPRGS (SEQ ID NO: 151, cleavable by thrombin),

[0758] TSGSGRSANARG (SEQ ID NO: 168, cleavable by uPA and MT-SP1),

[0759] TSQSGRSANQRG (SEQ ID NO: 169, cleavable by uPA and MT-SP1),

[0760] TSPSGRSAYPRG (SEQ ID NO: 170, cleavable by uPA and MT-SP1),

[0761] TSGSGRSATPRG (SEQ ID NO: 171, cleavable by uPA and MT-SP1),

[0762] TSQSGRSATPRG (SEQ ID NO: 172, cleavable by uPA and MT-SP1),

[0763] TSASGRSATPRG (SEQ ID NO: 173, cleavable by uPA and MT-SP1),

[0764] TSYSGRSAVPRG (SEQ ID NO: 174, cleavable by uPA and MT-SP1),

[0765] TSYSGRSANFRG (SEQ ID NO: 175, cleavable by uPA and MT-SP1),

[0766] TSSSGRSATPRG (SEQ ID NO: 176, cleavable by uPA and MT-SP1),

[0767] TSTTGRSASPRG (SEQ ID NO: 177, cleavable by uPA and MT-SP1), and

[0768] TSTSGRSANPRG (SEQ ID NO: 178, cleavable by uPA and MT-SP1).

[0769] The sequences shown in Table 1 can also be used as protease cleavage sequences.

[0770] [Table 1]

[0771] Protease cleavage sequence (cleaved by uPA and MT-SP1)

[0772]

[0773]

[0774]

[0775]

[0776]

[0777]

[0778]

[0779]

[0780]

[0781]

[0782] The following sequences can also be used as protease cleavage sequences:

[0783] X1-X2-X3-X4-X5-X6-X7-X8(SEQ ID NO:833)

[0784] wherein X1 to X8 each represent a single amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 represents R; X5 is an amino acid selected from A, D, E, F, G, H, I , K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0785] The following sequences can also be used as protease cleavage sequences:

[0786] X1-X2-X3-X4-X5-X6-X7-X8(SEQ ID NO:834)

[0787] wherein X1 to X8 each represent a single amino acid, X1 is an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W, and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W, and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L , M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from the group consisting of A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X8 is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0788] The following sequences can also be used as protease cleavage sequences:

[0789] X1-X2-X3-X4-X5-X6-X7-X8(SEQ ID NO:835)

[0790] wherein X1 to X8 each represent a single amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, F, L, M, P, Q, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y. wherein X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0791] The following sequences can also be used as protease cleavage sequences:

[0792] X1-X2-X3-X4-X5-X6-X7-X8(SEQ ID NO:836)

[0793] wherein X1 to X8 each represent a single amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, wherein X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0794] The following sequences can also be used as protease cleavage sequences:

[0795] X1-X2-X3-X4-X5-X6-X7-X8(SEQ ID NO:837)

[0796] wherein X1 to X8 each represent a single amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, G, H, wherein X is an amino acid selected from the group consisting of A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0797] The following sequences can also be used as protease cleavage sequences:

[0798] X1-X2-X3-X4-X5-X6-X7-X8(SEQ ID NO:838)

[0799] wherein X1 to X8 each represent a single amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; , F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from E, F, K, M, N, P, Q, R, S and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

[0800] The following sequences can also be used as protease cleavage sequences:

[0801] X1-X2-X3-X4-X5-X6-X7-X8(SEQ ID NO:839)

[0802] wherein X1 to X8 each represent a single amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, wherein X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0803] The following sequences can also be used as protease cleavage sequences:

[0804] X1-X2-X3-X4-X5-X6-X7-X8(SEQ ID NO:840)

[0805] wherein X1 to X8 each represent a single amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, wherein X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X is an amino acid selected from the group consisting of A, D, E, F, G, I, K, N, T, and W.

[0806] The following sequences can also be used as protease cleavage sequences:

[0807] X1-X2-X3-X4-X5-X6-X7-X8(SEQ ID NO:841)

[0808] wherein X1 to X8 each represent a single amino acid, X1 is an amino acid selected from A, G, I, P, Q, S and Y; X2 is an amino acid selected from K or T; X3 is G; X4 is R; X5 is S; X6 is A; X7 is an amino acid selected from H, I and V; and X8 is an amino acid selected from H, V and Y.

[0809] The following sequences can also be used as protease cleavage sequences:

[0810] X1-X2-X3-X4-X5-X6-X7-X8(SEQ ID NO:842)

[0811] wherein X1 to X8 each represent a single amino acid, X1 is Y; X2 is an amino acid selected from S and T; X3 is G; X4 is R; X5 is S; X6 is an amino acid selected from A and E; X7 is an amino acid selected from N and V; and X8 is an amino acid selected from H, P, V and Y.

[0812] The following sequences can also be used as protease cleavage sequences:

[0813] X1-X2-X3-X4-X5-X6-X7-X8-X9(SEQ ID NO:843)

[0814] wherein X1 to X9 each represent a single amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0815] The following sequences can also be used as protease cleavage sequences:

[0816] X1-X2-X3-X4-X5-X6-X7-X8-X9(SEQ ID NO:844)

[0817] wherein X1 to X9 each represent a single amino acid, X1 is an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and X9 is an amino acid selected from A, G, H, I, L and R.

[0818] The following sequences can also be used as protease cleavage sequences:

[0819] X1-X2-X3-X4-X5-X6-X7-X8-X9(SEQ ID NO:845)

[0820] wherein X1 to X9 each represent a single amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, F, L, M, P, Q, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S , T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and X9 is an amino acid selected from A, G, H, I, L and R.

[0821] The following sequences can also be used as protease cleavage sequences:

[0822] X1-X2-X3-X4-X5-X6-X7-X8-X9(SEQ ID NO:846)

[0823] wherein X1 to X9 each represent a single amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R , S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and X9 is an amino acid selected from A, G, H, I, L and R.

[0824] The following sequences can also be used as protease cleavage sequences:

[0825] X1-X2-X3-X4-X5-X6-X7-X8-X9(SEQ ID NO:847)

[0826] wherein X1 to X9 each represent a single amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, G, H, I, K, L, M, N, Q , R, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0827] The following sequences can also be used as protease cleavage sequences:

[0828] X1-X2-X3-X4-X5-X6-X7-X8-X9(SEQ ID NO:848)

[0829] wherein X1 to X9 each represent a single amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from E, F, K, M, N, P, Q, R, S, and W; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0830] The following sequences can also be used as protease cleavage sequences:

[0831] X1-X2-X3-X4-X5-X6-X7-X8-X9(SEQ ID NO:849)

[0832] wherein X1 to X9 each represent a single amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K , L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, F, G, L, M, P, Q, V, and W; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0833] The following sequences can also be used as protease cleavage sequences:

[0834] X1-X2-X3-X4-X5-X6-X7-X8-X9(SEQ ID NO:850)

[0835] wherein X1 to X9 each represent a single amino acid, X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K , L, M, N, P, Q, R, S, T, V, W, and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X8 is an amino acid selected from A, D, E, F, G, I, K, N, T, and W; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0836] The following sequences can also be used as protease cleavage sequences:

[0837] X1-X2-X3-X4-X5-X6-X7-X8-X9(SEQ ID NO:851)

[0838] wherein X1 to X9 each represent a single amino acid, X1 is an amino acid selected from A, G, I, P, Q, S, and Y; X2 is an amino acid selected from K or T; X3 is G; X4 is R; X5 is S; X6 is A; X7 is an amino acid selected from H, I, and V; X8 is an amino acid selected from H, V, and Y; and X9 is an amino acid selected from A, G, H, I, L, and R.

[0839] The following sequences can also be used as protease cleavage sequences:

[0840] X1-X2-X3-X4-X5-X6-X7-X8-X9(SEQ ID NO:852)

[0841] wherein X1 to X9 each represent a single amino acid, X1 is Y; X2 is an amino acid selected from S and T; X3 is G; X4 is R; X5 is S; X6 is an amino acid selected from A and E; X8 is an amino acid selected from H, P, V and Y, and X9 is an amino acid selected from A, G, H, I, L and R.

[0842] The following sequences can also be used as protease cleavage sequences:

[0843] X10-X11-X1-X2-X3-X4-X5-X6-X7-X8(SEQ ID NO:1062)

[0844] wherein X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, wherein X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0845] The following sequences can also be used as protease cleavage sequences:

[0846] X10-X11-X1-X2-X3-X4-X5-X6-X7-X8(SEQ ID NO:1063)

[0847] wherein X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, wherein X is an amino acid selected from the group consisting of A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0848] The following sequences can also be used as protease cleavage sequences:

[0849] X10-X11-X1-X2-X3-X4-X5-X6-X7-X8(SEQ ID NO:1064)

[0850] wherein X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, F, L, M, P, Q, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F , G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

[0851] The following sequences can also be used as protease cleavage sequences:

[0852] X10-X11-X1-X2-X3-X4-X5-X6-X7-X8(SEQ ID NO:1065)

[0853] wherein X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E , F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

[0854] The following sequences can also be used as protease cleavage sequences:

[0855] X10-X11-X1-X2-X3-X4-X5-X6-X7-X8(SEQ ID NO:1066)

[0856] wherein X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from wherein X is an amino acid selected from the group consisting of A, D, E, G, H, I, K, L, M, N, Q, R, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y.

[0857] The following sequences can also be used as protease cleavage sequences:

[0858] X10-X11-X1-X2-X3-X4-X5-X6-X7-X8(SEQ ID NO:1067)

[0859] wherein X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R ; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from E, F, K, M, N, P, Q, R, S and W; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

[0860] The following sequences can also be used as protease cleavage sequences:

[0861] X10-X11-X1-X2-X3-X4-X5-X6-X7-X8(SEQ ID NO:1068)

[0862] wherein X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is an amino acid selected from A, D, F, G, L, M, P, Q, V and W; and X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y.

[0863] The following sequences can also be used as protease cleavage sequences:

[0864] X10-X11-X1-X2-X3-X4-X5-X6-X7-X8(SEQ ID NO:1069)

[0865] wherein X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and X8 is an amino acid selected from A, D, E, F, G, I, K, N, T and W.

[0866] The following sequences can also be used as protease cleavage sequences:

[0867] X10-X11-X1-X2-X3-X4-X5-X6-X7-X8(SEQ ID NO:1070)

[0868] wherein X1 to X11 each represent a single amino acid, X10 is an amino acid sequence selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, G, I, P, Q, S and Y; X2 is an amino acid selected from K or T; X3 is G; X4 is R; X5 is S; X6 is A; X7 is an amino acid selected from H, I and V; and X8 is an amino acid selected from H, V and Y.

[0869] The following sequences can also be used as protease cleavage sequences:

[0870] X10-X11-X1-X2-X3-X4-X5-X6-X7-X8(SEQ ID NO:1071)

[0871] wherein X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is Y; X2 is an amino acid selected from S and T; X3 is G; X4 is R; X5 is S; X6 is an amino acid selected from A and E; X7 is an amino acid selected from N and V; and X8 is an amino acid selected from H, P, V and Y.

[0872] The following sequences can also be used as protease cleavage sequences:

[0873] X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9(SEQ ID NO:1072)

[0874] wherein X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I , K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and X9 is an amino acid sequence selected from A, G, H, I, L and R.

[0875] The following sequences can also be used as protease cleavage sequences:

[0876] X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9(SEQ ID NO:1073)

[0877] wherein X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, E, F, G, H, K, M, N, P, Q, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L , M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and X9 is an amino acid sequence selected from A, G, H, I, L and R.

[0878] The following sequences can also be used as protease cleavage sequences:

[0879] X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9(SEQ ID NO:1074)

[0880] wherein X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, F, L, M, P, Q, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, L, wherein X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X is an amino acid sequence selected from the group consisting of A, G, H, I, L, and R.

[0881] The following sequences can also be used as protease cleavage sequences:

[0882] X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9(SEQ ID NO:1075)

[0883] wherein X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, E, F, H, I, K, L, M, N, P, Q, R, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, F, G, H, I, K, wherein X is an amino acid selected from the group consisting of A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X is an amino acid sequence selected from the group consisting of A, G, H, I, L, and R.

[0884] The following sequences can also be used as protease cleavage sequences:

[0885] X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9(SEQ ID NO:1076)

[0886] wherein X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E, G, H, wherein X is an amino acid selected from the group consisting of A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X is an amino acid sequence selected from the group consisting of A, G, H, I, L, and R.

[0887] The following sequences can also be used as protease cleavage sequences:

[0888] X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9(SEQ ID NO:1077)

[0889] wherein X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, E , F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X6 is an amino acid selected from E, F, K, M, N, P, Q, R, S and W; X7 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X8 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and X9 is an amino acid sequence selected from A, G, H, I, L and R.

[0890] The following sequences can also be used as protease cleavage sequences:

[0891] X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9(SEQ ID NO:1078)

[0892] wherein X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, wherein X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and X is an amino acid sequence selected from the group consisting of A, G, H, I, L, and R.

[0893] The following sequences can also be used as protease cleavage sequences:

[0894] X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9(SEQ ID NO:1079)

[0895] wherein X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, D, E, F, G, H, I, K, M, N, P, Q, S, T, W and Y; X2 is an amino acid selected from A, D, E, F, H, K, L, M, P, Q, S, T, V, W and Y; X3 is an amino acid selected from A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; X4 is R; X5 is an amino acid selected from A, D, wherein X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; X is an amino acid selected from the group consisting of A, D, E, F, G, I, K, N, T, and W; and X is an amino acid sequence selected from the group consisting of A, G, H, I, L, and R.

[0896] The following sequences can also be used as protease cleavage sequences:

[0897] X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9(SEQ ID NO:1080)

[0898] wherein X1 to X11 each represent a single amino acid, X10 is an amino acid sequence selected from I, T and Y; X11 is S; X1 is an amino acid selected from A, G, I, P, Q, S and Y; X2 is an amino acid selected from K or T; X3 is G; X4 is R; X5 is S; X6 is A; X7 is an amino acid selected from H, I and V; X8 is an amino acid selected from H, V and Y; and X9 is an amino acid selected from A, G, H, I, L and R.

[0899] The following sequences can also be used as protease cleavage sequences:

[0900] X10-X11-X1-X2-X3-X4-X5-X6-X7-X8-X9(SEQ ID NO:1081)

[0901] wherein X1 to X11 each represent a single amino acid, X10 is an amino acid selected from I, T and Y; X11 is S; X1 is Y; X2 is an amino acid selected from S and T; X3 is G; X4 is R; X5 is S; X6 is an amino acid selected from A and E; X7 is an amino acid selected from N and V; X8 is an amino acid selected from H, P, V and Y; and X9 is an amino acid selected from A, G, H, I, L and R.

[0902] Except using above-mentioned protease cleavage sequence, can also obtain new protease cleavage sequence by screening.For example, based on the result to the crystal structure analysis of known protease cleavage sequence, can explore new protease cleavage sequence by the interaction of active residue / recognition residue and this enzyme that changes cleavage sequence.Can also explore new protease cleavage sequence by the interaction between the sequence that changes and this protease that checks amino acid in the known protease cleavage sequence.As another example, can explore protease cleavage sequence by checking the peptide library that protease and use in vitro display method (for example phage display and ribosome display) show, or with the interaction that is fixed on the peptide array on chip or bead.

[0903] The interaction between a protease cleavage sequence and a protease can be examined by testing the protease for cleavage of the sequence in vitro or in vivo.

[0904] The cleavage fragments after protease treatment can be separated and quantified by electrophoresis such as SDS-PAGE to evaluate the protease cleavage sequence, the activity of the protease, and the cleavage ratio of the molecule into which the protease cleavage sequence has been introduced. Non-limiting embodiments of the method for evaluating the cleavage ratio of a molecule into which a protease cleavage sequence has been introduced include the following method: For example, when the cleavage ratio of an antibody variant into which a protease cleavage sequence has been introduced is evaluated using recombinant human u-plasminogen activator / urokinase (human uPA, huPA) (R&D Systems; 1310-SE-010) or recombinant human matriptase / ST14 catalytic domain (human MT-SP1, hMT-SP1) (R&D Systems; 3946-SE-010), 100 μg / mL of the antibody variant is reacted with 40 nM huPA or 3 nM hMT-SP1 in PBS at 37°C for 1 hour, and then capillary electrophoresis immunoassay is performed. Capillary electrophoresis immunoassay can be performed using Wes (Protein Simple), but the present method is not limited thereto. As an alternative to capillary electrophoresis immunoassay, SDS-PAGE or the like can be performed for separation, followed by Western blotting for detection. This method is not limited to these methods. Before and after cleavage, the light chain can be detected using an anti-human λ chain HRP-labeled antibody (abcam: ab9007), but any antibody that can detect cleavage fragments can be used. The area of ​​each peak obtained after protease treatment is output using software for Wes (Compass for SW; Protein Simple), and the cleavage ratio (%) of the antibody variant can be determined by the following formula:

[0905] (Peak area of ​​cleaved light chain) x 100 / (Peak area of ​​cleaved light chain + Peak area of ​​uncleaved light chain)

[0906] If protein fragments can be detected both before and after protease treatment, the cleavage ratio can be determined. This can be done not only for antibody variants but also for various protein molecules into which the protease cleavage sequence has been introduced.

[0907] The in vivo cleavage ratio of a molecule into which a protease cleavage sequence has been introduced can be determined by administering the molecule to an animal and detecting the administered molecule in a blood sample. For example, an antibody variant into which a protease cleavage sequence has been introduced is administered to a mouse, and plasma is collected from its blood sample. According to methods known to those skilled in the art, the antibody is purified from the plasma using Dynabeads Protein A (Thermo; 10001D), and then a capillary electrophoresis immunoassay is performed to evaluate the protease cleavage ratio of the antibody variant. Capillary electrophoresis immunoassay can be performed using Wes (Protein Simple), but the method is not limited thereto. As an alternative to the capillary electrophoresis immunoassay, SDS-PAGE or the like can be performed for separation, followed by detection using Western blotting. The method is not limited to these methods. The light chain of the antibody variant collected from the mouse can be detected using an anti-human λ chain HRP-labeled antibody (abcam; ab9007), but any antibody that can detect cleavage fragments can be used. Once the area of ​​each peak obtained by capillary electrophoresis immunoassay is output using the software of Wes (Compass for SW; Protein Simple), the proportion of the remaining light chain can be calculated as [peak area of ​​light chain] / [peak area of ​​heavy chain] to determine the proportion of full-length light chain that remains uncleaved in the mouse. If protein fragments collected from living organisms can be detected, the in vivo cleavage efficiency can be determined. Not only can the cleavage ratio of the antibody variant be determined, but also the cleavage ratio of various protein molecules into which the protease cleavage sequence has been introduced can be determined. Calculating the cleavage ratio by the above method makes it possible, for example, to compare the in vivo cleavage ratios of antibody variants into which different cleavage sequences have been introduced, as well as to compare the cleavage ratios of individual antibody variants between different animal models (e.g., normal mouse models and tumor-transplanted mouse models).

[0908] For example, the protease cleavage sequences shown in Table 1 are all newly discovered by the present inventors. Polypeptides containing these protease cleavage sequences can be used as protease substrates that are hydrolyzed by the action of proteases. Therefore, the present invention provides protease substrates comprising sequences selected from SEQ ID NOs: 833-852 and 1062-1081, and the sequences listed in Table 1. The protease substrates of the present invention can be used, for example, as libraries, from which proteins with properties that meet the desired objectives can be selected for incorporation into the polypeptides of the present invention. Specifically, in order to selectively cleave the polypeptides of the present invention by proteases localized in lesions, the sensitivity of the substrate to the protease can be assessed. When the polypeptides of the present invention are administered in vivo, the molecule can come into contact with various proteases before reaching the lesion. Therefore, the molecule should preferably be sensitive to the proteases localized in the lesion and have as high a resistance as possible to other proteases. In order to select the desired protease cleavage sequence based on the desired objective, each protease substrate can be comprehensively analyzed in advance for its sensitivity to various proteases to determine its protease resistance. Based on the obtained protease resistance profile, it is possible to identify a protease cleavage sequence with the necessary sensitivity and resistance.

[0909] Alternatively, the polypeptide into which the protease cleavage sequence has been incorporated is subjected not only to the enzymatic action of the protease before reaching the lesion, but also to various environmental stresses, such as pH changes, temperature, and oxidation / reduction stress. Based on comparative information about resistance to these external factors between protease substrates, a protease cleavage sequence with the desired properties can be selected.

[0910] In one embodiment of the present invention, a flexible linker is further attached to one or both ends of the protease cleavage sequence. The flexible linker at one end of the protease cleavage sequence may be referred to as a first flexible linker, and the flexible linker at the other end may be referred to as a second flexible linker. In a specific embodiment, the protease cleavage sequence and the flexible linker have any of the following general formulas:

[0911] (protease cleavage sequence),

[0912] (first flexible linker)-(protease cleavage sequence),

[0913] (protease cleavage sequence)-(second flexible linker), and

[0914] (First flexible linker)-(Protease cleavage sequence)-(Second flexible linker).

[0915] The flexible linker according to the present embodiment is preferably a peptide linker. The first flexible linker and the second flexible linker are each independently and arbitrarily present, and are identical or different flexible linkers in which each linker contains at least one flexible amino acid (Gly, etc.). The flexible linker comprises, for example, a sufficient number of residues for the protease cleavage sequence (arbitrarily selected from amino acids such as Arg, Ile, Gln, Glu, Cys, Tyr, Trp, Thr, Val, His, Phe, Pro, Met, Lys, Gly, Ser, Asp, Asn, Ala, in particular Gly, Ser, Asp, Asn and Ala, in particular Gly and Ser, especially Gly, etc.) to obtain the desired protease accessibility.

[0916] The flexible linker that is applicable to the two ends of the protease cleavage sequence is generally such a flexible linker: it improves the contact (access) of protease to the protease cleavage sequence and improves the cleavage efficiency of the protease. Suitable flexible linkers can be easily selected, and preferably can be selected from different lengths, for example 1 amino acid (Gly etc.) to 20 amino acids, 2 amino acids to 15 amino acids or 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids or 7 amino acids to 8 amino acids. In some embodiments of the present invention, the flexible linker is a peptide linker of 1 to 7 amino acids.

[0917] Examples of the flexible linker include, but are not limited to, glycine polymer (G)n, glycine-serine polymer (including, for example, (GS)n, (GSGGS: SEQ ID NO: 27)n, and (GGGS: SEQ ID NO: 28)n, wherein n is an integer of at least 1), glycine-alanine polymer, alanine-serine polymer, and other flexible linkers well known in the art.

[0918] Among them, glycine polymers and glycine-serine polymers have attracted attention because these amino acids are relatively unstructured and can easily function as neutral tethers between components.

[0919] Examples of flexible linkers composed of glycine-serine polymers include, but are not limited to:

[0920] Ser

[0921] Gly·Ser(GS)

[0922] Ser·Gly(SG)

[0923] Gly·Gly·Ser(GGS)

[0924] Gly·Ser·Gly(GSG)

[0925] Ser·Gly·Gly(SGG)

[0926] Gly·Ser·Ser(GSS)

[0927] Ser·Ser·Gly(SSG)

[0928] Ser·Gly·Ser(SGS)

[0929] Gly·Gly·Gly·Ser(GGGS,SEQ ID NO:28)

[0930] Gly·Gly·Ser·Gly(GGSG,SEQ ID NO:29)

[0931] Gly·Ser·Gly·Gly(GSGG,SEQ ID NO:46)

[0932] Ser·Gly·Gly·Gly(SGGG,SEQ ID NO:47)

[0933] Gly·Ser·Ser·Gly(GSSG,SEQ ID NO:48)

[0934] Gly·Gly·Gly·Gly·Ser(GGGGS,SEQ ID NO:49)

[0935] Gly·Gly·Gly·Ser·Gly(GGGSG,SEQ ID NO:33)

[0936] Gly·Gly·Ser·Gly·Gly(GGSGG,SEQ ID NO:30)

[0937] Gly·Ser·Gly·Gly·Gly(GSGGG,SEQ ID NO:32)

[0938] Gly·Ser·Gly·Gly·Ser(GSGGS,SEQ ID NO:27)

[0939] Ser·Gly·Gly·Gly·Gly(SGGGG,SEQ ID NO:51)

[0940] Gly·Ser·Ser·Gly·Gly(GSSGG,SEQ ID NO:52)

[0941] Gly·Ser·Gly·Ser·Gly(GSGSG,SEQ ID NO:31)

[0942] Ser·Gly·Gly·Ser·Gly(SGGSG,SEQ ID NO:53)

[0943] Gly·Ser·Ser·Ser·Gly(GSSSG,SEQ ID NO:34)

[0944] Gly·Gly·Gly·Gly·Gly·Ser(GGGGGS,SEQ ID NO:50)

[0945] Ser·Gly·Gly·Gly·Gly·Gly(SGGGGG,SEQ ID NO:54)

[0946] Gly·Gly·Gly·Gly·Gly·Gly·Ser(GGGGGGS,SEQ ID NO:55)

[0947] Ser·Gly·Gly·Gly·Gly·Gly·Gly(SGGGGGG,SEQ ID NO:56)

[0948] (Gly·Gly·Gly·Gly·Ser(GGGGS,SEQ ID NO:49))n

[0949] (Ser·Gly·Gly·Gly·Gly(SGGGG,SEQ ID NO:51))n

[0950] In this specification, "association" may refer to, for example, the state in which two or more polypeptide regions interact with each other. Typically, hydrophobic bonds, hydrogen bonds, ionic bonds, and the like are formed between the desired polypeptide regions to form an association product. As an example of common association, antibodies, such as natural antibodies, are known to retain a paired structure of a heavy chain variable region (VH) and a light chain variable region (VL) through non-covalent bonds therebetween.

[0951] In some embodiments of the present invention, the inhibitory domain of the transport moiety is associated with the antigen binding domain. The inhibitory domain can constitute a portion of the transport moiety or can constitute the entire transport moiety. From another perspective, the inhibitory domain can also be defined as the portion of the transport moiety that is associated with the antigen binding domain.

[0952] In a more specific embodiment, the antigen binding domain and the inhibitory domain form an association such as that found between an antibody VH and an antibody VL, wherein the antigen binding domain is a single domain antibody and the inhibitory domain is VL, VH or VHH. In a further specific embodiment, the antigen binding domain and the inhibitory domain form an association such as that found between an antibody VH and an antibody VL, wherein the antigen binding domain is a single domain antibody and the inhibitory domain is VL, VH or VHH, and in the associated state thus formed, the inhibitory domain conformationally inhibits the binding of the antigen binding domain to the antigen, or conformationally changes the antigen binding site of the antigen binding domain so that the antigen binding activity of the single domain antibody is inhibited by VL, VH or VHH. In embodiments using VHH as a single domain antibody, it is believed that when CDR3 (the primary antigen binding site of VHH) or its adjacent sites are present at the interface associated with the inhibitory domain, the binding of VHH to the antigen is subject to conformational inhibition by the inhibitory domain.

[0953] The association of the antigen binding domain and the inhibitory domain can be eliminated, for example, by cleaving the cleavage site. The elimination of this association can be used interchangeably with, for example, the elimination of a state in which two or more polypeptide domains interact with each other. The interaction between two or more polypeptide domains can be completely eliminated, or the interaction between two or more polypeptide domains can be partially eliminated.

[0954] In this specification, "interface" generally refers to the surface where two regions associate or interact with each other. The amino acid residues forming the interface are generally one or more amino acid residues contained in each polypeptide region that undergo association, and more preferably refer to amino acid residues that are in proximity with each other and participate in the interaction during association. Specifically, the interaction includes non-covalent bonds, such as hydrogen bonds, electrostatic interactions, or salt bridge formation, between amino acid residues that are in proximity with each other during association.

[0955] In this specification, "interface-forming amino acid residues" specifically refers to amino acid residues contained in the polypeptide region that constitutes the interface. As an example, the polypeptide region that constitutes the interface refers to the polypeptide region responsible for selective intramolecular or intermolecular binding in antibodies, ligands, receptors, substrates, etc. Specific examples of such polypeptide regions in antibodies may include heavy chain variable regions and light chain variable regions. In some embodiments of the present invention, examples of such polypeptide regions may include antigen-binding domains and inhibitory domains.

[0956] Examples of amino acid residues that form an interface include, but are not limited to, amino acid residues that are in close proximity to each other during association. For example, amino acid residues that are in close proximity to each other during association can be found by analyzing the conformation of the polypeptides and examining the amino acid sequences of the regions of the polypeptides that form an interface during polypeptide association.

[0957] In some embodiments of the present invention, the amino acid residues involved in association in the antigen-binding domain, or the amino acid residues involved in association in the inhibitory domain, can be changed to promote the association of the antigen-binding domain with the inhibitory domain. In further specific embodiments, the amino acid residues in the antigen-binding domain that form the interface with the inhibitory domain, or the amino acid residues in the inhibitory domain that form the interface with the antigen-binding domain can be changed. In a preferred embodiment, the amino acid residues forming the interface can be changed by introducing mutations into the interface amino acid residues so that the two or more amino acid residues forming the interface have different charges. Changes in amino acid residues that result in different charges include: positively charged amino acid residues are changed to negatively charged amino acid residues or uncharged amino acid residues, negatively charged amino acid residues are changed to positively charged amino acid residues or uncharged amino acid residues, and uncharged amino acid residues are changed to positively charged or negatively charged amino acid residues. Such amino acid changes are carried out to promote association and are not limited by the position of the amino acid change or the type of amino acid, as long as the purpose of promoting association can be achieved. Examples of changes include, but are not limited to, substitutions.

[0958] In some embodiments of the present invention, the VHH used as an antigen binding domain is associated with a VL as an inhibitory domain. The amino acid residues involved in the association with VL in VHH may refer to, for example, amino acid residues that form the interface between VHH and VL. Examples of amino acid residues involved in the association with VL in VHH include, but are not limited to, amino acid residues at positions 37, 44, 45, and 47 (J. Mol. Biol. (2005) 350, 112-125). The activity of VHH is inhibited by promoting the association between VHH and VL. Similarly, the amino acid residues involved in the association with VHH in VL may refer to, for example, amino acid residues that form the interface between VHH and VL.

[0959] The amino acid residues involved in the association with the VL in the VHH can be changed to promote the association between the VHH and VL. Examples of such amino acid substitutions include, but are not limited to, F37V, Y37V, E44G, Q44G, R45L, H45L, G47W, F47W, L47W, T47W, and / or S47W. Instead of changing every residue in the VHH, a VHH originally having amino acid residues 37V, 44G, 45L, and / or 47W can also be used.

[0960] Amino acid residues involved in association with VHH may be changed in VH instead of VHH amino acids, and amino acid changes may also be introduced into both VHH and VL as long as the purpose of promoting the association between VHH and VL can be achieved.

[0961] In some alternative embodiments of the present invention, the antigen-binding domain and the inhibitory domain can be associated with each other by using VHH as the antigen-binding domain and using VH or VHH as the inhibitory domain. The amino acid residues in the VHH used as the antigen-binding domain that participate in the association with the VH or VHH used as the inhibitory domain can be identified and changed to promote the association of the antigen-binding domain VHH with the inhibitory domain VH or VHH. In addition, the amino acid residues in the VH or VHH used as the inhibitory domain that participate in the association with the VHH used as the antigen-binding domain can be identified and changed.

[0962] When a single-domain antibody other than VHH is used as the antigen-binding domain, the amino acid residues involved in the association in the antigen-binding domain or inhibitory domain can also be identified and changed similarly to the above.

[0963] In some embodiments of the invention, the transport moiety and the antigen binding domain are fused via a linker. In more specific embodiments, the transport moiety and the antigen binding domain are fused via a linker that contains a cleavage site. In alternative specific embodiments, the transport moiety and the antigen binding domain are fused via a linker, and the resulting fusion protein contains a cleavage site.

[0964] 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 fusion protein formed contains a cleavage site. In a specific embodiment, one to several N-terminal amino acids of the transport portion and / or one to several C-terminal amino acids of the antigen binding domain are changed, 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, the cleavage site can be formed, for example, by converting the four C-terminal amino acids of the antigen binding domain to an LSGR sequence and converting the four N-terminal amino acids of the transport portion to an SDNH sequence.

[0965] In some embodiments of the 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 can be placed at any position in the polypeptide, as long as the antigen binding domain is released by protease cleavage and does not lose its antigen binding activity after release.

[0966] In some embodiments of the present invention, the transport moiety comprises an antibody constant region, and the N-terminus of the antibody constant region and the C-terminus of the antigen binding domain are fused with or without a linker.

[0967] In a specific embodiment, the protease cleavage sequence is located within the antibody constant region contained in the transport portion. In this case, the protease cleavage sequence can be located within the antibody constant region so that the antigen-binding domains are released by protease cleavage. In a specific embodiment, the protease cleavage sequence is located within the antibody heavy chain constant region contained in the transport portion, more specifically located on a side closer to the antigen-binding domains than the amino acid at position 140 (EU numbering) in the antibody heavy chain constant region, preferably on a side closer to the antigen-binding domains than the amino acid at position 122 (EU numbering) in the antibody heavy chain constant region. In an alternative embodiment, the protease cleavage sequence is located within the antibody light chain constant region contained in the transport portion, and more specifically located on a side closer to the antigen-binding domains than the amino acid at position 130 (EU numbering) (Kabat numbering position 130) in the antibody light chain constant region, preferably on a side closer to the antigen-binding domains than the amino acid at position 113 (EU numbering) (Kabat numbering position 113) in the antibody light chain constant region.

[0968] In some embodiments of the 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 moiety are fused with or without a linker.

[0969] In a specific embodiment, the protease cleavage sequence is located within a single domain antibody. In a more specific embodiment, the single domain antibody is a single domain antibody prepared from VH or VHH, and the protease cleavage sequence is located on the side of the transport portion closer to the amino acid at position 35b (Kabat numbering) in the single domain antibody, preferably on the side of the transport portion closer to the amino acid at position 95 (Kabat numbering) in the single domain antibody, and more preferably on the side of the transport portion closer to the amino acid at position 109 (Kabat numbering) in the single domain antibody. In an alternative specific embodiment, the single domain antibody is a single domain antibody prepared from VL, and the protease cleavage sequence is located on the side of the transport portion closer to the amino acid at position 32 (Kabat numbering) in the single domain antibody, preferably on the side of the transport portion closer to the amino acid at position 91 (Kabat numbering) in the single domain antibody, and more preferably on the side of the transport portion closer to the amino acid at position 104 (Kabat numbering) in the single domain antibody.

[0970] In some embodiments of the present invention, the transport moiety comprises an antibody constant region, the antigen binding domain is a single domain antibody, and the antibody constant region and the single domain antibody are fused with or without a linker. In more specific embodiments, the N-terminus of the antibody constant region and the C-terminus of the single domain antibody are fused with or without a linker. In alternative specific embodiments, the C-terminus of the antibody constant region and the N-terminus of the single domain antibody are fused with or without a linker.

[0971] In a specific embodiment, the protease cleavage sequence is located within the antibody constant region contained in the transport portion. In a more specific embodiment, the protease cleavage sequence is located on the side of the antibody heavy chain constant region that is closer to the single domain antibody than the amino acid at position 140 (EU numbering), preferably on the side of the antibody heavy chain constant region that is closer to the single domain antibody than the amino acid at position 122 (EU numbering). In an alternative specific embodiment, the protease cleavage sequence is located on the side of the antibody light chain constant region that is closer to the antigen binding domain than the amino acid at position 130 (EU numbering) (Kabat numbering position 130), preferably on the side of the antibody light chain constant region that is closer to the antigen binding domain than the amino acid at position 113 (EU numbering) (Kabat numbering position 113).

[0972] In a specific embodiment, the protease cleavage sequence is located within a single domain antibody. In a more specific embodiment, the single domain antibody is a single domain antibody prepared by VH or by VHH, and the protease cleavage sequence is located on the side of the antibody constant region that is closer to the amino acid at position 35b (Kabat numbering) in the single domain antibody, preferably on the side of the antibody constant region that is closer to the amino acid at position 95 (Kabat numbering) in the single domain antibody, and more preferably on the side of the antibody constant region that is closer to the amino acid at position 109 (Kabat numbering) in the single domain antibody. In an alternative specific embodiment, the single domain antibody is a single domain antibody prepared by VL, and the protease cleavage sequence is located on the side of the antibody constant region that is closer to the amino acid at position 32 (Kabat numbering) in the single domain antibody, preferably on the side of the antibody constant region that is closer to the amino acid at position 91 (Kabat numbering) in the single domain antibody, and more preferably on the side of the antibody constant region that is closer to the amino acid at position 104 (Kabat numbering) in the single domain antibody.

[0973] In certain embodiments, the protease cleavage sequence is located near the boundary between the antigen binding domain and the transport portion. The phrase "near the boundary between the antigen binding domain and the transport portion" refers to a portion that is located upstream or downstream of the site of attachment between the antigen binding domain and the transport portion and does not significantly affect the secondary structure of the antigen binding domain.

[0974] In a more specific embodiment, the antigen binding domain is linked to the antibody constant region contained in the transport portion, and the protease cleavage sequence is located near the boundary between the antigen binding domain and the antibody constant region. The phrase "near 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 prepared from VH or VHH and is linked to the antibody heavy chain constant region, the phrase "near the boundary between the antigen binding domain and the antibody constant region" can refer to between amino acid position 101 (Kabat numbering) of the single domain antibody and amino acid position 140 (EU numbering) of the antibody heavy chain constant region, and preferably can refer to between amino acid position 109 (Kabat numbering) of the single domain antibody and amino acid position 122 (EU numbering) of the antibody heavy chain constant region. When the antigen-binding domain is a single-domain antibody prepared from VH or VHH and is linked to an antibody light chain constant region, the phrase "near the boundary between the antigen-binding domain and the antibody light chain constant region" may refer to between amino acid position 101 (Kabat numbering) of the single-domain antibody and amino acid position 130 (EU numbering) (Kabat numbering position 130) of the antibody light chain constant region, and preferably may refer to between amino acid position 109 (Kabat numbering) of the single-domain antibody and amino acid position 113 (EU numbering) (Kabat numbering position 113) of the antibody light chain constant region. When the antigen-binding domain is a single-domain antibody prepared from VL, the phrase "near the boundary between the antigen-binding domain and the antibody constant region" refers to between amino acid position 96 (Kabat numbering) of the single-domain antibody and a designated position of the antibody constant region, and preferably between amino acid position 104 (Kabat numbering) of the single-domain antibody and a designated position of the antibody constant region.

[0975] In some embodiments of the present invention, the polypeptide is an IgG antibody-like molecule. Examples of such embodiments include, but are not limited to, embodiments in which the transport portion comprises an IgG antibody constant region, a single domain antibody serving as the antigen-binding domain replaces the VH of the IgG antibody, and the antigen-binding activity is suppressed by the VL; embodiments in which the transport portion comprises an IgG antibody constant region, a single domain antibody serving as the antigen-binding domain replaces the VL of the IgG antibody, and the antigen-binding activity is suppressed by the VH; and embodiments in which the transport portion comprises an IgG antibody constant region, a single domain antibody serving as the antigen-binding domain replaces one of the VH and VL of the IgG antibody, and another single domain antibody that suppresses the antigen-binding activity of the antigen-binding domain replaces the other domain of the IgG antibody.

[0976] As used herein, the term "IgG antibody-like molecule" is used to define a molecule comprising a portion that is structurally substantially similar to the constant domain or constant region of an IgG antibody, and a portion that is structurally substantially similar to the variable domain or variable region of an IgG antibody, wherein the molecule has a conformation substantially similar to that of an IgG antibody. In IgG antibody-like molecules, domains similar to antibody CH1 and domains similar to CL can be used interchangeably; that is, as long as there is an interaction between the domains similar to the interaction between CH1 and CL of an IgG antibody, the domain connected to the portion similar to the hinge region of an antibody can be the antibody CH1 domain or the antibody CL domain. However, in the present specification, an "IgG antibody-like molecule" may or may not exhibit antigen-binding activity while retaining a structure similar to that of an IgG antibody.

[0977] The polypeptide may comprise one or more antigen-binding domains. One or more inhibitory domains may inhibit the antigen-binding activity of the multiple antigen-binding domains. The multiple antigen-binding domains may each be associated with an inhibitory domain. The multiple antigen-binding domains may each be fused to a transport moiety. The multiple antigen-binding domains may each be releasable from the polypeptide. The cleavage sites for releasing the multiple antigen-binding domains may be multiple cleavage sites corresponding to the number of antigen-binding domains.

[0978] When the polypeptide is an IgG antibody-like molecule, the antigen binding domains can be established in the portions corresponding to the two variable regions of the IgG antibody, such as Figure 7 As shown. With reference to the present invention, those skilled in the art will understand such embodiments. The antigen-binding domains incorporated into the two arms can have the same antigen-binding specificity or different antigen-binding specificities. With reference to the present invention, those skilled in the art will understand such embodiments. Clearly, these embodiments are within the scope of the present invention.

[0979] In some embodiments of the present invention, the antigen binding domain is further connected to the second antigen binding domain. Examples of the second antigen binding domain include, but are not limited to, single domain antibodies, antibody fragments, a module of about 35 amino acids contained in the cell membrane protein avimer referred to as the A domain (WO2004 / 044011 and WO2005 / 040229), an adnectin containing 10Fn3 domains, which is derived from a protein binding domain of the glycoprotein fibronectin expressed on the cell membrane (WO2002 / 032925), an affibody containing an IgG binding domain scaffold, wherein the IgG binding domain scaffold constitutes a three-helix bundle consisting of 58 amino acids of protein A (WO1995 / 001937), DARPins (designed ankyrin repeat proteins), which are molecular surface exposed structures of ankyrin repeats (AR). The present invention relates to a novel polypeptide chain containing a plurality of antigen-binding domains, each having a 33-amino acid residue structure folded into a turn subunit, two antiparallel helices and a loop (WO2002 / 020565), an anticalin having four loop regions connecting eight antiparallel chains, which bend toward the central axis at one end of a barrel-like structure, the barrel structure being highly conserved in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) (WO2003 / 029462), and a concave region in a horseshoe-shaped internal parallel plate structure composed of repetitive leucine-rich repeat (LRR) modules of variable lymphocyte receptors (VLRs) that do not have an immunoglobulin structure, as found in the acquired immune system of jawless vertebrates such as lampreys or hagfishes (WO2008 / 016854). In a preferred embodiment, the second antigen-binding domain has an antigen-binding specificity different from that of the first 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.

[0980] In some more specific embodiments, the antigen binding domain and the second antigen binding domain are single domain antibodies with different antigen binding specificities, and the connected antigen binding domain and the second antigen binding domain can be released from the polypeptide, and the antigen binding domain and the second antigen binding domain form a bispecific antigen binding molecule after release. Examples of such bispecific antigen binding molecules include, but are not limited to, bispecific antigen binding molecules having an antigen binding domain that specifically binds to a target cell antigen and a second antigen binding domain that specifically binds to an immune cell surface antigen, bispecific antigen binding molecules having an antigen binding domain and a second antigen binding domain that binds to different subunits of the same antigen, and bispecific antigen binding molecules having an antigen binding domain and a second antigen binding domain that binds to different epitopes of the same antigen. Such bispecific antigen binding molecules can recruit immune cells to the vicinity of target cells and are therefore considered useful in treating diseases caused by target cells.

[0981] The antigen-binding activity of the second antigen-binding domain may or may not be inhibited by the transport moiety. The second antigen-binding domain may or may not be associated with a partial structure of the transport moiety. In particular, when the antigen-binding domain and the second antigen-binding domain differ in antigen-binding specificity, the antigen-binding domain in the unreleased state cannot exert antigen-binding activity, e.g. Figure 8 As shown, even if the antigen-binding activity of the second antigen-binding domain is not inhibited and even if the second antigen-binding domain is not associated with a partial structure of the transport moiety, the bispecific antigen-binding molecule comprising an antigen-binding domain linked to the second antigen-binding domain cannot function to bispecifically bind to two antigens.

[0982] Figure 8 An exemplary format is shown in which the antigen binding domain is further linked to a second antigen binding domain.

[0983] In this specification, the term "specificity" refers to the property that one of the specific binding molecules does not substantially bind to molecules other than one or more of its binding partner molecules. This term is also used when the antigen-binding domain is specific for an epitope contained in a specific antigen. This term is also used when the antigen-binding domain is specific for a specific epitope among multiple epitopes contained in an antigen. Herein, the term "substantially does not bind" is determined according to the method described in the section on binding activity, and means that the binding activity of the specific binding molecule for molecules other than its binding partner is 80% or less of its binding activity for its binding partner molecule, typically 50% or less, preferably 30% or less, and particularly preferably 15% or less.

[0984] The present invention also relates to a pharmaceutical composition (medicament) comprising the polypeptide of the present invention and a pharmaceutically acceptable carrier.

[0985] As used herein, "treatment" (and its grammatical derivatives, such as "treat" and "treating") refers to clinical intervention intended to alter the natural course of the individual being treated, and can be performed both for prevention and during the course of a clinical pathological condition. Desirable effects of such treatment include, but are not limited to, preventing the development or recurrence of a disease, alleviating symptoms, alleviating any direct or indirect pathological effects of a disease, preventing metastasis, reducing the rate of progression of a disease, remission or alleviation of the disease condition, and improving or enhancing prognosis. In some embodiments, the polypeptides of the invention are used to delay the onset of a disease or to delay the progression of a disease.

[0986] In the present invention, a pharmaceutical composition generally refers to a drug used to treat or prevent a disease or for testing or diagnosis. In the present invention, the term "pharmaceutical composition comprising a polypeptide" is used interchangeably with "a method for treating a disease, comprising administering a polypeptide to a subject to be treated," and is used interchangeably with "use of a polypeptide for preparing a medicament for treating a disease." Furthermore, the term "pharmaceutical composition comprising a polypeptide" is used interchangeably with "use of a polypeptide for treating a disease."

[0987] The pharmaceutical compositions of the present invention can be formulated using methods known to those skilled in the art. For example, the pharmaceutical compositions can be administered parenterally in the form of injections of sterile solutions or suspensions containing water or any other pharmaceutically acceptable liquid. The pharmaceutical compositions can be formulated by, for example, combining the polypeptide with a pharmacologically acceptable carrier or medium, particularly sterile water or physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring, excipient, carrier, preservative, adhesive, and the like, and mixing them into a unit dosage form suitable for generally accepted pharmaceutical practice. The amount of active ingredient in these formulations is adjusted so as to provide an appropriate volume within a specified range.

[0988] The sterile composition for injection can be prepared using a carrier such as distilled water for injection according to conventional pharmaceutical practice. Examples of injectable aqueous solutions include isotonic solutions containing physiological saline, glucose or other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol and sodium chloride). The aqueous solution can be used in combination with an appropriate solubilizing agent, for example, an alcohol (e.g., ethanol), a polyol (e.g., propylene glycol, polyethylene glycol) or a nonionic surfactant (e.g., Polysorbate 80 (trademark), HCO-50, etc.).

[0989] Examples of oil solutions include sesame oil and soybean oil. Oil solutions can also be used in combination with benzyl benzoate and / or benzyl alcohol as solubilizers. Oil solutions can be supplemented with buffers (e.g., phosphate buffered saline and sodium acetate buffered solutions), soothing agents (e.g., procaine hydrochloride), stabilizers (e.g., benzyl alcohol and phenol), and antioxidants. The prepared injection solution is usually placed in a suitable ampoule.

[0990] The pharmaceutical compositions of the present invention are preferably administered parenterally. For example, the compositions may be administered in parenteral, nasal, pulmonary, or transdermal dosage forms. The pharmaceutical compositions can be administered systemically or topically, for example, by intravenous, intramuscular, intraperitoneal, or subcutaneous injection.

[0991] The method of administration can be appropriately selected according to the patient's age and symptoms. The dosage of the pharmaceutical composition containing the polypeptide can be set within the range of, for example, 0.0001 mg to 1000 mg per kilogram of body weight per administration. Alternatively, the dosage of the pharmaceutical composition containing the polypeptide can be set to, for example, a dosage of 0.001 mg to 100,000 mg per patient. However, the present invention is not necessarily limited to these numerical values. Although the dosage and administration method vary according to the patient's weight, age, symptoms, etc., those skilled in the art can set an appropriate dosage and administration method taking these conditions into consideration.

[0992] The present invention also relates to a method for preparing a polypeptide comprising a transport portion having an inhibitory domain and an antigen binding domain

[0993] The method for preparing the polypeptide of the present invention is a method comprising the following: obtaining an antigen-binding domain having antigen-binding activity; connecting the antigen-binding domain to a transport portion so that the antigen-binding activity of the antigen-binding domain is inhibited by the inhibitory domain to form a polypeptide precursor, and further inserting a cleavage site into the polypeptide precursor, or changing a part of the polypeptide precursor into a cleavage site. The method for introducing the cleavage site can be the insertion of any cleavage site and the change of a part of the polypeptide precursor, as long as the cleavage site can be introduced into the polypeptide precursor. Alternatively, the altered site can be introduced into the polypeptide precursor by a combination of the two methods. For those skilled in the art, with reference to this specification, such embodiments should be apparent and are included within the scope of the present invention.

[0994] Another method for preparing the polypeptide of the present invention is a method comprising: obtaining an antigen-binding domain having antigen-binding activity; and linking the antigen-binding domain to a transport moiety via a cleavage site, such that the antigen-binding activity of the antigen-binding domain is inhibited by an inhibitory domain, thereby forming a polypeptide. When the antigen-binding domain is linked to the transport moiety via a cleavage site, the cleavage site may be sandwiched between the antigen-binding domain and the transport moiety, or a portion of the antigen-binding domain and / or a portion of the transport moiety may be altered and used as part of the cleavage site.

[0995] "Inserting" amino acid sequence A into amino acid sequence B means dividing amino acid sequence B into two parts without deletion and connecting the two parts with amino acid sequence A (i.e., generating the following amino acid sequence: "first half amino acid sequence B-amino acid sequence A-second half amino acid sequence B"). "Introducing" amino acid sequence A into amino acid sequence B means dividing amino acid sequence B into two parts and connecting the two parts with amino acid sequence A. This not only encompasses "inserting" amino acid sequence A into amino acid sequence B as described above, but also encompasses connecting the two parts with amino acid sequence A after deleting one or more amino acid residues of amino acid sequence B (including residues adjacent to amino acid sequence A) (that is, replacing a portion of amino acid sequence B with amino acid sequence A).

[0996] In embodiments using a single domain antibody as the antigen binding domain and a protease cleavage sequence as the cleavage site, methods for producing the polypeptide are as follows.

[0997] In one embodiment of the present invention, a method for preparing a polypeptide comprising a trafficking moiety having an inhibitory domain and an antigen binding domain is a method comprising the following steps:

[0998] (a) obtaining a single domain antibody that binds to a target antigen;

[0999] (b) linking the single domain antibody obtained in step (a) to a transport moiety such that the antigen binding activity of the single domain antibody is inhibited by the inhibitory domain of the transport moiety to form a polypeptide precursor; and

[1000] (c) Introducing a protease cleavage sequence into the polypeptide precursor.

[1001] In one embodiment of the present invention, a method for preparing a polypeptide comprising a trafficking moiety having an inhibitory domain and an antigen binding domain is a method comprising the following steps:

[1002] (a) obtaining a single domain antibody that binds to a target antigen;

[1003] (b) linking the single domain antibody obtained in step (a) to a transport moiety such that the antigen binding activity of the single domain antibody is inhibited by the inhibitory domain of the transport moiety to form a polypeptide precursor; and

[1004] (c) A protease cleavage sequence is introduced near the boundary between the single domain antibody and the transport moiety.

[1005] In one embodiment of the present invention, a method for preparing a polypeptide comprising a trafficking moiety having an inhibitory domain and an antigen binding domain is a method comprising the following steps:

[1006] (a) obtaining a single domain antibody that binds to a target antigen; and

[1007] (b) linking the single domain antibody obtained in step (a) to a transport moiety via a protease cleavage sequence, such that the antigen binding activity of the single domain antibody is inhibited by the inhibitory domain of the transport moiety, to form a polypeptide.

[1008] In certain embodiments, the method for preparing a polypeptide comprising a trafficking moiety having an inhibitory domain and an antigen binding domain is a method of preparation further comprising the steps of:

[1009] (d) confirming that the binding activity of the single domain antibody incorporated into the polypeptide or incorporated into the polypeptide precursor to the target antigen is weakened or lost.

[1010] In the present invention, the phrase "binding activity is weakened" means that the binding activity to the target antigen is reduced compared to before ligation, and the degree of this reduction is not limited.

[1011] In certain embodiments, the method for preparing a polypeptide comprising a trafficking moiety having an inhibitory domain and an antigen binding domain is a method of preparation further comprising the steps of:

[1012] (e) releasing the single domain antibody by protease cleavage of the protease cleavage sequence and confirming that the released single domain antibody binds to the antigen.

[1013] In one embodiment of the present invention, the method for preparing a polypeptide is a preparation method comprising the following steps, wherein the polypeptide is an IgG antibody-like molecule comprising a transport portion having an inhibitory domain and an antigen binding domain:

[1014] (a) obtaining a single domain antibody that binds to a target antigen;

[1015] (b) allowing the single domain antibody obtained in step (a) to associate with VL as a substitute for VH of an IgG antibody, or allowing the single domain antibody to bind to VH as a substitute for VL of an IgG antibody, so that the antigen-binding activity of the single domain antibody is suppressed, to form an IgG antibody-like molecule precursor comprising the single domain antibody; and

[1016] (c) Introducing a protease cleavage sequence into an IgG antibody-like molecule precursor comprising a single domain antibody.

[1017] In one embodiment of the present invention, the method for preparing a polypeptide is a preparation method comprising the following steps, wherein the polypeptide is an IgG antibody-like molecule comprising a transport portion having an inhibitory domain and an antigen binding domain:

[1018] (a) obtaining a single domain antibody that binds to a target antigen;

[1019] (b) allowing the single domain antibody obtained in step (a) to associate with VL as a substitute for VH of an IgG antibody, or allowing the single domain antibody to bind to VH as a substitute for VL of an IgG antibody, so that the antigen-binding activity of the single domain antibody is suppressed, to form an IgG antibody-like molecule precursor comprising the single domain antibody; and

[1020] (c) A protease cleavage sequence is introduced near the boundary between the single domain antibody and the antibody constant region in the IgG antibody-like molecule precursor.

[1021] In one embodiment of the present invention, the method for preparing a polypeptide is a preparation method comprising the following steps, wherein the polypeptide is an IgG antibody-like molecule comprising a transport portion having an inhibitory domain and an antigen binding domain:

[1022] (a) obtaining a single domain antibody that binds to a target antigen; and

[1023] (b) connecting the single domain antibody obtained in step (a) as a substitute for IgG antibody VH or VL to the IgG antibody heavy chain constant region or light chain constant region through a protease cleavage sequence, so that the antigen binding activity of the single domain antibody is inhibited to form an IgG antibody-like molecule comprising the single domain antibody.

[1024] In certain embodiments, the method for preparing a polypeptide is a method for preparing a ...

Claims

1. A polypeptide comprising an antigen binding domain and a transport portion, wherein the transport portion has an inhibitory domain that inhibits the antigen binding activity of the antigen binding domain, and the antigen binding domain has a shorter half-life than the transport portion, and wherein the polypeptide has a protease cleavage sequence comprising one or more sequences selected from the group consisting of: SEQ ID NO:678,171,673,371,693,389,801,813,492,802,814,493,168-170,172-178,208-370,372-388,390-491,494-672,674-677,679-692,694-800,803-812,815, and 1052 to 1055, wherein the polypeptide releases the antigen binding domain when cleaved by a protease, wherein the antigen binding domain comprises a single domain antibody and the inhibitory domain of the transport portion is VHH, antibody VH or antibody VL, wherein the transport portion comprises an antibody constant region, wherein the N-terminus of the antibody constant region of the transport portion and the C-terminus of the antigen binding domain are fused with or without a linker, wherein the protease cleavage sequence is located near the boundary between the antigen binding domain and the antibody constant region.

2. A polypeptide comprising an antigen binding domain and a transport portion, wherein the transport portion has an inhibitory domain that inhibits the antigen binding activity of the antigen binding domain, and the antigen binding domain has a shorter half-life than the transport portion, and wherein the polypeptide has a protease cleavage sequence comprising one or more sequences selected from the following: SEQ ID NO: 853-951, wherein the polypeptide releases the antigen binding domain when cleaved by a protease, wherein the antigen binding domain comprises a single domain antibody, and the inhibitory domain of the transport portion is VHH, antibody VH or antibody VL, wherein the transport portion comprises an antibody constant region, wherein the N-terminus of the antibody constant region of the transport portion and the C-terminus of the antigen binding domain are fused with or without a linker, wherein the protease cleavage sequence is located near the boundary between the antigen binding domain and the antibody constant region.

3. A polypeptide according to claim 1 or 2, wherein the inhibition of the antigen-binding activity of the antigen-binding domain by the inhibitory domain in a state where the protease cleavage sequence has been cleaved by a protease is weaker than the inhibition of the antigen-binding activity of the antigen-binding domain by the inhibitory domain in a state where the protease cleavage sequence has not been cleaved.

4. The polypeptide according to claim 1 or 2, wherein the molecular weight of the antigen binding domain is 60 kDa or less, and the molecular weight of the antigen binding domain is less than that of the transport portion, or 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 claim 1 , wherein the antigen-binding domain has higher antigen-binding activity in a state in which it is released from the polypeptide than in a state in which it is not released from the polypeptide.

6. The polypeptide of claim 1 or 2, wherein the antigen binding activity of the antigen binding domain is inhibited by association of the inhibitory domain of the transport moiety with the antigen binding domain.

7. The polypeptide of claim 1 or 2, wherein the protease cleavage sequence is cleaved by a protease such that the association of the inhibitory domain of the transport moiety with the antigen binding domain is abolished. The polypeptide according to claim 1 or 2, wherein the protease is a cancer tissue-specific protease or an inflammatory tissue-specific protease.

9. The polypeptide according to claim 1, wherein the antibody constant region of the polypeptide is an IgG antibody constant region.

10. The polypeptide according to claim 9, wherein the polypeptide is an IgG antibody-like molecule.

11. The polypeptide of claim 1 or 2, wherein a second antigen binding domain is further linked to the antigen binding domain.

12. The polypeptide of claim 11, wherein the first antigen binding domain and the second antigen binding domain form a bispecific antigen binding molecule when released.

13. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 12.

14. A method for producing the polypeptide according to any one of claims 1 to 12.

15. A polynucleotide encoding the polypeptide according to any one of claims 1 to 12.

16. A vector comprising the polynucleotide according to claim 15.

17. A host cell comprising the polynucleotide according to claim 15, or the vector according to claim 16.

18. A method for preparing the polypeptide according to any one of claims 1 to 12, comprising the step of culturing the host cell according to claim 17.

19. A method for preparing a polypeptide according to any one of claims 1 to 12, comprising the following steps: (a) obtaining a single domain antibody that binds to a target antigen; (b) linking the single domain antibody obtained in step (a) to a transport moiety such that the antigen binding activity of the single domain antibody is inhibited by the inhibitory domain of the transport moiety to form a polypeptide precursor; and (c) Introducing a protease cleavage sequence into the polypeptide precursor.

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